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JULY/JUILLET 2017, VOL. 81, NO. 3

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Published quarterly by the Canadian Veterinary Medical AssociationPublication trimestrielle de l’Association canadienne des médecins vétérinaires

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ARTICLES

Porcine reproductive and respiratory syndrome virus (PRRSV) in pig meat Philippe Raymond, Christian Bellehumeur, Malliga Nagarajan, Diane Longtin, Alexandra Ferland, Peter Müller, Rachel Bissonnette, Carole Simard . . . . . . . . . . . . . . . . . 162

Development of porcine circovirus 2 (PCV2) open reading frame 2 DNA vaccine with different adjuvants and comparison with commercial PCV2 subunit vaccine in an experimental challenge Changhoon Park, Jiwoon Jeong, Kyuhyung Choi, Su-Jin Park, Ikjae Kang, Chanhee Chae . . . . . . . . . . . . . 171

Retrospective study of the relationship of Torque teno sus virus 1a and Torque teno sus virus 1b with porcine circovirus associated diseaseAlejandro Vargas-Ruiz, Hugo Ramírez-Álvarez, José I. Sánchez-Betancourt, Víctor Quintero-Ramírez, Ignacio C. Rangel-Rodríguez, Joel A. Vázquez-Perez, Lucia A. García-Camacho . . . . . . . . . . . . . . . . . . . . . . . . 178

Identification of the linear ligand epitope on classical swine fever virus that interacts with porcine kidney 15 cellsYin Mei, Feng Yue, Hong-mei Ning, Juan-juan Zhou, Xuan-nian Wang . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186

Direct repeat unit (dru) typing and antimicrobial resistance of methicillin-resistant Staphylococcus pseudintermedius isolated from dogs in Atlantic Canada Matthew E. Saab, J. Scott Weese, J.T. McClure. . . . . . . . 192

The impact of carboplatin and toceranib phosphate on serum vascular endothelial growth factor (VEGF) and metalloproteinase-9 (MMP-9) levels and survival in canine osteosarcomaTracy L. Gieger, Julie Nettifee-Osborne, Briana Hallman, Chad Johannes, Dawn Clarke, Michael W. Nolan, Laurel E. Williams . . . . . . . . . . . . . . 199

Oxidative stress and food supplementation with antioxidants in therapy dogsSara Sechi, Filippo Fiore, Francesca Chiavolelli, Corrado Dimauro, Anna Nudda, Raffaella Cocco . . . . 206

Evaluation of transmission infrared spectroscopy and digital and optical refractometers to identify low immunoglobulin G concentrations in alpaca serumIbrahim Elsohaby, Jennifer J. Burns, Christopher B. Riley, J. Trenton McClure . . . . . . . . . . . . 217

Cystic duct pressures after ligation with a novel absorbable device in an ex vivo caprine cholecystectomy modelAndrea J. Sundholm Tepper, Odd V. Höglund, Bonnie G. Campbell, Chi-Ya Chen, Boel A. Fransson . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223

SHORT COMMUNICATIONS/COMMUNICATIONS BRÈVES

Evaluation of serum symmetric dimethylarginine in dogs with heartworm infectionBom-Sul Choi, Hyeongsun Moon, Sang-IL Suh, Changbaig Hyun . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228

Maternal and fetal arterial blood gas data in normotensive, singleton, isoflurane anesthetized sheep at 124–126 days of gestationClaire M. Loughran, Matthew W. Kemp, Gabrielle C. Musk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231

Towards an improved estimate of antimicrobial use in animals: Adjusting the “population correction unit” calculation Brian R. Radke . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235

JULY/JUILLET 2017, VOL. 81, NO. 3

Article

162 The Canadian Journal of Veterinary Research 2017;81:162–170

I n t r o d u c t i o nPorcine reproductive and respiratory syndrome (PRRS) is among

the most economically significant swine infectious diseases (1,2). The role of pig meat and swill feeding in PRRS virus (PRRSV) trans-mission is questioned as international trade in pork expands (3). The enveloped virus is between 50 and 65 nm in diameter and is classified in the Arteriviridae family within the order Nidovirales. It contains a single-stranded positive-sense RNA of approximately

15 kb in length that encodes at least 9 open reading frames (ORF) (4). There are 2 recognized PRRSV genotypes: the North American (genotype 2, VR-2332 prototype) and the European (genotype 1, Lelystad prototype). Both have similar genomic organizations, but are genetically and antigenically distinct (5). In Canada, the North American genotype has been reported in lineages typical of vac-cines (Ingelvac PRRS ATP, Ingelvac PRRS MLV) and of the MN-184 wild types (6,7). Porcine reproductive and respiratory syndrome is mainly characterized by reproductive failure in sows and respiratory

Porcine reproductive and respiratory syndrome virus (PRRSV) in pig meatPhilippe Raymond, Christian Bellehumeur, Malliga Nagarajan, Diane Longtin,

Alexandra Ferland, Peter Müller, Rachel Bissonnette, Carole Simard

A b s t r a c tPorcine reproductive and respiratory syndrome, caused by the porcine reproductive and respiratory syndrome virus (PRRSV), is an economically important disease in the swine industry. Previous studies demonstrated the presence of the virus in pig meat and its transmissibility by oral consumption. This study further analyzed the infectivity of PRRSV in commercial pig meat. Fresh bottom meat pieces (n = 1500) randomly selected over a period of 2 y from a pork ham boning plant located in Quebec, Canada, were tested by reverse transcriptase polymerase chain reaction (RT-PCR). Each trimmed meat was stored in the plant freezer, subsampled weekly for up to 15 wk, and tested with quantitative RT-PCR to determine the viral load. Meat infectivity was evaluated using specific pathogen-free piglets, each fed with approximately 500 g of meat at the end of the storage time. Genotype-specific RT-PCR confirmed the presence of PRRSV mainly during cold weather in 0.73% of the fresh meat pieces. Wild and vaccine strains of genotype 2 were detected. Porcine reproductive and respiratory syndrome virus nucleic acid was stable in meat stored at around 220°C during the 15 wk. Serological and molecular analysis showed the transmission of infection by a majority of PRRSV positive meat pieces (5/9) fed orally to naïve recipients. The results confirmed a low prevalence of PRRSV in market’s pig meat, and virus transmissibility by oral consumption to naïve recipients even after several weeks of storage in a commercial freezer. It occurred mainly with meat harboring the highest PRRSV RNA copies, in the range of 109 copies per 500 g of meat, with both wild type and vaccine-related strains.

R é s u m éLe syndrome reproducteur et respiratoire porcin, causé par le virus du syndrome respiratoire et reproducteur porcin (vSRRP), est une maladie ayant un impact économique important pour l’industrie porcine. Des études antérieures ont démontré la présence du virus dans la viande de porc ainsi que sa transmissibilité par ingestion. La présente étude poursuit l’analyse de l’infectiosité du vSRRP dans la viande commerciale de porc. Des coupes de fesses de porc fraîches (n = 1500) sélectionnées aléatoirement sur une période de deux ans dans une usine de désossage située au Québec (Canada), ont été testées en utilisant une transcription réverse suivie d’une amplification en chaîne par polymérase (RT-PCR). Chaque pièce de viande parée a été entreposée dans les congélateurs à l’usine, échantillonnée hebdomadairement pendant 15 semaines, et testée par RT-PCR quantitatif afin de calculer la charge virale. Le potentiel infectieux a été évalué sur des porcelets exempts d’agent pathogène spécifique qui ont été nourris avec approximativement 500 g de viande à la fin de la période d’entreposage. Une RT-PCR spécifique au génotype a confirmé la présence du vSRRP principalement durant les temps froids, dans 0,73 % des pièces de viandes fraîches. Des souches sauvages et vaccinales du génotype 2 ont été détectées. L’acide nucléique du virus du syndrome respiratoire et reproducteur porcin est demeuré stable dans la viande durant la période d’entreposage de 15 semaines à 220 °C. L’analyse sérologique et moléculaire a démontré la transmission de l’infection par une majorité des pièces de viande positives au vSRRP (5/9) chez les porcelets naïfs ayant consommé la viande. Les résultats confirment la faible prévalence du vSRRP dans la viande distribuée sur le marché ainsi que la transmissibilité du virus par consommation orale chez des hôtes naïfs même après plusieurs semaines d’entreposage dans un congélateur commercial. La transmission s’est produite surtout avec les viandes ayant un nombre de copies d’ARN de vSRRP plus élevés, environ 109 copies par 500 g de viande, associées à des souches de type tant sauvage que vaccinal.

(Traduit par les auteurs)

Canadian Food Inspection Agency, Saint-Hyacinthe Laboratory, Saint-Hyacinthe, Quebec J2S 8E3.

Address all correspondence to Dr. Philippe Raymond; telephone: (450) 768-6808; fax: (450) 768-6767; e-mail: [email protected]

Received July 7, 2016. Accepted January 17, 2017.

2000;64:0–00 The Canadian Journal of Veterinary Research 163

illness in pigs of all ages. These clinical signs vary markedly between herds and depend on the virulence of the infecting strain. In eastern Europe and China, new highly pathogenic subtypes of PRRSV have been reported (8–10).

Porcine reproductive and respiratory syndrome virus is highly contagious in pig herds. It can be transmitted by direct contact or by infectious aerosols. Vaccines have been used to limit the propa-gation of the disease, but are often found inefficient (11). The virus can also persist for months in the target cells of the monocyte and macrophage lineages of an infected animal, despite the immune protection (11,12). Transient detection of PRRSV in pig meat follow-ing experimental transmission has been observed (12). The virus was isolated in vitro from 6 out of 1049 sample pools of fresh meat (13), while others failed to detect PRRSV in 472 pig carcasses tested by PCR (14).

In countries currently free of PRRS, the risk of importing the PRRS virus in fresh pork is a concern. Some early import risk analyses for chilled or frozen meats have concluded that virally infected pig meat could represent a source for the introduction of the virus in PRRS-free countries (15,16). These analyses were notably based on a study from Lelystad, the Netherlands, showing the transmission of PRRSV through oral uptake of infected porcine muscular tissues by naïve recipients (17,18). In 2003, Magar and Larochelle (6) led a study to investigate whether pig meat could harbor PRRSV and if so, whether viruses in positive meats could infect the animals when fed to SPF pigs. In their study, analyses and bioassays were performed with meat conserved at ultra-low temperatures. It was argued that these experi-ments were performed in optimal conditions for virus survival in meat (19–21). For instance, van der Linden et al (18) showed that freezing meat at 223°C for 10 d and then thawing it decreased the virus titers in the majority of PRRSV infected muscle samples. The present study was undertaken to reproduce conditions in commercial settings under which meat would be cut, packaged, and stored in a frozen state for many weeks, similar to overseas import or export conditions. It pro-vides additional knowledge on PRRSV prevalence, survival in pig meat, meat viral load, and transmission from meat to naïve piglets.

M a t e r i a l s a n d m e t h o d s

Pork samplesPork carcasses were from market pigs killed at 3 abattoirs from

the province of Quebec, Canada. Carcasses were transported in refrigerated trucks and received at a pork ham boning plant (PHBP) located in St. Hyacinthe, Quebec, 2 to 5 d after slaughter. Between 5 and 17 fresh bottom meat pieces of around 900 to 1100 g each were selected randomly from the cutting and trimming lines 2 to 3 d a week (117 visits). The meat pieces could not be associated with a specific abattoir once on the cutting and trimming line. Each meat piece was identified using a unique identification number (ID). Approximately 2 g of muscle tissue was subsampled from each meat piece with a sterile scalpel at 3 distant locations and pooled in a 50 mL sterile tube. These subsamples corresponded to week 0 (T0). Meat subsamples were kept on ice until their arrival at the CFIA Saint Hyacinthe Laboratory for RNA extraction a few hours later. At the PHBP, each of the selected fresh bottom meat pieces was put

in a labeled plastic bag and stored immediately in cardboard boxes among commercial meats in the company’s freezer, maintained between 224°C and 221°C. Based on the reverse transcriptase polymerase chain reaction (RT-PCR) assays, 1 PRRSV non-confirmed, 9 PRRSV positive, and 2 RT-PCR PRRSV negative frozen meat pieces were selected for further analysis. In storage follow-up experiments, they were subsampled weekly at the PHBP from T1 up to 15 consecu-tive weeks (Tmax). Subsamples were collected in each frozen meat piece near the first sampling locations using a clean and sterilized bit and a drill. Approximately 2 g of frozen meat were subsampled and kept on ice until processed at the lab a few hours later. The bottom frozen meat pieces were immediately put back in their boxes in the company’s freezer, among commercial meats. At the last sampling time (Tmax), leftover frozen meats were transported in a frozen state and stored at 270°C, until used in viral transmission bioassays.

Viral transmission bioassaysAll animals were treated according to the policy and guidelines

of the Canadian Council on Animal Care (CCAC) and the Animal Care Committee of our laboratory. For the feeding trials, 9 PRRSV RT-PCR confirmed positive and one non-confirmed pig meat sample were fed to the piglets. These bioassays were adapted from Magar and Larochelle (6) using the same facilities, biosecurity measures, and animal adaption protocol. Each meat sample was used to feed 2 specific pathogen-free (SPF) piglets 5 to 6 wk of age. Each pig pair was housed in a separate cubicle. These piglets were provided by the CFIA Ottawa Laboratory (Fallowfield, Ontario). The meat samples were thawed at 4°C overnight, weighed, cut into small pieces (around 2 cm3) with sterile scalpels, and divided in 4 equal portions of approximately 250 g each. Piglets were fed a portion on 2 consecutive days. The portion used to feed the pigs on the second day was kept at 4°C overnight. In an alternative protocol, 3 meat samples (ID 1311, 1332, and 1424) were also divided into 4 equal por-tions of around 250 g. While 2 portions were stored at 4°C overnight, the other 2 were stored at room temperature (RT, between 18°C to 23°C). A pig was fed on 2 consecutive days with the meat sample stored at 4°C while the paired animal, located in a separate cubicle, received the other part of the same meat sample stored at RT. Each trial included either 1 or 2 control pigs maintained on a standard pig diet with no thawed meat throughout the experiments.

Enzyme-linked immunosorbent assay (ELISA)Blood samples were collected on a regular basis from the jugu-

lar vein of each piglet with a 20-G needle. Throat mucus samples were collected with minitip flocked swabs (Millipore; Billerica, Massachusetts, USA) to follow PRRSV infection in piglets. Both were collected at arrival and at 0, 7, 14, 21, and 28 d after feeding. Only the throat samples from the first 3 bioassays were tested. Serum and throat samples were used to detect PRRSV by molecular assays. Serum samples were tested for the presence of antibodies to PRRSV in piglets via ELISA (HerdCheck; IDEXX Laboratories, Westbrook, Maine, USA) as recommended by the manufacturer.

RNA extractionAll meat subsamples from the PHBP were homogenized on the

same collecting day as previously described (22). Supernatant


aliquots were stored at 270°C until nucleic acid extraction. RNA was extracted from 100 mL of the meat homogenate using the RNeasy Mini Kit according to the manufacturer’s protocol (Qiagen, Hilden, Germany). Total RNA was eluted with 40 μL of RNase-free water. A Nanodrop-1000 (Thermo Scientific, Wilmington, Delaware, USA) was used to quantify total RNA in each sample (OD 260 nM). For the meat-fed piglets, RNA was extracted from 140 mL of serum sample using the QIAamp Viral RNA Mini Kit (Qiagen). RNA was also extracted from throat swab samples using RNeasy Mini Kit with 0.6 mL RLT buffer. All RNA extracts were stored at 270°C prior to the analyses. The OR7 RT-PCR analyses were conducted within a week while samples from decay were combined on the same plate and tested by OR7 qRT-PCR at the end of the storage period. Phylogeny analyses were conducted on RNA extract stored up to 3 y.

RT-PCRThe RT-PCR for the detection of the ORF-7 gene of both American

and European strains was performed on 1500 fresh meat samples collected randomly as a screen test (T0). A 3 mL volume of RNA extract was mixed with 0.48 mM of primers P1 and P2 developed previously and tested using the Qiagen One-Step RT-PCR Kit and the GeneAmp PCR System 9770 (Applied Biosystems, Carlsbad, California, USA) in the presence of RNasin (Promega, Madison, Wisconsin, USA) (23). The RT-PCR included a reverse transcription step at 50°C for 30 min and a PCR amplification step with enzyme activation at 95°C for 15 min followed by 34 cycles of 94°C for 30 s, 57°C for 30 s, and 72°C for 30s, and a final elongation step at 72°C for 7 min. When analyzed on ethidium bromide agarose gel (2%) a specific band of 303 bp was expected for genotype 2, whereas a band of 291 bp was expected for the genotype 1. A sample having a band of the expected molecular weight was declared PRRSV presumptive, if the positive and negative controls were valid. Each presumptive positive RNA sample was retested with distinct ORF-7 RT-PCR using similar RT-PCR mix and amplification conditions, but different primer sets. These tests were also done to determine the genotype of the virus. The primer set PR15M (GGTAAGATCATCGCTCAGCA) and PR16M (GACACAATTGCCGCTCACTA) was developed in this study and was specific to genotype 2 (ORF-7 RT-PCR) (data not shown). This primer set amplified a region that corresponds to bp position 14969 to 15116 of NVSL (AY545985.1). To detect genotype 1, P4 and P2 primers were used (23). Each presumptive positive was also tested with PRRSV primers UN17F and UNI17R used in the study of Magar and Larochelle (6). A presumptive meat was con-firmed PRRSV positive using at least 2 distinct extracts tested with ORF-7 RT-PCR assays, otherwise it was declared non-confirmed. This approach was used to reduce the number of false positives. Meat harboring a virus level close to the LOD might have been classified as non-confirmed in this process as well as some cross-contaminated pieces.

RNA transcriptAn RNA transcript was produced from the PRRSV NVSL strain

97-7895 using the PR29F (TTAATACGACTCACTATAGGGTGGG TAAGATCATCGCTCAGCA) and PR30R (TTGACGACAGACA CAATTGCCGCT) primer set as well as the MEGAshortscript T7 Kit, and purified with the MEGAclear Kit (Applied Biosystems/Ambion,

Mississauga, Ontario). The RNA transcript was quantified with the Nanodrop-1000.

SYBR Green quantitative Real-Time RT-PCR (qRT-PCR)

To assess viral load before the transmission experiment at T10 or Tmax in meats and to evaluate viral decay over time from T1 to Tmax, viral RNA copies were estimated by one-step SYBR Green qRT-PCR assay (ORF-7 qRT-PCR) using the QuantiTect SYBR Green RT-PCR Kit (Qiagen) an the RNA transcript standard. The PR15M and PR16M primers and the MX4000 or MX3005p real-time thermocyclers (Agilent Technologies, Santa Clara, California, USA) were used for these assays. The qRT-PCR included a reverse transcription step at 50°C for 30 min and a PCR amplification step with enzyme activa-tion at 95°C for 15 min. These were followed by 40 cycles of 95°C for 15 s, 57°C for 30 s, and 72°C for 30 s, and finally a dissociation cycle. All samples from the same animal (T0 to Tmax) were analyzed on the same plate to avoid inter-assay variability. In the presence of a specific melting temperature [Mt (78.5°C to 80.5°C)], the sample was declared positive. Samples were declared positive but not quantifi-able for PRRSV when non-specific Mt was detected in addition to the specific one. These results were indicative of a simultaneous non-specific amplification and were detected only below 100 viral RNA copies/mL, which represents the limit of quantification (LOQ). No specific amplification was detected more than 95% of the time below the limit of detection (LOD) of 10 viral RNA copies/mL. The number of viral RNA copies per mg of each quantifiable subsample tested by qRT-PCR was calculated according to the standard curve multiplied by the RNA extract volume (40 mL), divided by the extraction effi-cacy factor (7%) and by the weight of the meat homogenate sample (20 mg per extract), and normalized using the total RNA extracted.

Porcine circovirus (PCV) detectionFor the detection of PCV nucleic acid in meat extract, DNA was

extracted using the DNeasy Tissue Kit (Qiagen) and recovered with 50 μL of elution buffer. A volume of 3 μL was tested using the Platinum Taq DNA Polymerase Kit (Invitrogen), 1.5 mM MgCl2, and 0.2 mM of each VCP5F (AGTGAGCGGGAAAATGCA) and VCP6R (CACACAGTCTCAGTAGATCATCC) primers. This primer set amplified a region that corresponds to bp position 515 to 741 of postweaning multisystemic wasting syndrome PCV (AF027217.1). The PCR was achieved after 2 min of incubation at 95°C followed by 35 cycles at 95°C for 1 min, 56°C for 1 min, and 72°C for 1 min. The PCR products were kept at 4°C until agarose gel evaluation. Positive results for the PCR were further confirmed using the same PCR conditions with primer set VCP22F (TGGCCCGCAGTATTCTGATT) and VCP23R (CAGCTGGGACAGCAGTTGAG) that amplified a region that correspond to bp position 790 and 861 of postweaning multisystemic wasting syndrome PCV.

Virus decayTo estimate virus decay over time, a ratio of viral RNA copy

(Cx/C1) was calculated for each quantifiable subsample of a storage follow-up series, where Cx represents the copy numbers/mL for a specific subsample from week 2 to week 10 (Tx), and C1 represents the value obtained at week one (T1). The degradation rate “k” was


determined by plotting the natural logarithm transformation of the ratio of viral RNA copies (Cx/C1) versus time (weeks). Virus half-life was then calculated using the equation virus half-life = (ln2)/k. Statistical analyses were performed on the linear regression model by ANOVA using the F statistic (Minitab release 16 software). The T0 and T1 values of a same series were also compared using a paired t-test, with P , 0.05 being statistically significant.

Meat viral loadThe meat viral load was estimated at T10 or Tmax by multiplying the

normalized PRRSV RNA copies per mg by the total meat quantity given to 1 pig for the bioassay (around 500 g).

Phylogeny analysisPhylogeny analysis was done using the nucleotide sequence of

an amplified PRRSV ORF-5 region of NVSL. The RT-PCR was done using primer set P420 and P620 (24) and the One-Step RT-PCR Kit as described. The nested PCR was done using primer set 5FN and 5DN (25) and Platinum TAQ DNA Polymerase Kit (Invitrogen, Carlsbad, California, USA). The ORF-5 cDNA sequences were aligned using the neighbor-joining method with Clone Manager (Scientific & Educational Software, Cary, North Carolina, USA). An ORF-5 sequence of 544 bp was used for the alignment, which corresponded to bp positions 13811 to 14354 of NVSL. Genbank reference strains used for the alignment were: 17198-6 (EF442776.1), 2000-54471A (EU556182.1), 34075-NE (U66380.1), 98-6470-1 (AF339493.1), CH-1a (AY032626.1), FJ-1 (AY881994.1), HB-1 sh/2002 (DQ642048.1), IA-27 (EU758940.1), IAF-EXP91 (L40898.1), IAF-Klop (U64928.1), Ingelvac ATP MLV (DQ988080.1), Ingelvac RespPRRS MLV (AF066183.4), Lelystad (M96262.2), Lena (JF802085.1), MD-001 (AF121131.1), MN184 (EF442777.1), NADC-8 (AF396835.1), PA8 (AF176348.2), PrimePAC (AF066384.1), PRRSV0003749 (DQ477778.1), SDSU73 (EF442775.1), and VR-2332 (AY150564.1).

Re s u l t s

PRRSV in pig meatOverall, 1500 fresh meat samples were collected randomly at the

PHBP over a 2-year period. All meat samples were screened for the presence of PRRSV using the RT-PCR targeting both American and European strains. Among meat samples, 16 (1.1%) were found presumptive positive for PRRSV with a typical amplified frag-ment size of around 303 bp, a very faint band for some samples. Presumptive positives were tested with distinct ORF-7 RT-PCR to confirm the positive results and to define the PRRSV genotypes. Out of 16 presumptive PRRSV positive cases, 11 were confirmed positive with at least a second set of ORF-7 primers. Those confirmed posi-tives represented 0.73% of the overall meat tested. All confirmed positives were typical of the North American strains. From these, 9 were selected to further estimate the viral decay and to determine the infectivity of the stored meat at Tmax by feeding piglets during bioassays. Two samples (ID 245 and 587) were not selected because they were positive for PCV type 2 in RT-PCR (data not shown). A non-confirmed and 2 RT-PCR negative meat samples were also kept for the viral decay study. During the study, 3 confirmed PRRSV posi-

tive meats were detected in April (3/98), 2 in September (2/120), 2 in October (2/225), 1 for each January (1/91), February (1/123), May (1/112), and November (1/97), and none during the months of March (0/86), June (0/120), July (0/194), August (0/184), and December (0/50).

Estimation of PRRSV load and viral decay in pig meat

To estimate viral decay, qRT-PCR was performed on meat sub-samples maintained in the PHBP freezer and collected weekly for up to 15 wk (Table I). During this follow-up series, only 4 meat samples had an average PRRSV RNA concentration above the LOQ. For meats ID 715 and 1311, the number of viral RNA copies in almost all the subsamples could not be estimated (, LOQ) although specific RT-PCR products were detected for PRRSV (10/10 and 9/11, respec-tively) confirming their positive status. For sample 1424, only 3 out of 10 subsamples were positive and values were below the LOQ. No follow-up subsamples were positive for ID 574 and ID 597 from T1 to Tmax. Sample ID 340 and both negative controls remained negative throughout the storage period.

To estimate the variation of virus decay over time, a ratio of normalized viral RNA copies of each quantifiable subsample of a follow-up series was calculated for samples ID 84, 115, 319, and 1332 (Table I). No significant decay in viral RNA concentrations of PRRSV was observed during the storage period. Subsamples from ID 84 were tested in 3 separate experiments to estimate the inter-assay coefficient of variation (CV%) of the meat extract viral RNA copies/mL. It was estimated at 42%, showing significant variation from one qRT-PCR test to another for the same sample.

The meat viral load was estimated at T10 or Tmax, based on the viral RNA copies detected per mg of meat (Table I). The extraction efficacy factor was found to be extremely low for the internal extraction con-trol that was used. Only 7% of the input virus in the homogenized meat subsamples was recovered after RNA extraction of the muscle tissues. Recovery rates between 1% and 12% were also reported in previous studies using process control viruses with mouse norovirus, mengo, and hepatitis E virus (26). Numbers vary according to the method, the matrix, and the control virus. Pork meat usually has a high fat content that can interfere with nucleic acid extraction and inhibit the subsequent detection step. Nevertheless, low recovery yields are associated with high inter-assay variations. Based on these results, each piglet consumed approximately 109.2 to 109.8 viral RNA copies of PRRSV for every 500 g of meat it was fed. This range was estimated to be equivalent to 105.0 to 105.6 viral RNA copies per mL after extraction.

Experimental transmissionResults of PRRSV and antibody detection from pigs fed with

PRRSV positive meat samples are summarized in Table II. Overall, a total of 18 piglets were fed in 5 different bioassays using 2, 3, 1, 2, and 1 positive PRRSV meat samples, respectively. Two piglets were fed with a non-confirmed PRRSV meat sample. A total of 9 control piglets were included in the bioassays. Meat samples were gener-ally readily consumed after much chewing. Usually, all meat pieces were eaten by the pigs within 30 min to 2 h. At one occasion, the sample was smaller and the pigs receiving the sample were fed


with a total of 386 g instead of ~500 g (ID 715, Table II). During the 28-day observation period, all pigs appeared healthy. Positive transmission was observed with 5 confirmed PRRSV positive meat samples (5/9, 55.6%). Transmission in PRRSV confirmed positive cases was detected at 7 d post-exposure (DPE) by PCR analysis of the serum and throat samples and at 14 DPE by serology. None of the control piglets were shown to be infected. The meat viral load was an important factor for transmission. Oral transmission was detected with all meat samples (4/4) with quantifiable viral load. The pair of piglets fed with meat sample ID 1332 was found positive with both meat pieces stored at RT and at 4°C. Positive transmission was also detected with only 1 of the 2 piglets exposed to sample ID 1424. This sample remained non-quantifiable throughout the follow-up in the freezer and only a limited set of subsamples at T2, T3, and T9 were found to be qRT-PCR positive (, LOQ). For this animal, viral transmission occurred only with the sample that was thawed at 4°C. The pair of piglets that was fed with meat sample ID 1311 was also apparently exposed to a lower virus load and was not infected. Average PRRSV RNA copies in meat sample ID 1311 was below the limit of quantification except at T0 and T5. The pair of piglets that consumed the non-confirmed positive meat sample ID 340 was not infected either.

Homology of strainsStrains from positive transmission bioassays were successfully

sequenced and matched both before and after transmission (5/5). However, not all 11 PRRSV cases confirmed to be positive were successfully sequenced. The ORF-5 targeted sequence of 544 bp was obtained for 6 isolates only. Two isolates were only partially sequenced (ID 245 and 715). The quality of ID 587 ORF-5 virus

sequence remains low. Two viruses could not be amplified by nested RT-PCR despite several attempts (ID 597 and 574). These difficulties could have been attributed to the low concentration of PRRSV RNA in meat samples (i.e., , LOQ) or strain variability. Based on the PRRSV ORF-5 sequence homology, 2 of the positive PRRSV transmis-sible meats (ID 319 and 1332) showed a close relationship (. 97%) with modified live virus Ingelvac RespPRRS MVL vaccine and the strains of lineage 5 over 544 bp (Figure 1) (27). The meat sample ID 715, confirmed positive but negative in the transmission bioas-say, was also closely related to RespPRRS MVL vaccine type (98%) and was 100% homologous to meat sample ID 1332 over a smaller sequence stretch of 457 bp. Four confirmed positive PRRSV meats (ID 84, 115, 1311, and 1424) were related (~ 90%) to wild-type strains of lineage 1 such as MN184 and IAF-Klop (Figure 1). The meat ID 245 was also closely related to the lineage 1 genotype. The meat ID 245 ORF-5 sequence was closely related to meat 84 (100% over 478 bp). Sample IDs 245 and 715 were not included in the ORF-5 phylogram because it was not possible to cover the same ORF-5 region with these samples. The control strain used during the RT-PCR (NVSL) did not show a high homology with the detected sequences (, 90%). The current study confirmed the presence of 3 Ingelvac RespPRRS MVL related vaccine-type and 5 wild-type strains in confirmed PRRSV positive meats.

D i s c u s s i o nThe percentage of presumptive RT-PCR positive PRRSV meat

samples was very similar to the ones reported previously in meat samples from Quebec (1.07% versus 1.2%) (6). However, only 11/16 were confirmed. In contrast, others have not found PRRSV by

Table I. Variations of porcine reproductive and respiratory syndrome virus (PRRSV) concentrations in selected meat samples stored at 220°C over time

Follow-up positiveMeat Weeks at Normalized PRRS viral RNA copies per mg resultsIDa 220°Cb T0 T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 Tmax

b Averagec (T1 to Tmax) 84 15 104.8 104.5 104.1 104.5 104.3 104.5 104.7 104.5 104.7 104.4 104.4 104.1 104.5 11/11 115 13 103.7 104.0 103.9 104.0 104.4 103.8 103.6 103.5 1 103.7 103.5 1 103.9 11/11 319 11 103.5 103.7 103.5 103.2 1 103.8 103.9 103.5 103.4 104.1 103.7 103.9 103.7 11/11 340 11 0 0 NT 0 0 0 0 0 0 0 0 0 0 0/10 574 11 1 0 0 0 0 0 0 0 0 0 0 0 , LOQ 0/11 597 11 1 0 0 0 0 0 0 0 0 0 0 0 , LOQ 0/11 715 11 1 1 1 1 1 1 1 NT 1 1 1 1 , LOQ 10/101311 11 103.6 1 1 0 1 103.7 1 1 1 1 1 0 , LOQ 9/111332 11 103.9 103.5 103.7 103.5 103.7 1 103.8 104.0 103.8 103.7 103.8 103.7 103.7 11/111400* 11 0 0 0 0 0 0 0 0 0 0 0 0 0 0/111414* 11 0 0 0 0 0 0 0 0 0 0 0 0 0 0/111424 11 0 0 1 1 0 0 0 0 0 1 0 NT , LOQ 3/10a PRRSV negative control (*).b The total period of time the meat was stored at 220°C (Tmax).c The average PRRS viral RNA copy per mg calculated from meat subsample collected from week 1 (T1) to the end of the storage period (Tmax) and normalized based on the RNA extract concentration.1 — Positive samples that are detected but are below the limit of quantification in quantitative RT-PCR; , LOQ — Average value below the limit of quantification of 103.5 RNA copies per mg; ID — meat identification number; NT — samples not tested.


RT-PCR in carcasses at slaughter (14). The prevalence, the meat sampling changes and the sensitivity of the RT-PCR assays could explain the PRRSV detection variation observed between studies. In addition, meat is a heterogeneous matrix composed of various ele-ments such as fat, muscles, nerves, and vessels. In actively infected animals, PRRSV is not expected to be evenly distributed in meat but rather, located in residual blood (12,28). It is thus expected to be unequally distributed among subsamples collected to conduct the studies, leading to significant variations. In the current study, the levels of viral RNA were relatively low in general and close to the limit of detection of the molecular assays. The impact of assay sensitivity and low recovery yields on prevalence estimates is not

negligible in such cases (19). The aging of the screening ORF7 prim-ers is probably not a major issue based on their homologies with the reference PRRSV strains of the 2008 circulating lineages (27). Other factors associated with the sampling scheme could have influenced prevalence estimates, such as the type of meats, the handling of the carcasses before packaging, and the seasons during which the samplings were performed. Despite having taken the necessary precautions, samples collected at the PHBP could have been cross-contaminated in the processing line or during sample analysis. The confirmation steps used herein reduce the probability of overestimat-ing the presence of the virus in pig meat and underestimating their transmission in the bioassays.

Table II. Porcine reproductive and respiratory syndrome virus (PRRSV) detection in pigs experimentally fed with PRRSV positive meat samples

Estimated Meat meat Meat weight viral load Bioassay RT-PCR/ELISAd (Days post exposure)Pig ID (g)b (PRRSV copy)c (trial #) °Ca 27 0 7 14 21 28844-02 84 253/249 109.8 1 4 2/2 2/2 1/2 1/1 1/1 2/1844-07 84 251/251 109.8 1 4 2/2 2/2 1/2 1/1 1/1 1/1844-04 319 250/250 109.6 1 4 2/2 2/2 1/2 1/1 1/1 1/1844-06 319 250/250 109.6 1 4 2/2 2/2 1/2 1/1 1/1 2/1844-01 340 246/246 0 1 4 2/2 2/2 2/2 2/2 2/2 2/2844-09 340 245/250 0 1 4 2/2 2/2 2/2 2/2 2/2 2/2844-08 Ctrl 0/0 — 1 — 2/2 2/2 2/2 2/2 2/2 2/2844-05 Ctrl 0/0 — 1 — 2/2 2/2 2/2 2/2 2/2 2/2848-09 574 250/250 1 2 4 2/2 2/2 2/2 2/2 2/2 2/2848-02 574 250/250 1 2 4 2/2 2/2 2/2 2/2 2/2 2/2848-06 597 250/237 1 2 4 2/2 2/2 2/2 2/2 2/2 2/2848-07 597 250/233 1 2 4 2/2 2/2 2/2 2/2 2/2 2/2848-10 715 251/135 1 2 4 2/2 2/2 2/2 2/2 2/2 2/2848-04 715 251/136 1 2 4 2/2 2/2 2/2 2/2 2/2 2/2848-05 Ctrl 0/0 — 2 — 2/2 2/2 2/2 2/2 2/2 2/2848-08 Ctrl 0/0 — 2 — 2 /2 2/2 2/2 2/2 2/2 2/2852-04 115 250/238 109.2* 3 4 2/2 2/2 1/2 NT NT 1/1852-05 115 251/239 109.2* 3 4 2/2 2/2 1/2 1/1 NT 1/1852-01 Ctrl 0/0 — 3 — 2/2 2/2 2/2 2/2 2/2 2/2874-04 1311 250/250 1 4 4 2/2 2/2 2/2 2/2 2/2 2/2874-02 1311 250/250 1 4 RT 2/2 2/2 2/2 2/2 2/2 2/2875-01 1424 250/250 1 4 4 2/2 2/2 1/2 1/1 1/1 1/1874-03 1424 250/250 1 4 RT 2/2 2/2 2/2 2/2 2/2 2/2874-01 Ctrl 0/0 — 4 — 2/2 2/2 2/2 2/2 2/2 2/2875-02 Ctrl 0/0 — 4 — 2/2 2/2 2/2 2/2 2/2 2/2879-03 1332 250/250 109.5 5 4 2/2 2/2 1/2 1/1 1/1 1/1879-02 1332 250/250 109.5 5 RT 2/2 2/2 1/2 1/1 1/1 1/1878-07 Ctrl 0/0 — 5 — 2/2 2/2 2/2 2/2 2/2 2/2879-01 Ctrl 0/0 — 5 2 2/2 2/2 2/2 2/2 2/2 2/2a The meat was stored at 4°C or room temperature (RT) for 24 and 48 h after storage at the PHBP.b Each meat sample was divided in 2 equal parts The quantity of meat sample given on both days to each piglet.c The total PRRS viral load in equivalent viral copies calculated using the ORF7 qRT-PCR results from aliquots of the meat homogenate stored at 220°C at Tmax. Value calculated using concentration at T10 (*). Confirmed positive samples that are below the limit of quantification in quantitative RT-PCR(1).d Serum test results from ORF7-RT-PCR genotype 2 assays and HerdCheck ELISA results either positive (1) or negative (2).ID — meat identification number; NT — not tested.


No PRRSV positive meat was detected for 3 consecutive months during summer, although 33% of the meat samples were collected during those months. Although more sampling would be needed to confirm this trend, those results suggest that fewer pigs at the age of slaughter are infected with PRRSV or the level of infection is lower during summer. A study conducted by Larochelle et al (29) in Quebec reported that about 75% of field cases of PRRSV between 1998 and 2002 were submitted from November to April, suggesting a higher prevalence of disease during colder months. Other studies have also shown more frequent mechanical transmissions of PRRSV in cold weather than in warm weather (30,31). Considering that the

stability of PRRS virions decreases when the temperature increases (12,32–34), these trends seem to confirm the lower prevalence of PRRSV during the summer months, suggesting a lower risk in oral transmission.

Similarly, the standard meat storage temperature at the packag-ing center might be too low to affect PRRS viral decay in frozen meat within the study time frame. The 15-week follow-up study was designed to provide insight on the virus integrity in meat throughout the period of time needed for transportation and storage during exportation overseas. No significant decay of viral RNA was detected in our study, but that could be the result of the subsamples

Figure 1. Phylogram of porcine reproductive and respiratory syndrome (PRRS) viral ORF-5 cDNA sequence and confirmed positive meat samples (in bold). The X-axis represents the percentage divergence. The numbered lineages follow the classification described previously (27). Internal labels represent the percentage of 1000 trees that support the clustering, only bootstrap values . 50% are shown.


heterogeneity, the variations in inter-assays, and the poor extraction efficiency. It could also be the result of the study time frame and the RNA stability. Indeed, the data are compatible with the reported absence of PRRSV titer variation after 10 wk at 220°C in cell culture media (12), while others have reported a decrease in viral infectiv-ity over time with storage temperature increase (220°C to 30°C) in absence of viral RNA concentration variation (34,35).

Several studies have analyzed the oral transmission of PRRSV to pigs in order to assess the risk associated with swill feeding (36). This study is the first to report on the PRRSV RNA concentrations in pig meat collected at the PHBP, that are associated with positive transmission after storage in commercial conditions and thawing. For meat viral loads above 109 total PRRSV RNA copies/500 g of meat, the transmission rate was high (100%) but these samples were still relatively rare in the survey. The transmission rate of positive PRRSV meat samples was slightly lower than the one reported by Magar and Larochelle (6) (5/9 versus 7/11) in the same region. These results are surprisingly similar considering the differences between both studies, including the meat cuts, the storage temperature, and the variation in viral load.

The transmission rates observed with frozen packaged meat from the PHBP were relatively low when taking into account the prevalence of confirmed positive PRRSV meat products. The product of the prevalence and the transmission rate in this study (0.4% for 500 g) was in the same range as a model developed to describe the probability that meat imported from a country where PRRSV is pres-ent will contain an infectious dose of PRRSV after shipping (0.18% for 1 kg) (21). Several parameters, which could have influenced the transmission of PRRSV, were not considered in our study and require further investigation. For instance, scrap sizes (500 g versus 10 g) or disposal conditions (prolonged RT), which were not estimated or barely explored, should have an impact on the meat viral load and the transmission rates (37). Legislations, regulations, and training on swill feeding, must also be considered but were outside of the scope of this study. For example, swill feeding is not allowed in Canada. In addition, although the reversion of live attenuated vaccine strains to virulence under field conditions occurs (38), the inclusion of vac-cine strains or vaccine strain derivatives in risk assessments requires balancing their benefits. In the current study, close to a third of the sequenced strains were related to vaccine types. These numbers are similar to the ones reported previously in Canada (6,7). The results indicate that the likelihood of meat from the PHBP contains wild strains of PRRSV and is infectious, is low. On the other hand, the elimination of vaccine strains from herds could require an equal amount of effort as the elimination of the wild type strains. The viral and clinical outcomes in piglets orally infected with pig meat and maintained in natural conditions, are still unknown.

In summary, low residual quantities of PRRSV are found in a small percentage of pig meat collected at the pork ham boning plant. The values observed were very similar to and/or slightly lower than the ones reported previously for the province of Quebec. No statistically significant degradation of the PRRSV RNA was observed at 220°C during 15 wk. Previously frozen PRRSV-positive meat was able to infect naïve pigs by oral exposure. Transmission was more efficient for the meat samples containing a quantifiable viral load, in the range of 109 genomic equivalents. However, the prevalence of these

meat samples with a high PRRSV viral load remained low, especially when considering field strains only.

A c k n o w l e d g m e n t sThis research was made possible through the financial support of

the Canadian Food Inspection Agency, the Canadian Pork Council, and Olymel. We would like to acknowledge Dr. Bianca Morel and her team for their assistance in the serological assays and sampling, Drs. Fatima Belayat and Yves Robinson for their assistance with autopsies, M. René Mineau for his support in the animal facilities, Ms Sylvianne Paul for her technical assistance, the CFIA Ottawa Fallowfield Laboratory and M. Steve Smith for providing SPF piglets, Dr. Carl Gagnon for consultation, and M. André Perron for revision of the manuscript.

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Article


I n t r o d u c t i o nPorcine circovirus 2 (PCV2), the smallest nonenveloped, single-

stranded, circular DNA virus in nature, belongs to the genus Circovirus of the family Circoviridae (1). It has 2 major open reading frames (ORFs): ORF1 encodes the replication-associated protein, and ORF2 encodes the capsid protein (2). This virus is considered a caus-ative agent of several diseases and syndromes, collectively referred to as porcine circovirus-associated diseases (PCVAD) (3), among which postweaning multisystemic wasting syndrome (PMWS) is the most important (3).

Commercial PCV2 vaccines were introduced into the international market in 2006 (4). Their efficacy is evaluated on the basis of the induction of protective immunity and reduction of PCV2 viremia (4). High levels of PCV2 viremia have been shown to be associated with the development of PCVAD (5–8). The induction of a humoral response (neutralizing antibodies) and a cell-mediated response [interferon-g-secreting cells (IFN-g-SCs)] contributes to reducing PCV2 viremia and subsequently controlling PCV2 infection (9).

Experimental PCV2 vaccines have also been studied during the past 10 y (10). A PCV2 DNA vaccine holds promise for elicit-ing protective humoral and cell-mediated immune responses and

Development of porcine circovirus 2 (PCV2) open reading frame 2 DNA vaccine with different adjuvants and comparison with commercial

PCV2 subunit vaccine in an experimental challengeChanghoon Park, Jiwoon Jeong, Kyuhyung Choi, Su-Jin Park, Ikjae Kang, Chanhee Chae

A b s t r a c tThe objective of this study was to compare the protection against challenge with porcine circovirus 2 (PCV2) induced by an experimental vaccine based on open reading frame (ORF) 2 of PCV2 DNA plus an adjuvant (aluminum hydroxide, cobalt oxide, or liposome) and a commercial PCV2 subunit vaccine. A total of 35 colostrum-fed, cross-bred, conventional piglets were randomly divided into 7 groups. The commercial vaccine was more efficacious against PCV2 challenge than the 4 experimental vaccines according to immunologic, virologic, and pathological outcomes. The pigs inoculated with the experimental vaccine containing the liposome adjuvant had significantly higher levels (P , 0.05) of neutralizing antibodies and interferon-g-secreting cells, and significantly lower levels (P , 0.05) of PCV2 viremia than the pigs inoculated with the other experimental vaccines. The pigs inoculated with the experimental vaccines containing either the liposome adjuvant or the cobalt oxide adjuvant had significantly lower lymphoid lesion scores (P , 0.05) than the pigs in the group inoculated with the PCV2 DNA vaccine dissolved in phosphate-buffered saline. Liposome proved to be a potent adjuvant that efficiently enhanced both humoral and cellular immune responses induced by the PCV2 DNA vaccine.

R é s u m éL’objectif de la présente étude était de comparer la protection contre une infection défi avec le circovirus porcin de type (CVP2) induite par un vaccin expérimental à base du cadre de lecture ouvert (ORF) 2 de l’ADN de CVP2 plus un adjuvant (hydroxyde d’aluminium, oxyde de cobalt, ou liposome) et un vaccin CVP2 sous-unitaire commercial. Un total de 35 porcelets croisés, conventionnels et nourris au colostrum ont été séparés de manière aléatoire en sept groupes. Le vaccin commercial était plus efficace contre l’infection par CVP2 que les quatre vaccins expérimentaux en fonction des résultats immunologiques, virologiques, et pathologiques. Les porcs inoculés avec le vaccin expérimental contenant l’adjuvant liposome avaient des titres significativement (P , 0,05) plus élevés d’anticorps neutralisants et un nombre significativement (P , 0,05) moindre de cellules secrétant de l’interféron-g et une virémie à CVP2 que les porcs inoculés avec les autres vaccins expérimentaux. Les porcs inoculés avec les vaccins expérimentaux contenant l’adjuvant liposome ou oxyde de cobalt avaient des scores de lésions lymphoïdes significativement (P , 0,05) plus faibles que les porcs dans le groupe inoculé avec le vaccin ADN CVP2 dissout dans de la saline tamponnée. Les liposomes se sont avérés être un adjuvant puissant qui a augmenté de manière efficace autant la réponse immunitaire humorale que cellulaire induite par le vaccin à ADN CVP2.

(Traduit par Docteur Serge Messier)

Seoul National University, College of Veterinary Medicine, Department of Veterinary Pathology, 1 Gwanak-ro, Gwanak-gu, 08826, Seoul, Republic of Korea.

Changhoon Park and Jiwoon Jeong contributed equally to this work.

Address all correspondence to Dr. Chanhee Chae; telephone: 182-2-880-1277; fax: 182-2-871-5821; e-mail: [email protected]

Received October 7, 2016. Accepted January 11, 2017.


reducing PCV2 viremia in mouse models of PCV2 infection (11–15). In contrast, a PCV2 DNA vaccine was not able to reduce PCV2 viremia efficiently in pigs (16), and protective immunity has not been investigated.

Commercially available PCV2 subunit vaccines are still effective in protecting against concurrent PCV2b and the emerging PCV2b mutant strain (9,17). However, it is necessary to develop a vaccine that is more cost-effective than the current PCV2 vaccines. Perhaps a DNA vaccine is the best candidate. Such vaccines have often been disappointing when tested in pigs because of the relatively poor induction of protective immunity (18). One possible approach to improve the immune responses is the use of different adjuvants. Therefore, the objective of this study was to compare the protection against PCV2 challenge induced by a commercial PCV2 vaccine and an experimental PCV2 ORF2 DNA vaccine containing an adjuvant: aluminum hydroxide, cobalt oxide, or liposome.


Preparation of an ORF-2-based plasmid and DNA vaccine

The entire ORF2 sequence was amplified from the genomic DNA of PCV2b SNUVR000463 [GenBank (National Center for Biotechnology Information, Bethesda, Maryland, USA) acces-sion no. KF871068] with the use of specific primers as previously described (14) and subcloned into the mammalian expression vec-tor pCI-neo (Promega, Madison, Wisconsin, USA) to construct the recombinant expression plasmid for ORF2 (pORF2). To ascertain Cap protein expression in porcine cells, PCV-free porcine kidney (PK)-15 cells were transfected with pORF2 with the use of the reagent Lipofectamine 2000 (Invitrogen, Carlsbad, California, USA) according to the manufacturer’s instructions. After a 48-hour incu-bation the cells were fixed and examined by the indirect immuno-fluorescent antibody (IFA) technique with the use of a mouse mono-clonal antibody against porcine PCV2 ORF2 (Rural Technologies, Brookings, South Dakota, USA) and goat IgG against mouse antigen conjugated with fluorescein isothiocyanate (Kirkegaard and Perry Laboratories, Gaithersburg, Maryland, USA) as the primary and secondary antibodies, respectively. Finally, the pORF2 was purified with an EndoFree Plasmid Giga Kit (Qiagen, Valencia, California, USA) for use as a DNA vaccine.

Preparation of the various forms of experimental vaccine

Equal volumes of phosphate-buffered saline (PBS; 10 mM, pH 7.4) and pORF2 DNA (2 mg/mL in 2 3 PBS) were mixed on a magnetic plate stirred at 45 3 g for 24 h, and the resultant vaccine was admin-istered to pigs within 4 h of formulation.

Equal volumes of aluminum hydroxide gel (Rehydragel LV; General Chemical, Berkeley Heights, New Jersey, USA) and pORF2 DNA (2 mg/mL in 2 3 PBS) were mixed on a magnetic plate stirred at 45 3 g for 24 h, and the resultant vaccine was administered to pigs within 4 h of formulation.

To add cobalt oxide as an adjuvant, we used a slight modification of previously described methods (19,20). Briefly, cobalt oxide (Co3O4)

nanoparticles in double-ionized water (500 mg/mL) were disrupted with a probe sonicator (220 to 240 V, 10.3A; Philip Harris Scientific, Hyde, England) at 40% power for 1 min to break up aggregation. Then equal volumes of pORF2 DNA (2 mg/mL in 2 3 PBS) were added and mixed by vortexing for 5 min. The hydrodynamic size and zeta potential were measured with a Zetasizer (Nano-ZS90; Malvern, Malvern Hills, England). Finally, a 1-mL dose containing 500 mL of pORF2 DNA (2 mg/mL in PBS) and 500 mL of nanoparticle adjuvant (500 mg/mL) was prepared and stored at 4°C before use.

For the preparation of liposome nanoparticles to be used as an adjuvant, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (Avanti Polar Lipids, Alabaster, Alabama, USA), dihexadecyl phosphate (Sigma-Aldrich, St. Louis, Missouri, USA), and cholesterol (Avanti Polar Lipids) were mixed at a ratio of 3.18:0.55:1.55 (w/w/w) accord-ing to techniques previously described (21). Briefly, lipid films were created in a glass vial by evaporating the organic solvent [a mixture of chloroform and methanol at a ratio of 6.5:3.5 (v/v)] under a steady stream of nitrogen gas. Traces of organic solvent were removed by keeping the films in a vacuum desiccator overnight. The lipid films were then hydrated for 12 h by adding pORF2 DNA (1 mg/mL in PBS). Next, the suspensions were sonicated in a bath-type sonicator (Branson Ultrasonics Corporation, Danbury, Connecticut, USA) for 10 min and then extruded through 400-, 200-, and 100-nm membrane filters (Hamilton Company, Reno, Nevada, USA) and stored at 4°C before use. The hydrodynamic size and zeta potential were measured with a Zetasizer.

In-vitro transfection with the PCV2 molecular DNA clone

To test the infectivity of the clone in vitro, PCV-free PK-15 cells were grown in 8-well chamber slides as previously described (22). Briefly, when the PK-15 cells were about 85% confluent they were transfected with the clone alone or with different adjuvants (1:1 mixture) as described. Mock-transfected cells with empty pCI-neo vector were included as controls. Three days after transfection the cells were fixed with a solution containing 80% acetone and 20% methanol at 4°C for 20 min, and an IFA assay using a PCV2-specific rabbit polyclonal antiserum (Veterinary Diagnostic Laboratory, Iowa State University, Ames, Iowa, USA) was done to determine the in-vitro infectivity of the molecular DNA clone.

AnimalsA total of 56 colostrum-fed, cross-bred, conventional piglets born

to PCV2-unvaccinated sows were purchased at 5 d of age from a commercial farm. All the piglets were seronegative for PCV2, porcine reproductive and respiratory syndrome virus (PRRSV), and Mycoplasma hyopneumoniae and did not have PCV2 or PRRSV viremia according to real-time polymerase chain reaction (PCR). Twenty-one pigs were used for a negative-control study, and the remaining 35 were used as experimental subjects. All of the methods were approved by the Seoul National University Institutional Animal Care and Use Committee.

Negative-control studyThe 21 pigs were randomly divided into 7 groups (3 pigs/group)

by means of the random-number generation function of Excel


(Microsoft Corporation, Redmond, Washington, USA). The pigs were vaccinated with only null plasmid, only an adjuvant (alumi-num hydroxide, cobalt oxide, or liposome), or null plasmid plus an adjuvant. The negative-control vaccine was prepared the same as the PCV2 DNA vaccine but with a null plasmid instead of the ORF2-based plasmid DNA. The pigs in each group were housed in the same pen within the same room. Blood samples were collected at −42 (7 d of age), −28 (21 d of age), 0 (49 d of age), 7, 14, 21, 42, and 70 (119 d of age) d after challenge. All the pigs were euthanized for necropsy 70 d after challenge, and superficial inguinal lymph nodes were collected for histopathological and immunohistochemical study.

Experimental designThis study used a randomized, blinded, controlled design. The

35 experimental pigs were randomly divided into 7 groups (5 pigs/group) by means of the random-number generation function of Excel (Table I). Groups 1 to 4 received the PCV2 ORF2 DNA vaccine dissolved in PBS or the PCV2 ORF2 DNA vaccine with aluminum hydroxide, cobalt oxide, or liposome as adjuvant, respectively, as two 1.0-mL doses injected intramuscularly in the right side of the neck at 7 and 21 d of age. The dose (1.0-mL) of pORF2 (1 mg) in each vaccine with an adjuvant was administered to the pigs as previously described (13). The pigs in group 5 were injected with a commercial PCV2 subunit vaccine (CircoFLEX; Boehringer Ingelheim Vetmedica, St. Joseph, Missouri, USA) at 21 d of age (−28 d after challenge) according to the manufacturer’s recommendations. At 49 d of age the pigs in the vaccinated groups (groups 1 to 5) and the unvaccinated, challenged group (group 6) were inoculated intranasally with 2 mL each of PCV2b [strain SNUVR000463, 5th passage; 1.0 3 105 median tissue culture infectious doses (TCID50) per milliliter]. The pigs in group 7 remained unvaccinated and unchallenged and served as negative controls.

All pigs were commingled and randomly assigned to 1 of 2 rooms. At the time of challenge the negative-control group was moved to a separate, similar room. The pigs in each group were housed in the same pen within the same room. Blood samples were collected at −42 (7 d of age), −28 (21 d of age), 0 (49 d of age), 7, 14, 21, 42, and 70 (119 d of age) d after challenge. All the pigs were euthanized for necropsy at 70 d after challenge, and superficial inguinal lymph nodes were collected for histopathological and immunohistochemi-cal study.

Quantification of PCV2 DNA in bloodDNA was extracted from serum by means of the QIAamp DNA

mini kit (Qiagen, Crawley, England) and used to determine the PCV2 genomic DNA copy numbers by real-time PCR with PCV2b-specific primers as previously described (23).

Serum antibody assayThe serum samples were assayed by a serum virus neutralization

test (24). Neutralizing antibody titers were expressed as the recipro-cal of the highest serum dilution that completely blocked infection of the PCV-free PK-15 cells with the challenge PCV2b strain.

Enzyme-linked immunospot (EliSpot) assayThe numbers of PCV2-specific IFN-g-SCs stimulated by the chal-

lenge PCV2b strain were determined in peripheral blood mono-nuclear cells (PBMCs) as previously described (25) with slight modifications. Briefly, 100 mL containing 5 3 105 PBMCs in RPMI 1640 medium supplemented with 10% fetal bovine serum (HyClone Laboratories, SelectScience, Bath, England) was seeded onto plates that had been coated with IFN-g monoclonal antibody against porcine antigen (5 mg/mL; MABTECH, Mariemont, Ohio, USA) and incubated with the PCV2b challenge isolate at a multiplicity

Table I. Experimental design and scoresa for lymphoid lesions and porcine circovirus 2 (PCV2) antigen in the different groups of piglets

Lymphoid PCV2 PCV2 lesion antigen Group PCV2 vaccineb challenge score score1 DNA vaccine–PBS Yes 1.00 6 0.45c 10.2 6 3.6d

2 DNA vaccine–aluminum hydroxide Yes 0.71 6 0.70c,d 9.7 6 2.7d

3 DNA vaccine–cobalt oxide Yes 0.57 6 0.73d,e 9.2 6 1.7b

4 DNA vaccine–liposome Yes 0.43 6 0.49d,e 9.0 6 2.6d

5 Commercial vaccine Yes 0.29 6 0.49e 7.8 6 1.8d

6 No Yes 2.00 6 0.53f 24.7 6 8.7e

7 No No 0.11 6 0.26g 0d

a Lymphoid lesions were scored as follows: 0 — no lymphoid depletion or granulomatous replacement; 1 — mild lymphoid depletion; 2 — moderate lymphoid depletion; and 3 — severe lymphoid depletion and granulomatous replacement. The PCV2 antigen score was determined by means of the Image J 1.45s Program of the US National Institutes of Health, Bethesda, Maryland, USA (http://imagej.nih.gov/ij/); the number of PCV2-positive cells per unit area (0.25 mm2) was counted. Different letters in superscript indicate statistically significant differences (P , 0.05) among groups.b DNA vaccine dissolved in phosphate-buffered saline (PBS) or prepared with an adjuvant; the commercial vaccine was CircoFLEX (Boehringer Ingelheim Vetmedica, St. Joseph, Missouri, USA).


of infection (ratio of the number of virions added per cell to the number of cells) of 0.05, phytohemagglutinin (10 mg/mL; Roche Diagnostics GmbH, Mannheim, Germany) as a positive control, or PBS as a negative control for 20 h at 37°C in a 5% humidified CO2 atmosphere. The wells were then washed 5 times with PBS (200 mL per well). Thereafter the procedure was conducted with the commer-cial EliSpot Assay Kit (MABTECH) according to the manufacturer’s instructions. Spots on the membranes were read by the automated AID EliSpot Reader (AID GmbH, Strassberg, Germany). The results were expressed as the numbers of IFN-g-SCs per million PBMCs.

Histopathological and immunohistochemical study

For morphometric analysis of histopathological lesions and determination of the PCV2 antigen score in lymph nodes, sections of 3 superficial inguinal lymph nodes were examined blindly (22,26). The lymphoid lesion scores ranged from 0 to 3: 0 — no lymphoid depletion or granulomatous replacement; 1 — mild lymphoid deple-tion; 2 — moderate lymphoid depletion; and 3 — severe lymphoid depletion and granulomatous replacement. The PCV2 antigen scores were determined by means of the Image J 1.45s Program of the US National Institutes of Health, Bethesda, Maryland, USA (http://imagej.nih.gov/ij/); the number of PCV2-positive cells per unit area (0.25 mm2) was counted.

Statistical analysisBefore statistical analysis the real-time PCR and neutralizing anti-

body data were transformed to log10 and log2 values, respectively. The normality of the distribution of the data for the examined vari-ables was evaluated by the Shapiro–Wilk test. Continuous data (for PCV2 DNA, PCV2 serologic findings, and PCV2-specific IFN-g-SCs) were analyzed with 1-way analysis of variance (ANOVA) followed by Tukey’s multiple-comparison test at each time point. Repeated-measures ANOVA followed by a post-hoc test for least significant dif-ferences was used to assess the mean difference in continuous data over time within the same group. Discrete data (lymphoid lesion score and PCV2 antigen score) were analyzed by Mann–Whitney tests. A value of P , 0.05 was considered to be significant.

Re s u l t sThe IFA assay with PCV2-specific antibody confirmed that the

molecular DNA clone alone and with different adjuvants was infec-tious in vitro: about 10% to 15% of the PK-15 cells were transfected.

From −42 to 0 d after challenge PCV2 DNA was not detected in any of the serum samples from the 7 groups. From 0 to 7 d after challenge the PCV2 genomic copy numbers in the serum increased significantly (P , 0.05) in the 5 vaccinated groups as well as the unvaccinated, challenged group. From 21 to 42 d after challenge the copy numbers decreased significantly (P , 0.05) in the pigs vac-cinated with CircoFLEX. From 14 to 70 d after challenge the copy numbers were significantly lower (P , 0.05) in the vaccinated groups than in the unvaccinated, challenged group. At 7, 42, and 70 d after challenge the pigs vaccinated with CircoFLEX had significantly lower (P , 0.05) copy numbers than the pigs vaccinated with PCV2 DNA. At 7, 14, 21, and 42 d after challenge the pigs receiving the

vaccine with liposome adjuvant had significantly lower (P , 0.05) copy numbers than the pigs receiving the other variations of the PCV2 DNA vaccine. At 21 d after challenge the pigs receiving the vaccine with aluminum hydroxide adjuvant had significantly lower (P , 0.05) copy numbers than the pigs receiving the vaccine with PBS (Figure 1). No genomic copies of PCV2 were detected in the serum from the unvaccinated, unchallenged pigs throughout the experi-ment or in the pigs receiving only the null plasmid, only an adjuvant, or the null plasmid plus an adjuvant in the negative-control study.

There was a significant difference (P , 0.05) in the log2- transformed neutralizing antibody titers (Figure 2A) among the groups: the titers increased significantly in the pigs receiving the PCV2 DNA vaccine with liposome or CircoFLEX from −28 to 0 d after challenge, as well as in all the vaccinated pigs and the unvac-cinated, challenged pigs from 0 to 7 d after challenge. Significantly higher titers were induced in the pigs receiving the PCV2 DNA vaccine with liposome or CircoFLEX than in all the other challenged groups at 0, 7, and 14 d after challenge. At 21, 42, and 70 d after challenge the titers were significantly higher in pigs vaccinated with CircoFLEX than in pigs receiving the PCV2 DNA vaccine and the unvaccinated, challenged pigs. At 21 d after challenge the titers were significantly higher in the pigs receiving the PCV2 DNA vaccine with liposome than in the pigs receiving the other PCV2 DNA vaccines and the unvaccinated, challenged pigs. No neutralizing antibodies were detected in the unvaccinated, unchallenged pigs throughout the experiment or in any of the pigs in the negative-control study.

The numbers of PCV2-specific IFN-g-SCs (Figure 2B) increased significantly (P , 0.05) in the pigs receiving the PCV2 DNA vac-cine with adjuvant or CircoFLEX from −28 to 0 d after challenge, as well as in the pigs receiving the PCV2 DNA vaccine with PBS and the unvaccinated, challenged pigs from 0 to 7 d after chal-lenge. The numbers decreased significantly (P , 0.05) in all the challenged groups from 42 to 70 d after challenge. The numbers were significantly higher (P , 0.05) in the pigs receiving the PCV2 DNA– liposome and CircoFLEX vaccines than in the pigs in the other vaccine groups as well as the unvaccinated, challenged group at 0, 7, and 14 d after challenge. At 0 and 14 d after challenge the numbers were significantly higher (P , 0.05) in the pigs receiving the PCV2 DNA–aluminum hydroxide and PCV2 DNA–cobalt oxide vaccines than in the pigs receiving the PCV2 DNA–PBS vaccine and the unvaccinated, challenged pigs. At 0 d after challenge the numbers were significantly higher (P , 0.05) in the pigs receiving the PCV2 DNA–PBS vaccine than in the pigs in the unvaccinated, challenged group. At 7 d after challenge the numbers were significantly higher (P , 0.05) in the pigs receiving the PCV2 DNA vaccine–cobalt oxide than in the pigs receiving the PCV2 DNA vaccine–PBS and the unvaccinated, challenged pigs. No PCV2-specific IFN-g-SCs were detected in the unvaccinated, unchallenged pigs throughout the experiment. In addition, no PCV2-specific IFN-g-SCs were detected in any of the pigs in the negative-control study.

The scores for lymphoid lesions and PCV2 antigen (Table I) were significantly lower (P , 0.05) in the vaccinated groups than in the unvaccinated, challenged group. The pigs in the CircoFLEX group had significantly lower (P , 0.05) lymphoid lesion scores than the pigs in the PCV2 DNA vaccine groups and the unvaccinated, chal-lenged group. The pigs in the PCV2 DNA vaccine–cobalt oxide


and PCV2 DNA vaccine–liposome groups had significantly lower (P , 0.05) lymphoid lesion scores than the pigs in the PCV2 DNA vaccine–PBS group. In addition, no lymphoid lesions or PCV2 antigens were detected in any lymph nodes from the pigs in the negative-control study.

D i s c u s s i o nIn this study PCV2-specific neutralizing antibodies and IFN-g-SCs

were induced only in pigs vaccinated with the PCV2 ORF2 DNA vaccines with adjuvant and not in the pigs vaccinated with only null plasmid, only an adjuvant, or null plasmid and an adjuvant. Transfection efficiency of the plasmid is critical for the immunogenic-ity of a DNA vaccine. In the present study the adjuvants (aluminum hydroxide, cobalt oxide, and liposome) could have affected the transfection efficiency of the DNA vaccine but did not. Therefore, the differences in immunogenicity of the vaccines with the various adjuvants were due to the different effects of each adjuvant on the immune responses of the pigs.

Liposome is a potent adjuvant that could efficiently enhance the neutralizing-antibody immune responses induced by a PCV2 DNA vaccine. Aluminum hydroxide remains the primary adjuvant used in veterinary vaccines. As expected, in the present study the vaccine with aluminum hydroxide gel induced the lowest levels of PCV2-specific IFN-g-SCs compared with the other vaccines with adjuvant. In addition, the vaccine with cobalt oxide nanoparticles was not able to induce higher levels of humoral and cell-mediated immune responses than the vaccine with the liposome adjuvant. These results indicate that liposome may be a good candidate for use as an adjuvant for a PCV2 DNA vaccine.

Induction of humoral and cell-mediated immunity is likely to con-tribute to a reduction of PCV2 viremia (5,6,8,27), which plays a key role in the development of PCVAD (8,27). In this study the humoral immunity induced by the PCV2 DNA vaccine with liposome con-tributed to a reduction in PCV2 viremia, as has been reported for

commercial vaccines in pigs challenged with PCV2 (25,28,29). The PCV2 DNA vaccine with liposome induced higher levels of neutral-izing antibodies and PCV2-specific IFN-g-SCs than the DNA vaccine alone or with other adjuvants and thus greater protective immunity and a greater reduction in PCV2 viremia. Therefore, the liposome adjuvant is particularly promising for PCV2 DNA vaccination.

As expected, induction of protective immunity and reduction of PCV2 viremia by the commercial PCV2 subunit vaccine based on ORF2 was even better than with the ORF2-based DNA vaccine. These results agree with previous findings in which a prototypic PCV2 subunit vaccine was more efficient in reducing PCV2 viremia in pigs than a PCV2 DNA vaccine (16). However, these results are in contrast with a murine model in which protective humoral immunity induced by a PCV2 DNA vaccine was better than that induced by a PCV2 subunit vaccine (14). We have no clear explanation for this discrepancy, but it may be due to a difference between mice and pigs in activation of dendritic cells by a PCV2 DNA vaccine, as has previously been reported (18).

The PCV2 DNA vaccine used in this study was administered twice to the experimental subjects, at 7 and 21 d of age, since the first vaccination did not provoke sufficient immune response, but the commercial vaccine was administered only once, at 21 d of age, according to the manufacturer’s recommendation. It is not too early to vaccinate at 1 wk, because a previous study showed immune responses to PCV2 vaccine in 5-day-old pigs (30).

Although DNA vaccines have generally been regarded as effective in smaller animals such as mice, but less efficient in larger species

Figure 1. Mean numbers of genomic copies of porcine circovirus 2 (PCV2) DNA in serum, transformed to log10, after challenge with a PCV2b strain (SNUVR000463) in groups of piglets receiving an experimental PCV2 DNA vaccine dissolved in phosphate-buffered saline () or prepared with an adjuvant [aluminum hydroxide gel (), cobalt oxide nanoparticles (), or liposome ()], receiving the commercial vaccine CircoFLEX (Boehringer Ingelheim Vetmedica, St. Joseph, Missouri, USA) (), or remaining unvaccinated (), as well as in the serum of unvaccinated, unchallenged piglets (). Different superscript letters indicate statistically significant differences (P , 0.05) among groups.

Days after challenge

Log 10

PC

V2 g

enom

ic c

opie

s/m

L

Figure 2. Humoral and cell-mediated immune responses in the groups. A — titers of PCV2-specific neutralizing antibody (NA), transformed to log2. B — numbers of PCV2-specific interferon-g-secreting cells (IFN-g-SCs) per 106 peripheral blood mononuclear cells (PBMCs). Group symbols and superscript numbers as in Figure 1.



PC

V2 s

peci

fic IF

N-g

-SC

/106

PB

MC

Log 2

PC

V2-s

peci

fic N

A t

iters

A

B


such as pigs and humans (18), the contrary has been shown as well (31,32). DNA vaccination can stimulate both humoral and cellular immune responses in pig models (14), and the potential utility of plasmid DNA as a component of a PCV2 vaccine is currently an area of active investigation. An additional possible approach to improve immune responses is by the use of different adjuvants. In the present study the pigs vaccinated with the 1-dose commercial PCV2 vaccine exhibited significantly lower levels of PCV2 viremia than the pigs vaccinated with the 2-dose experimental PCV2 DNA vaccine in this study. In the field, swine producers prefer to use a 1-dose PCV2 vaccine because it requires less labor and reduces stress in the animals. Further work is needed to develop a 1-dose experimental PCV2 DNA vaccine that is comparable in efficacy to the 1-dose commercial PCV2 vaccine.

A c k n o w l e d g m e n t sThis research was supported by contract research funds from

the Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea, and by the BK 21 Plus Program (grant 5360-20150100) for Creative Veterinary Science Research, National Research Foundation of Korea.

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Article


I n t r o d u c t i o nTorque teno virus (TTV) is a circular single-stranded negative

sense DNA virus that enclosed 4 open reading frames (ORF); ORF1, ORF2, ORF 1/1, and ORF 2/2 (former ORF3), and a GC-rich region within an un-translated region (UTR), which was first discovered in human samples with post-transfusion hepatitis of unknown eti-ology in 1997 (1). Currently, based on the International Committee

on Taxonomy of Viruses (2), all human and animal TTV belong to Anelloviridae family. Genus Iotatorquevirus comprises of 2 species, TTSuV1a (former TTSuV1) and TTSuV1b (former TTSuV2). The phylogenetic relationship of incomplete sequences has proposed 4 biotypes (a, b, c, and d) of TTSuV1a, and 2 biotypes (a and b) of TTSuV1b (3,4). Genus Kappatorquevirus, however, includes only one species: Torque teno sus virus k2 (2). High prevalence of co-infection between TTSuV1a and TTSuV1b has been documented worldwide.

Retrospective study of the relationship of Torque teno sus virus 1a and Torque teno sus virus 1b with porcine circovirus associated disease

Alejandro Vargas-Ruiz, Hugo Ramírez-Álvarez, José I. Sánchez-Betancourt, Víctor Quintero-Ramírez, Ignacio C. Rangel-Rodríguez, Joel A. Vázquez-Perez, Lucia A. García-Camacho

A b s t r a c tGenus Iotatorquevirus consists of 2 species, Torque teno sus virus 1a and Torque teno sus virus 1b, which are ubiquitous in swine populations, and are widely reported in association with porcine circovirus associated disease (PCVAD). To evaluate the relationship with PCVAD, 100 formalin-fixed paraffin-embedded tissue samples were used to detect both Iotatorquevirus species by nested PCR and sequencing. Sixty-eight PCVAD cases were selected as well as 32 porcine circovirus type 2 (PCV2) non-affected cases. Overall, 33 of the 100 cases were positive for Torque teno sus virus 1a and 8 of 100 were positive for Torque teno sus virus 1b. Only 24 of 68 (35%) PCVAD cases were positive for Torque teno sus virus 1a; 39% (9/23) of post-weaning multisystemic wasting syndrome, and 33% (15/45) of PCV2-associated reproductive failure cases. Among PCV2 non-affected cases, 28% were positive for Torque teno sus virus 1a and 6% were positive for Torque teno sus virus 1b. Torque teno sus virus 1b was not detected in PCV2-associated reproductive failure cases. Regardless of the PCV2-status, a lower frequency of both Iotatorquevirus species was found than depicted in other reports and there was no statistical relationship with PCVAD (x2 , 0.01). Given the worldwide genomic variability of Iotatorquevirus species, it is feasible that species prevalent in Mexico share a lower nucleotide sequence identity, leading to different pathogenic potential.

R é s u m éLe genre Iotatorquevirus consiste en deux espèces, le virus Torque teno sus 1a et le virus Torque teno sus 1b, qui sont ubiquitaires dans la population porcine, et couramment rapportés en association avec la maladie associée au circovirus porcin (MACVP). Afin d’évaluer la relation avec MACVP, 100 échantillons de tissus fixés dans la formaline et enrobés de paraffine ont été utilisés pour détecter les deux espèces de Iotorquevirus par réaction d’amplification en chaîne par la polymérase nichée et séquençage. Soixante-huit cas de MACVP ont été sélectionnés ainsi que 32 cas non-affectés d’infection par le circovirus porcin de type (CVP2). Globalement, 33 des 100 cas étaient positifs pour le virus Torque teno sus 1a et 8 des 100 étaient positifs pour le virus Torque tenos sus 1b. Seulement 24 des 68 (35 %) cas de MACVP étaient positifs pour le virus Torque tenos sus 1a; 39 % (9/23) du syndrome de dépérissement post-sevrage, et 33 % (15/45) des cas de problèmes reproducteurs associés au CVP2. Parmi les cas non-affectés de CVP2, 28 % étaient positifs pour le virus Torque teno sus 1a et 6 % étaient positifs pour le virus Torque tenos sus 1b. Le virus Torque tenos sus 1b n’a pas été détecté dans les cas de problèmes reproducteurs associés au CVP2. Indépendamment du statu vis-à-vis le CVP2, une fréquence plus basse des deux espèces d’Iotatorquevirus fut trouvée comparativement à ce qui est décrit dans d’autres études et il n’y avait pas de relation statistiquement significative avec MACVP (x2 , 0,01). Étant donné la variabilité génomique mondiale des espèces d’Iotatorquevirus il est possible que les espèces prévalentes au Mexique partagent une plus faible identité de séquences nucléotidiques, entrainant ainsi un potentiel pathogène différent.


Department of Biological Sciences, College of Superior Studies (FESC), National University of Mexico (UNAM), Carretera Cuautitlán–Teoloyucan Km 2.5, Cuautitlán Izcalli, 54714, Estado de México (Vargas-Ruiz, Ramírez-Álvarez, Quintero-Ramírez, Rangel-Rodríguez, García-Camacho); Department of Swine Production, College of Veterinary Medicine, National University of Mexico (UNAM), Av. Universidad 3000, Col. Cd. Universitaria, Del. Coyoacán, CP 04510, Distrito Federal (Sánchez-Betancourt); Infectious Diseases Research Center, National Institute of Respiratory Diseases, Mexico City, Mexico (Vázquez-Perez).

Address all correspondence to Dr. Lucia A. García-Camacho; tel: 5623 1854; e-mail: [email protected]

Received September 22, 2016. Accepted January 20, 2017.


Iotatorquevirus species are ubiquitous in domestic and wild pigs, and have been identified in Europe (Hungary, Italy, France, and Spain), Asia (China, Korea, Japan, and Thailand), and North America (Canada and USA).

Transmission among pigs is horizontal and mainly via fecal-oral, but transmission through other routes may be important (3). It is unknown whether TTSuV1a and TTSuV1b infection promotes a specific disease as a primary agent or in co-infection with other pathogens (5). However, it has been suggested that in co-infections with other viruses, TTSuV1a might promote increased disease severity or virulence and TTSuV1b might be associated with repro-ductive failure (1). In this scenario, several studies have suggested an involvement of both species with porcine circovirus associated disease (PCVAD) since cases of post-weaning multisystemic wasting syndrome (PMWS) have shown a high prevalence of Iotatorquevirus species (6,7). Moreover, clinical manifestations and characteristic lesions of porcine dermatitis and nephropathy syndrome (PDNS) have been reproduced by TTSuV1a inoculation in gnotobiotic pigs (8). In fact, TTSuV1a has been proposed as the additional factor (X-factor) for the development of PCVAD (9). Porcine circovirus associated disease is economically important to the swine industry since it has an impact on production and reproduction parameters. Although all clinical presentations of PCVAD [PMWS, PDNS, por-cine circovirus 2-associated reproductive failure (PCV2-RF), and granulomatous enteritis] in Mexico have been confirmed by in situ hybridization (10), the prevalence of Iotatorquevirus species or their possible association with PCVAD has not been recorded. The aim of the present work was to identify TTSuV1a and/or TTSuV1b from well-documented cases of PCVAD in order to assess their potential relationship with the occurrence of PCVAD in an unvaccinated population.


Case selectionArchived formalin-fixed paraffin-embedded porcine tissues

(lymph node, spleen, tonsils, and fetal hearts) from 2001 to 2009 were selected from 100 swine cases. Sixty-eight cases were from non-vaccinated swine with confirmed PCVAD on the basis of clinical signs, characteristic microscopic lesions, and in situ hybridization (11). The cases were subdivided as follows: 23 PMWS-affected tis-sues (lymph nodes and spleen), depicting severe lymphoid deple-tion and granulomatous inflammation, and a diffuse pattern of PCV2-positive in situ hybridization (ISH) and 45 cases of PCV2-RF, consisting of fetal hearts with non-suppurative myocarditis as well as a random ISH pattern positive for PCV2. The PCVAD-positive cases were submitted from 13 states of the Mexican Republic with a high-density swine population (Figure 1). Additionally, 32 PCV2 non-affected cases were evaluated and consisted of 12 tissues (lymph node and tonsil) negative for PCV2 by ISH from age-matched, clini-cally normal, non-vaccinated pigs from a PCV2 non-affected farm according to clinical criteria (12) and 20 hearts from PCVAD non-infected aborted fetuses submitted for diagnosis. Each tissue was tested individually.

Primer designDue to genomic variability among available sequences, degen-

erate primers that target ORF1 of TTSuV1a and TTSuV1b were designed using computer software [Primer3 imput program (v.3.0.0; Institute for Biomedical Research, Boston, Massachusetts, USA) (13) and Bioedit software program (v7.2.5; Ibis Bioscience, Carlsbad, California, USA) (14)]. Ten TTSuV1a sequences from different

Figure 1. Geographical distribution of porcine population in the Mexican Republic. Mexican states with higher swine production (27). States from which samples were submitted.


countries available in the NCBI database were used to design the primers (GenBank accession numbers: HM633249, HM633253, HM633258, AY823990, HM633257, AB076001, GU188045, GU456383, GU456384, GQ120664). For TTSuV1b, 12 sequences available in the NCBI database from different countries were used to design the prim-ers (GenBank accession numbers: HM633230, JX173484, HQ204188, GU376737, KC461227, JQ782385, HM633218, GU456386, GU188046, GU570207, AY823991, NC014092). First-round reverse primer contains only 1 degenerate base. Primers were synthesized commercially (IDT Integrated DNA Technologies, Coralville, Iowa, USA).

Nested polymerase chain reaction (PCR)DNA extraction from all tissues was done separately using com-

mercial kits according to the manufacturer’s instructions (QIAamp DNAFFPE Tissue kit; Qiagen, Germany). Briefly, DNA was eluted in a volume of 200 mL molecular grade water, and stored at 220°C. The lowest limit of detection was determined by serial dilution (1:2) and was of 12.5 ng/mL. Nested 50-mL polymerase chain reaction (PCR) were done using the sets of degenerate primers (Table I) in a thermo-cycler (Eppendorf, Hamburg, Germany) containing 2.5 UTaq DNA

polymerase (GoTaq Flexi DNA polymerase; Promega Corporation, Madison, Wisconsin, USA) PCR buffer 13, magnesium chloride 2.25 mM (TTSuV1a)/1.5 mM (TTSuV1b), 0.2 mM of each deoxy-nucleotide (dNTP), 100 pmol of each primer, and 20 ng of template. The following thermal cycle was as follows: the initial activation step at 94°C for 3 min followed by 40 cycles of 1 min at 94°C, 1 min at 56°C (TTSuV1a) or 53°C (TTSuV1b) and 1 min at 72°C, finally last extension step of 10 min at 72°C. The PCR products were electro-phoretically separated in a 1.5% agarose gel stained with ethidium bromide. The gel was visualized under ultraviolet light (Apollo Instrumentation, Claremont, California, USA) and photodocumented (Doc-It System; UVP BioImaging Systems, Cambridge, UK).

SequencingTwo amplified products from each species were randomly

selected and purified from agarose gel using a commercial kit (Min Elute Gel Extraction kit; Qiagen) following the manufacturer’s instructions. The sequencing of the purified PCR products was done using high fidelity, processing, and specificity enzyme kits (Taq Platinum Polymerase; High Fidelity, Carlsbad, California,

Table I. Sequences of the primers utilized for nested polymerase chain reaction (PCR)

Species Sequence Primer LengthTTSuV1a 59-AACTGGCAGGACCACCTATG-39 Forward 481 bp 59-AGTGTBACHTCHCCACTYC-39 Reverse 59-AAAGAGACGCTATGGCTGGA-39 Forward nested 255 bp 59-TGYTTTTCWGTGTCCCAYTGC-39 Reverse nested

TTSuV1b 59-ATGCCTTACAGACGCTATC-39 Forward 605 bp 59-TGTGATGTTAATTTTGGTGGA-39 Reverse 59-AAGCTCCGGTCATACAATG-39 Forward nested 211 bp 59-GCTGTCCATATATTTCTCCAG-39 Reverse nestedSequences of the primers utilized the detection to the TTSuV1a and TTSuV1b for nested PCR. Bold letters indicate degenerate base. All primers were synthesized by another source (Integrated DNA Technologies, Coralville, Iowa, USA).

Figure 2. Iotatorquevirus species nested polymerase chain reaction (PCR) from porcine circovirus associated disease (PCVAD) positive cases. A — TTSuV1a nested PCR from FR-PCV2 positive cases. One hundred base pairs molecular weight marker (MWM). Lanes 1 positive control, lane 2 negative control, line 3, 4, and 5 showing 255 bp amplified products. B — TTSuV1b nested PCR from post weaning multisystemic wasting syndrome (PMWS) positive cases. One hundred base pairs MWM. Lanes 5 positive control, lane 7 negative control, line 8, 9 and 10 display 211 bp amplified products, 2% agarose gel.


USA). Internal primers were used to sequence partial sequences of ORF1, using a sequencer (Model 3100; Applied Biosystems, Foster, California, USA).

Data analysisNucleotide sequences were edited, aligned, and analyzed using

computer software (Bioedit software v7.2.0) (13). Phylogenetic analysis were done using molecular evolutionary genetics analysis version 6 (MEGA6) (15). The maximum likelihood tree was com-puted using Tamura-Neg Gamma distance. The test of phylogeny was carried out through bootstrap method with 1000 number of replication and the gaps/missing data treated with pairwise deletion. Two by four contingency frames were made based on PCR results to evaluate the relationship of TTSuV1a and TTSuV1b with PCVAD using a Chi-squared test at a trust interval of 99% (P , 0.01).

Re s u l t sThe PCR protocols using specific degenerate primers to amplify

TTSuV1a and TTSuV1b were optimized to obtain of 255 bp (Figure 2A), and 211 bp (Figure 2B), respectively, from PCVAD cases. The sequences of 2 nested products of each virus species proved to be specific (GenBank accession numbers: KU891810 and KU891811 TTSuV1a, KU891808 TTSuV1b and KU891809 TTSuVk2). At alignment, the amplified regions of each species were highly

conserved among available sequences. The topography of the phy-logenetic tree of TTSuV1a (Figure 3) revealed that the amplified sequences belong to the species 1a, conforming a well-defined cluster (Mexican, German, and Chinese sequences) standing supported with bootstrap values of 89. Conversely, the phylogenetic tree of TTSuV1b (Figure 4) showed that one Mexican sequence clustered with American and Chinese sequences, but another sequence was located in a different clade, revealing a major phylogenetic rela-tionship with TTSuVk2 Brazilian sequences (GenBank accession numbers: AY823991, NC014092).

In the current retrospective study, 100 cases were used (68 PCVAD-affected, and 32 PCVAD-non affected). Overall, 33% (33/100) were positive to TTSuV1a (TTSuV1a1) and 8% (8/100) were positive to TTSuV1b (TTSuV1b1). From all the PCVAD cases, 35% (24/68) were TTSuV1a1 and 9% (6/68) were TTSuV1b1, whereas 28% (9/32) and 6% (2/32) of the non-affected cases were positive to TTSuV1a and TTSuV1b, respectively (Table II). The frequency of PMWS-affected cases compared to age-matched clinically normal piglets is shown in Table III. Thirty-one percent (11/35) of total cases were TTSuV1a1 and 22.8% (8/35) were TTSuV1b1. Thirty-nine percent (9/23) of PMWS-affected cases were positive for TTSuV1a, 26% (6/23) were TTSuV1b1. Of the clinically normal pigs evaluated, 17% (2/12) were TTSuV1a1 and 17% (2/12) were TTSuV1b1. Co-infection was found only in PMWS cases, 3% of the total cases, and 9% (3/35) of PMWS cases. Concerning cases of reproductive failure in sows

Figure 3. Phylogenetic tree of the TTSuV1a showing the nucleotide sequences of different identified Torque teno virus with GenBank accession number, and country of origin. The sequences were distrib-uted in 3 TTSuV1a sequence groups ( Canadian, Chinese, American, Brazilian, and Hungarian; Mexican, German, and Chinese; Chinese and American; Spanish). TTSuV1b sequences ( Canadian, Chinese, American, and Brazilian), and TTV human sequences ( Italian and Japanese) are included. The Mexican sequences ( KU891810 and KU891811) belong to the 2 TTSuV1a positive cases described in this work. The length of the branch represents the genetic distance among the sequences.

Figure 4. Phylogenetic tree of the TTSuV1b showing the nucleotide sequences of different identified Torque teno virus with GenBank accession number, and country of origin. The sequences were dis-tributed in 2 TTSuV1b sequence groups ( Chinese, American, and Mexican; German, Spanish, Brazilian, Chinese, and Mexican). TTSuV1a sequences ( American, German, and Canadian) and TTV human sequences ( Italian) are included. The Mexican sequences ( KU891808 and KU891809) belong to the 2 TTSuV1b positive cases described in this work. The length of the branch represents the genetic distance among the sequences.


(Table IV), 34% (22/65) of cases and 33% (15/45) of PCV2-RF cases were TTSuV1a1. Similarly, 35% (7/20) of the reproductive failure cases not associated with PCV2 were positive for TTSuV1a1. No case of reproductive failure was positive for TTSuV1b. There was no statistical relationship between the manifestation of PCVAD and the presence of TTSuV1a or TTSuV1b by Ji2 test.

D i s c u s s i o nSeveral reports have suggested that co-infection of Iotatorquevirus

species with other viruses might increase severity of disease as a result of synergy (1,5,16). Consequently, both species have been the target of research for the study of multifactorial diseases, such as porcine respiratory disease complex (17–20) and PCVAD (6–8,21–23).

In natural cases of PMWS, a high frequency of co-infection between TTSuV1a and PCV2 has been described (6). In gnotobiotic

pig models, TTSuV1a was proposed to act as an aggravating factor of PMWS (7) and characteristic PDNS lesions were reproduced in PCV2-negative pigs after inoculation with TTSuV1a and porcine reproductive and respiratory syndrome virus (8). In PMWS-affected pigs, higher prevalence and increase viral load have been found with TTSuV1b than with TTSuV1a (6,22). In addition, a possible association of TTSuV1b with reproductive failure in sows has been proposed (1,24). Taken altogether, current information not only reveals high worldwide prevalence of Iotatorquevirus species but also a close association of its occurrence in cases of PCVAD. However, a relationship of both TTSuV1a and TTSuV1b with development of PCVAD is still unclear.

In the present study, the degenerate nested PCR proved to amplify TTSuV1a and TTSuV1b ORF1 specific sequences. The nested PCR results revealed that TTVSuV1a and TTSuV1b are widely distrib-uted in Mexican states with a high cluster of pig population, as it

Table III. Post-weaning multisystemic wasting syndrome (PMWS) nested polymerase chain reaction (PCR) results for open reading frame (ORF1) region of TTSuV1a and TTSuV1b

TTSuV1a1 TTSuV1a1 TTSuV1a2 TTSuV1a2

TTSuV1b1 TTSuV1b2 TTSuV1b1 TTSuV1b2 TotalPMWS-affected 3b 6 3 11 23Age-matchedclinically normal piglets 0 2 2 8 12

Totala 3 8 5 19 35a Not statistically different between PMWS-affected and age matched healthy piglets at P-value 0.01 (X2 test).b Co-infection only in PMWS-affected.

Table II. Overall nested polymerase chain reaction (PCR) results for open reading frame (ORF1) region of TTSuV1a and TTSuV1b (n = 100)


TTSuV1b1 TTSuV1b2 TTSuV1b1 TTSuV1b2 TotalPCVAD affected 3 21 3 41 68PCVAD non-affected 0 9 2 21 32

Totala 3 30 5 62 100a Not statistically different between porcine circovirus-associated disease (PCVAD)-affected and PCVAD non-affected at P-value 0.01 (X2 test).

Table IV. Reproductive failure nested polymerase chain reaction (PCR) results for open reading frame (ORF1) region of TTSuV1a and TTSuV1b


TTSuV1b1 TTSuV1b2 TTSuV1b1 TTSuV1b2 TotalPCV2-RF 0 15 0 30 45non PCV2-RF 0 7 0 13 20

Totala 0 22 0b 43 65a Not statistically different between porcine circovirus type 2-associated reproductive failure (PCV2-RF) and non-PCV2-RF at P-value 0.01 (X2 test).b Not TTSuV1b positive cases.


is described for PCV2 (25). Also, both species were amplified from cases of PCVAD. In the current scenario, only 35% of total cases were positive to TTSuV1a. Similar findings were obtained regard-less of presentation, 39% (9/23) of PMWS cases and 33% (15/45) of PCV2-RF cases (Table II). The global frequencies of the present work are far lower than reported in other countries, such as Spain (90%), Korea (85%), and China (80%), but comparable to frequencies in Thailand and the United States, which are reported to be 40% and 33%, respectively (26). In the same case series, frequencies in Canada were found to be highly variable (46% to 100%); it was suggested that differences in pig density might influence TTSuV1a prevalence (26). However, we observed a low prevalence of TTSuV1a from Mexican states with the highest proportion of swine farms (27). With regard to TTSuV1b, the gathered data of the present work showed more disparity since European countries have reported higher fre-quencies (6,21,22,28).

Detection of TTSuV1a and/or TTSuV1b has been strongly associ-ated with cases of PMWS, particularly in Europe. Among PMWS-affected pigs, TTSuV1a seroprevalence of 66% to 76% has been reported in Spain (6,22,23,28). Likewise, TTSuV1a frequencies of 77% and 71.4% were reported in Sweden (21) and Slovakia (29), respec-tively. Whereas TTSuV1a detection was 41% and 58% in Great Britain from fresh tissues and sera, respectively (30). Frequency of TTSuV1b among PMWS-affected cases, however, showed even more discrep-ancy since European countries, such as Spain, have reported frequen-cies of 91% (6,28) and 100% (22) that are consistent with the Sweden prevalence of 94% (21). Moreover, TTSuV1b occurrence of 71% and 64.3% were found in Great Britain (30) and Slovakia (29), respectively.

Altogether, PMWS-affected findings of the present work are not consistent with prevalence in Europe, but are comparable to TTSuV1a prevalence of 48%, 40%, and 30% reported in Brazil (31), Cuba (16), and Japan (18), respectively. However, the latter 2 studies were performed on emaciated pigs without further laboratory confir-mation of PCVAD status in affected tissues by ISH or immunohisto-chemistry. An explanation might be linked to geographic relationship within Asia and North America, but TTSuV1b prevalence of 94.7% found in Brazil (30) from PCV2-affected pigs is not in agreement with that hypothesis. Prevalence rates of 37.5% and 31% reported in Cuba (16) and Japan (18), respectively, are still closer to our findings.

Differences in Iotatorquevirus species prevalence rates among stud-ies might be related to target tissue. For instance, lower frequencies were found (41% for TTSuV1a and 79% for TTSuV1b) using pools of fresh lung, liver, kidney, spleen, and lymph node, but higher prevalence of both species (77% for TTSuV1a and 94% for TTSuV1b) was obtained from fresh lymph nodes (21). Such differences are most likely associated with PCV2-target tissues because lymph nodes and spleen are regarded as the main target of PMWS-affected pigs, displaying higher PCV2 loads (32). Therefore, testing fresh lymph node samples alone might increase the likelihood of detecting TTSuV species (6,22). In the current study, results were considerably lower compared to most reports despite the fact that severely and dif-fusely PCV2-affected lymphoid tissues were used. Consequently, the prevalence of TTSuV1a and TTSuV1b from PMWS-affected pigs appears to be low in Mexico.

Prevalence of Iotatorquevirus species in cases of PMWS is reported as higher than that of clinically normal pigs (1,21). Results indicate

that the prevalence is noticeably lower with PCVAD, however, though the same trend was noted, it is not statistically significant. Co-infection of both species in PMWS cases was much lower than that reported in Spain (76%) (22), but similar to that reported in Japan (10%) (18). However, in the current work, co-infection of age-matched clinically normal pigs was not observed (1,22,24).

Immune suppression caused by PCV2 infection has been sug-gested as a predisposing factor for the finding of TTSuV1b in PMWS cases because an increased viral load of TTSuV1b was seen in PMWS-affected pigs compared to the viral load of clinically normal pigs. Thus, clinically normal pigs might restrain infection with both Iotatorquevirus species and PCV2 (28). Such statements are in agree-ment with the data presented herein since co-infection, though in low proportion, was only found in PMWS-affected cases.

The role of Iotatorquevirus in reproductive failure has been pro-posed. To the authors’ knowledge, there are no reports of TTSuV1a nor TTSuV1b in cases of PCV2-RF. A higher seroprevalence of TTSuV1a (60% to 75%), compared to TTSuV1b (30% to 34%), has been detected in healthy sows (24,33). Litters from clinically nor-mal sows infected with TTSuV1a or TTSuV1b showed that 43% and 19% of piglets were TTSuV1a1 and TTSuV1b1, respectively (34). Likewise, 50% of stillbirths were TTSuV1a1 but 7% of still-births were TTSuV1b1, suggesting that both species may cause in utero infection (33). Viremia in sows is a prerequisite for vertical transmission, with the heart being a frequently affected tissue (17). Histopathology of fetal hearts from cases of reproductive failure in sows showed both non suppurative myocarditis and PCV2-specific ISH (35) in 39% of PCV2-RF and 47% of transplacental transmission. These tissues were included in the present work, revealing a low frequency of TTSuV1a and an absence of TTSuV1b with no signifi-cant statistical relation between the occurrence of PCV2-RF and the presence of TTSuV1a.

Using tissue pools from aborted fetuses, the frequency rates of TTSuV1a and TTSuV1b were 17% and 30%, respectively. Heart tissue was part of this tissue pool, but the precise site of infection could not be identified (36). Fetal hearts were one of the individual tissues evaluated in the current study, but other additional tissues that could potentially harbor Iotatorquevirus species were not evaluated, thus Iotatorquevirus species could not be definitively ruled out in cases of PCV2-RF. Nevertheless, since the heart is the main target organ for vertical transmission, preliminary findings suggest that participation of TTSuV1b regarding PCV2-RF is unlikely.

The current retrospective work is noteworthy because a non-vaccinated porcine population was evaluated from a time period prior to the start of through and worldwide immunization programs. Therefore, it may elucidate distinct viral dynamics as influenced by changing immune status and add to the understanding of the evolving nature of viruses. In addition, a TTSuVk2 sequence was amplified based on a new taxonomy (2). Thus, further classification of TTSuV1b is essential to separate it from TTSuVk2 species.

Taken together, the data reported herein are in agreement with a lack of relationship between Iotatorquevirus species and occurrence of PCVAD. Currently, a broad phylogenetic study is being done to ascertain prevailing swine Anelloviridae species in Mexico as well as its genomic variability that might account for a distinctive patho-genic potential.


A c k n o w l e d g m e n t sThe authors thank Karina Enriquez-Ramirez MSc. for her excellent

technical contribution to PCV2-in situ hybridization. The authors are grateful to the CONACYT scholarship program for its commitment to support graduate students. This work was financially supported by a grant from DGAPA-UNAM (PAPIIT IN203309).

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Article


I n t r o d u c t i o nA viral ligand generally is an epitope molecule that binds to a host

cell receptor; namely, the virus attachment protein (1). This receptor is a molecule on the surface of the host cell that recognizes the viral ligand and interacts with it to mediate viral invasion into the host cell (2,3). Binding of the viral ligand to its receptor is the decisive factor in virus–host specificity and tissue tropism. Therefore, full understanding of this interaction is critical to clarifying the patho-genic mechanisms of virus infection. Previous studies have reported on some classical swine fever virus (CSFV) receptors, such as heparin sulfate, mannose-6-phosphate, b-actin, low-density lipoprotein, 50-kDa membrane protein, and 60-kDa actin-binding membrane protein, that are involved in the process of CSFV infection (4–8). Research on CSFV ligands has confirmed that envelope glycoprotein Erns is a disulfide-linked homodimer known to possess ribonuclease activity. It lacks a typical transmembrane region and is found both on the surface of infected cells and in the culture medium. The interaction of Erns with cell surface glycoaminoglycans contributes in part to binding of the virus to susceptible cells (9–11). The envelope

glycoproteins E1 and E2 form a heterodimer that mediates invasion of CSFV into host cells (12,13). The structural protein E2 is a key determinant and major immunogen for viral entry and immunity. Surprisingly, the cellular membrane protein annexin 2 as the host protein can bind to CSFV E2 and promote viral growth in porcine kidney (PK)-15 cells (14).

Blocking the interaction of viral ligand and receptor could inhibit virus infection. However, the amino acid sequence of the viral ligand responsible for receptor binding is unknown. The purpose of this study was to provide a theoretical basis for further revealing the mechanism of CSFV infection, as well as for screening antiviral drugs and designing new vaccines from the perspective of the interaction between viral ligand and receptor.


Peptide synthesisThe bioinformatics software DNASIS MAX (Version 2.5; Informer

Technologies, Madrid, Spain) was used to analyze the amino acid

Identification of the linear ligand epitope on classical swine fever virus that interacts with porcine kidney 15 cells

Yin Mei, Feng Yue, Hong-mei Ning, Juan-juan Zhou, Xuan-nian Wang

A b s t r a c tBinding of the viral ligand to a specific receptor is the first step of virus entry into target cells. The envelope proteins Erns, E1, and E2 of classical swine fever virus (CSFV) are involved in the interaction with host cell receptors to mediate CSFV infection. The aim of this investigation was to identify epitopes that bind to porcine kidney (PK)-15 cells to prevent CSFV infection. Ten peptides representing Erns, E1, and E2 were synthesized. Immunohistochemical study showed that the SE24 peptide, which is derived from the E2 amino acid sequence, could effectively bind to PK-15 cells. Similarly, a flow cytometry assay demonstrated that SE24 binding to PK-15 cells could be blocked by CSFV. The binding of SE24 with PK-15 cells leads to decreased CSFV infection of PK-15 cells in a dose-dependent manner. These results suggest a potential new strategy for the prevention and control of CSFV infection that requires further investigation.

R é s u m éLa liaison d’un ligand viral à un récepteur spécifique est la première étape de l’entrée du virus dans les cellules cibles. Les protéines Erns, E1, et E2 de l’enveloppe du virus de la peste porcine classique (VPPC) sont impliquées dans l’interaction avec les récepteurs de la cellule hôte afin de médier l’infection par le VPPC. L’objectif de la présente étude était d’identifier les épitopes qui se lient aux cellules de rein de porcs (PK-15) afin de prévenir l’infection par le VPPC. Dix peptides représentant Erns, E1, et E2 ont été synthétisés. Des études immunohistochimiques ont montré que le peptide SE24, qui est dérivé de la séquence des acides aminés d’E2, pouvait effectivement se lier aux cellules PK-15. De manière similaire, une épreuve par cytométrie en flux a démontré que la liaison de SE24 aux cellules PK-15 pouvait être bloquée par le VPPC. La liaison de SE24 avec PK-15 amène une diminution de l’infection des cellules PK-15 d’une manière dose dépendante. Ces résultats suggèrent une nouvelle stratégie potentielle pour la prévention et la maîtrise de l’infection par le VPPC qui nécessite de plus amples études.


College of Veterinary and Animal Sciences, Henan Institute of Science and Technology, Xinxiang, Henan, 453003, China (Yin Mei, Hong-mei Ning); College of Life Science and Technology, Xinxiang University, Xinxiang, Henan, 453003, China (Feng Yue, Juan-juan Zhou, Xuan-nian Wang).

Address all correspondence to Dr. Xuan-nian Wang; telephone: 186-373-3682111; fax: 186-373-3683344; e-mail: [email protected]

Received September 20, 2016. Accepted February 13, 2017.


sequences of CSFV proteins Erns, E1, and E2. With standard operat-ing procedures 10 peptides to cover parts of these sequences were synthesized by the solid-phase peptide synthesis method with the 9-fluorenylmethyloxycarbonyl (Fmoc) amino acids attached to Wang resin (Gill Biochemical, Shanghai China). The Cys residue was added at its N terminal to couple the bifunctional reagent sulfosuccinimi-dyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC). Each synthesized peptide was cleaved from the resin with trifluoroacetic acid, precipitated with cold ether, and recovered by centrifugation.

After a wash with cold diethyl ether (at 4°C) to remove impurities, the peptide was dissolved in acetonitrile–water [1:1 (v/v)], which contained 1% acetic acid, and freeze dried. The characteristics of the 10 preliminarily purified synthetic peptides are provided in Table I.

Cells and virusesPorcine kidney-15 cells were maintained in Dulbecco’s modified

Eagle’s medium (DMEM; Gibco ThermoFisher Scientific) supple-mented with 10% fetal bovine serum (FBS) and incubated at 37°C

Table I. Characteristics of the synthetic peptides

Molecular Peptide Number of weight name Amino acid sequence Gene sequence amino acids (kDa)SE01 LATDTELKEGQGMMDASEGTNYTCCKLQRHEW ctggccacagacacggaactgaaagaaatacagggaatgatgga 47 5.82 NKHGWCNWYNIDPWI tgccagcgaggggacaaactatacgtgctgtaagttacagagaca tgaatggaacaaacatggatggtgtaactggtacaatatagaccc ctggata

SE02 LMNRTQANGAEGPPGKECAGTCRYDKDADING ttgatgaatagaacccaagcaaacttggcagaaggccctccggc 52 5.71 GTQARNRPTTLTGCKKGKNF caaggagtgcgctgtgacttgcaggtacgataaagatgctgacat caacgtggtcacccaggccagaaacaggccaacaaccctgacc ggttgcaagaaaggaaaaaatttt

SE03 TNTGENARQGAARGTSWLGRQLSTAGKRLEGR accaacactatagagaatgccagacagggagcagcgagggtaa 37 4.25 SKTWF catcttggctcgggaggcaactcagcactgccgggaagaggttgg agggtagaagcaaaacctggttt

SE11 YTNNCTPACLPKNTKGIGPGKFDTNAEDGKILHE tacactaacaactgcaccccggcttgcctccccaaaaatacaaag 38 4.17 MGGH ataataggccccggaaaatttgacactaacgcggaagacggaaa gattctccatgagatggggggtcac

SE12 IPQSYEEPEGCDTNQLNLTGELRTEDGIPSSVWN attcctcaatcccatgaagaacctgaaggctgcgacacaaaccag 50 5.83 GGKYGCGRPDWWPYET ctgaatctaacagtggaactcaggactgaagacgtaataccgtcat cagtctggaatgttggcaaatatgtgtgtgttagaccagactggtgg ccatatgaaacc

SE21 RLACKEDYRAISSTNEIGLLGAGGLTTTWKEYSH cggctagcctgcaaggaagattacaggtacgcaatatcatcgacc 45 5.16 DLQLNDGTGKA aatgagatagggctactcggggccgaaggtctcaccaccacctgg aaagaatacaaccacgatttgcaactgaatgacgggaccgttaag

SE22 FDGTNPSTEEMGDDFGFGLCPFDTSPGGKGKY ttcgacgggaccaacccatcaactgaggaaatgggagatgacttc 40 4.39 NTTLLNGS gggttcgggctgtgcccgttcgatacgagtcctgttgtcaagggaaa gtacaatacaaccttgttgaacggtagt

SE23 PTTLRTEGGKTFRREKPFPHRMDCGTTTGENED ccaacaactctgagaacagaagtggtaaagaccttcaggagaga 51 5.86 LFYCKLGGNWTCGKGEPV caagccctttccgcacagaatggattgtgcgaccaccacagtgga aaatggagatttattctactgtaagttggggggcaactggacatgtg tgaaaggtgaaccagtg

SE24 VHASDERLGPMPCRPKEIGSSAGPVRKTSCTF gtgcatgcatcagatgaaagactgggccctatgccatgcagaccc 50 5.73 NYAKTGKNKYYEPRDSYF aaagagattgtctctagtgcaggacctgtaaggaaaacttcctgta cattcaactacgcaaaaactttgaagaacaagtactatgagccca gggacagctacttc

SE25 QYMLKGEYQYWFDLDVTDRHSDYFAEFVVLVVV caatatatgcttaagggcgagtatcagtactggtttgacctggacgt 46 4.95 ALLGGRYVLWLIV gactgaccgccactcagattacttcgcagaatttgtcgtcttggtagt ggtagcactgttaggaggaagatatgtcctgtggctaatagtg


in 5% CO2. These cells are often used to propagate CSFV in vitro. The CSFV used was the Shimen strain, the standard virulent strain isolated from China in 1945 and grown in PK-15 cells.

Biotin labeling of the peptidesSulfo-NHS (hydroxysulfosuccinimide)–biotin (ThermoFisher

Scientific) was dissolved in double distilled water for a concentra-tion of 10 mmol/L. The peptides were dissolved in dimethylsulf-oxide (Aladdin Company, Shanghai, China) for a concentration of 40 mg/mL. A peptide–biotin solution was prepared by mixing 80 mL of sulfo-NHS–biotin and 50 mL of the peptide at room temperature for 30 min. After overnight incubation at 4°C the solution was adjusted to a concentration of 1 mg/mL with DMEM containing 10% FBS and frozen for storage.

Binding assayThe PK-15 cells were incubated in 96-well plates at 37°C in 5%

CO2 until 100% confluent and then incubated at 4°C for 1 h with the biotin-labeled peptides at concentrations of 0.2, 0.1, 0.05, and 0.025 mmol/L in aliquots of 100 mL per well. Three replicates of each peptide at each dilution ensured full binding of the peptide with the PK-15 cells. Biotin-labeled bovine serum albumin (BSA) was used as a negative control, and DMEM without biotin–peptide was used as a blank control. Unbound peptide was washed away with precooled 0.01 M phosphate-buffered saline (PBS; 0.02% KH2PO4, 0.29% NH2PO4, 0.02% KCl, and 0.8% NaCl), pH 7.4, contain-ing 0.05% Tween 20 (PBST) (Aladdin). The PK-15 cells were fixed

by 0.3% H2O2 in methanol (Sinopharm Chemical Reagent Company, Ningbo, China), 50 mL/well, for 10 min at 4°C, then were washed 3 times with PBST and blocked with 10% nonfat milk (Yili Industrial Group Company, Hohhot, China) in PBST, 200 mL/well, for 1 h at 37°C. The cells were again rinsed in PBST 3 times, and avidin labeled with horseradish peroxidase (Boster Biological Engineering, Wuhan, China) in 10% nonfat milk in PBST (1:500), 50 mL/well, was added. After incubation for 1 h at 37°C the supernatant was decanted and the plate washed with PBST 3 times. The AEC staining kit (Sigma-Aldrich, St. Louis, Missouri, USA) was used to detect binding of peptide to cell: 50 mL of substrate was applied to each well for 10 min at room temperature, and the cells were then observed through a microscope.

Peptide-binding and virus-blocking assaysThree groups were set up. In group A, biotin-labeled peptide,

0.2 mmol/L, was used to bind to PK-15 cells. In group B, biotin-labeled BSA was used as a negative control with PK-15 cells. In group C, the biotin-labeled peptide was used to bind to PK-15 cells after CSFV adsorption to the PK-15 cells. The biotin-labeled peptides, biotin-labeled BSA, and CSFV, 100 3 the median tissue culture infec-tious dose (TCID50), were incubated with 1 3 106 PK-15 cells for 1 h on ice. Three replicates were done in each group. In all 3 groups, 100 mL of avidin labeled with fluorescein isothiocyanate (1:500) was added and the mixture incubated for 30 min on ice. The supernatant was decanted, and the PK-15 cells were resuspended with 0.5 mL of prechilled PBS. After being mixed, the samples were assayed by

Figure 1. Binding of peptide SE24 to 100% confluent porcine kidney (PK)-15 cells when incubated at peptide concentrations of 0.2 (A), 0.1 (B), 0.05 (C), and 0.025 (D) mmol/L. Biotin-labeled bovine serum albumin (BSA) was used as a negative control (E) and Dulbecco’s modified Eagle’s medium as a blank control (F). The immunohistochemical staining procedure is detailed in Materials and methods. The staining was gradually lighter with the decrease in peptide concentration, which indicates that the capacity of peptide SE24 to bind with PK-15 cells is strong.


Figure 3. Rate of CSFV infection of PK-15 cells incubated with peptide SE24 at concentrations of 0.2 (A), 0.1 (B), 0.05 (C), 0.025 (D), 0.0125 (E), and 0.00625 (F) mmol/L. G — BSA (unrelated protein control). H — negative serum (isotype control). See Materials and methods for details. With a decrease in peptide concentration, the rate of CSFV infection gradually increased.

Figure 2. A — binding of biotin-labeled peptide SE24 to PK-15 cells. B — binding of biotin-labeled BSA to PK-15 cells. C — binding of biotin-labeled SE24 to PK-15 cells pretreated with classical swine fever virus (CSFV). Avidin labeled with fluorescein isothiocyanate was applied and flow cytometry used to detect the binding. See Materials and methods for details. The mean proportion of positive cells (6 standard deviation) decreased from 50.86% 6 3.30% to 39.43% 6 1.35% after CSFV pretreatment, indicating that the virus can block the binding of peptide SE24 to PK-15 cells.

Gm:17.58

CV:28.72

[251-1023]1465 (14.7%)

Gm:23.77

CV:24.12

[251-1023]5103 (51.0%)

M1 M2 M1 M2M1

M2

Gm:20.70

CV:25.42

[251-1023]3939 (39.4%)

FL1-H FL1-H FL1-H

Even

ts

Even

ts

Even

ts

64

0

64

0

64

0100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

A B C


Attune Acoustic Focusing Cytometer (Applied Biosystems, Waltham, Massachusetts, USA); 1 3 104 cells were counted for each sample.

Infection-inhibition assayThe PK-15 cells were cultured in 96-well plates until 70% to 80%

confluent and then incubated with peptide SE24 at concentrations of 0.2, 0.1, 0.05, 0.025, 0.0125, and 0.00625 mmol/L, for 100 mL/well, in 10% DMEM. Three replicates were done. For controls, BSA was used as an unrelated protein and negative serum was used as an isotype. After incubation at 37°C for 1 h the supernatant was decanted, and 10 3 TCID50 of CSFV was added to infect the cells. After 3 washes with 10% FBS in DMEM and refreshment with 5% FBS in DMEM, the cells were cultured at 37°C for 24 to 48 h.

For immunohistochemical staining the cells were rinsed in PBST 3 times for 5 min each time, then fixed by being incubated with 4% paraformaldehyde for 10 min at 4°C, and then rinsed again in PBST 3 times for 5 min each time. Nonspecific binding sites were then blocked by incubation of the cells with 10% skimmed milk powder for 1 h at 37°C followed by rinsing in PBST 3 times for 5 min each time. Next, rabbit anti-CSFV serum (1/200) was added and the mix-ture incubated for 30 min at 37°C and then rinsed in PBST 3 times for 5 min each time. Then goat IgG against rabbit antigen (1/40) was added and the mixture incubated for 30 min at 37°C and then rinsed in PBST 3 times for 5 min each time. Finally, the AEC kit was used again, with substrate applied for 10 min at room temperature. The number of infected cells was counted through a microscope.

Statistical analysisData were analyzed with the paired-samples t-test by means of

IBM SPSS Statistics for Windows, Version 19.0 (IBM Corporation, Armonk, New York, USA) and are presented as mean 6 standard deviation. P-values of less than 0.05 were considered statistically significant.

Re s u l t sThe binding-assay results showed that peptide SE24 could interact

with PK-15 cells at all the tested concentrations. Data for the other peptides are not shown. Positive cells stained brownish red (Figure 1, panels A to D). Neither the biotin-labeled BSA [negative control

(Figure 1, panel E)] nor the DMEM [blank control (Figure 1, panel F)] showed binding to the PK-15 cells. Peptide SE24, whose amino acid sequence is shown in Table I, is located on the E2 protein of CSFV and has a molecular weight of 5.73 kDa. Its binding occurred in a dose-dependent manner and was mainly on the cell membrane.

The ability of CSFV to block the binding of peptide SE24 to PK-15 cells was measured by flow cytometry (Figure 2). The mean proportion of positive cells and the mean fluorescence intensity of the binding were 50.86% 6 3.30% and 23.52% 6 1.24%, respec-tively, but when the PK-15 cells were pretreated with CSFV the means were significantly lower (P , 0.05), at 39.43% 6 1.35% and 20.26% 6 0.58%, respectively. The means for the negative-control group were significantly lower still (P , 0.05), at 14.50% 6 1.21% and 17.47% 6 0.71%, respectively. The infection-inhibition assay showed that peptide SE24, 0.2 mmol/L, could completely inhibit CSFV infection of PK-15 cells (Figure 3, panel A). With a decreasing concentration of the peptide, the number of infected cells increased (Figure 3, panels B to F), as did the infection rate (Table II). The infec-tion of PK-15 cells by CSFV was not inhibited by BSA, the negative control, at any concentration, and the cells stained reddish brown (Figure 3, panel G). With the blank control, no PK-15 cells stained reddish brown (Figure 3, panel H). With the SE24 concentration as the independent variable and the inhibition rate as the dependent variable, and log transformation of the data, Figure 4 shows graphi-cally that with a decrease in peptide concentration, the infection rate increased gradually.

D i s c u s s i o nAccording to previous studies, the envelope glycoproteins of

CSFV — namely, Erns, E1, and E2 — participate in virus adsorption and invasion (9–13). On this basis, 10 peptides were synthesized to

Table II. Inhibition by peptide SE24 of infection of porcine kidney 15 cells by classical swine fever virus

Infected cells;

Peptide concentration mean 6 standard deviationa

(mmol/L) Number Percentageb

0.2 0c 0c

0.1 260.00 6 9.00d 3.71 6 0.12d

0.05 286.00 6 10.53d 4.08 6 0.15e

0.025 566.33 6 22.30e 8.09 6 0.32f

0.0125 684.33 6 14.01f 9.78 6 0.20g

0.00625 874.67 6 21.39g 12.49 6 0.30h

a Different superscript letters in the same column indicate a significant difference (P , 0.05).b The total number of cells per well was 7.0 3 103.

Figure 4. Graphic representation of the decrease in the rate of CSFV infection of PK-15 cells when incubated with increasing concentrations of peptide SE24, until none of the cells were infected at a peptide concentration of 0.2 mmol/L. The infection rate (logy) and the peptide concentration (logx) were natural log-transformed.

logx

logy

2.4

2.2

2.0

1.8

1.6

1.4

25.0 24.5 24.0 23.5 23.0 22.5 22.0 21.5


cover parts of the amino acid sequences of these glycoproteins. The infection-inhibition assay confirmed the inhibitory effect of peptide SE24 on CSFV infection of the target cells, which was consistent with the blocking by CSFV of peptide SE24 binding to PK-15 cells. Surprisingly, our study showed that peptide SE24 and CSFV com-petitively bind to the same receptor on the membrane of PK-15 cells. The binding of peptide SE24 to PK-15 cells can block CSFV adsorp-tion but also suppress PK-15 cells already infected with CSFV. These findings suggest that peptide SE24 is a potential drug target for the prevention and treatment of classical swine fever.

The highest concentration of peptide SE24 used in the infection-inhibition assay, 0.2 mmol/L, almost entirely inhibited CSFV infec-tion of PK-15 cells. Water solubility and hydrophilicity can affect peptide activity. Thus, improving the solubility and hydrophilicity of the peptide would be very important in development of a peptide drug. Currently, the biologic activity and function of the polypep-tide, and shortening of its length, are being studied in our labora-tory. Now that we have shown in vitro that peptide SE24 effectively binds to target cells and inhibits infection of those cells by CSFV, further study is needed to verify the binding function and infection-inhibition activity of the peptide in vivo.

The peptide SE24 consists of 50 amino acids located on the CSFV E2 protein (amino acids 955 to 1004). Many experimental features of SE24 are consisted with E2 function. For instance, E2 is a deter-minant of cell culture tropism, and the E1 and E2 proteins formed a heterodimer that mediated invasion of host cells by CSFV (9,12,13). The E2 glycoprotein is likely to be involved in the host specificity of pestiviruses at their point of uptake into cells (15), and the E2 protein of CSFV can interact with swine host factors by the yeast 2-hybrid system (16). According to the prediction of E2 structure (17), peptide SE24 is located close to the transmembrane region rather than the antigen region. In theory, the viral ligand should be in the antigen region or the extracellular domain, which facilitates binding with the viral receptor. Whether peptide SE24 can be used as a viral ligand is unknown. The process by which viruses invade cells is complex. This study has suggested a novel approach for the prevention and treatment of viral diseases: inhibiting viral infection of host cells by blocking the virus receptor–ligand interaction.

A c k n o w l e d g m e n t sThis work was supported by the National Natural Science

Foundation of China (grant 31201877) and the Foundation of the Henan Educational Committee (grant 16A230001).

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Article


I n t r o d u c t i o nOver the last decade, methicillin-resistant Staphylococcus pseudin-

termedius (MRSP) has emerged as a major opportunistic pathogen in dogs, and as the leading cause of skin disease, otitis, surgical site infections, and wound infections (1,2). Methicillin-resistance in MRSP is mediated by the mecA gene, which codes for the expres-sion of a modified penicillin-binding protein, PBP29, and resistance to most b-lactam antimicrobials (2). Increasing antimicrobial resis-tance to antimicrobials other than b-lactams, has been reported in MRSP isolates, leaving few therapeutic options available in many instances (1–3). Methicillin-resistant Staphylococcus pseudintermedius has been implicated in hospital-acquired infections within veteri-

nary hospitals, and the potential of hospital-wide outbreaks should not be underestimated (1,4). Not only has MRSP been reported in the canine population, but also there have been several reports of humans infected or colonized with MRSP (5–11). Understanding the diversity and dissemination of MRSP is essential for develop-ing mitigation and control strategies and for future epidemiologic studies. Currently, there is no study characterizing MRSP isolates in Atlantic Canada.

A number of molecular tools have been reported to genetically dis-criminate among staphylococci. Most of these tools were first devel-oped and standardized for methicillin-resistant Staphylococcus aureus (MRSA), but few have been further refined for discriminating MRSP (1,12–16). While pulsed-field gel electrophoresis (PFGE) remains the

Direct repeat unit (dru) typing and antimicrobial resistance of methicillin-resistant Staphylococcus pseudintermedius isolated

from dogs in Atlantic CanadaMatthew E. Saab, J. Scott Weese, J.T. McClure

A b s t r a c tThere are few reports investigating the characterization of methicillin-resistant Staphylococcus pseudintermedius (MRSP) in dogs in Canada and none from Atlantic Canada. The objectives of this study were to strain type MRSP isolates cultured at a regional diagnostic laboratory using direct repeat unit (dru) typing and to describe their antimicrobial resistance profiles. Ninety-four isolates recovered from dogs between 2010 and 2012 had dru typing, cluster analysis, and antimicrobial susceptibility testing done. The majority of isolates belonged to type dt11a (30.9%), dt10h (24.5%), dt9a (18.1%), and dt11af (10.6%) with the remaining 15.9% of isolates distributed among 13 dru types. The predominant dru types identified were similar in Ontario; however, cluster 9a appears to be less common in Atlantic Canada. A significant difference in the distribution of clusters among Atlantic provinces was detected (P = 0.01). Resistance to $ 2 non-b-lactam antimicrobials was observed in 71.4% of the isolates. The MRSP isolates from this study were notably less resistant than those reported in the literature. A more comprehensive study of the MRSP dru types could help further elucidate the distribution of this pathogen in Canada.

R é s u m éIl y a peu de publications rapportant la caractérisation des isolats de Staphylococcus pseudintermedius résistant à la méticilline (SPRM) chez les chiens au Canada et aucun ne provenant des provinces maritimes. Les objectifs de la présente étude était de typer les isolats de SPRM cultivés à un laboratoire de diagnostic régional en utilisant le typage direct des unités répétées (dur) et de décrire les profils de résistance antimicrobienne. Quatre-vingt-quatorze isolats canins obtenus entre 2010 et 2012 furent soumis au typage du dur, une analyse de regroupement, et un antibiogramme. La majorité des isolats appartenait au type dt11a (30,9 %), dt10h (24,5 %), dt9a (18,1 %), et dt11af (10,6 %) et le 15,9 % des isolats restant étaient distribués parmi 13 types dur. Les types prédominants de dur identifiés étaient similaires à ceux de l’Ontario, Canada, bien que le regroupement 9a semble moins fréquent dans les provinces maritimes. Une différence significative dans la distribution des regroupements entre les provinces maritimes a été détectée (P = 0,01). Une résistance à $ 2 antimicrobiens n’appartenant pas à la classe des b-lactames fut observée chez 71,4 % des isolats. Les isolats de SPRM de la présente étude était sensiblement moins résistant que ceux rapportés dans la littérature. Une étude plus complète des types dur des SPRM pourrait aider à élucider un peu plus la distribution de cet agent pathogène au Canada.


Diagnostic Services, Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, Prince Edward Island (Saab); Department of Health Management, Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, Prince Edward Island (Saab, McClure); Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario (Weese).

Address all correspondence to Mr. Matthew Saab; e-mail: [email protected]

Received December 20, 2016. Accepted February 9, 2017.


gold standard for MRSA outbreak investigations, it has been shown to be less useful for MRSP investigations (12,14,17,18). Historically, multi-locus sequence typing (MLST) has been the primary tool for investigating the population genetic structure of MRSP, and 2 major sequence types (ST) have been identified (1,16,19). Both PFGE and MLST are laborious, costly, and require the use of reference strains, thus making them impractical tools for implementation in smaller diagnostic laboratories.

The use of a single-locus marker for isolate discrimination has also been explored for MRSA, and provides a less expensive and less laborious approach to strain typing (20,21). Sequence analysis of the staphylococcal protein A region (spa typing) has been shown to have greater discriminatory power than MLST and PFGE for MRSA, but it is less successful for strain typing all MRSP isolates (14,20–22). The mec-associated direct repeat unit (dru) typing was first demon-strated to be useful in discriminating highly-clonal MRSA isolates in Scotland (13), and has more recently been successfully applied to MRSP isolates from Canada, the United States (US), Europe, and Australia (22–24). In a previous study, significant associations between dru cluster and MLST were established, in which cluster 9a was associated with ST71 (the “International clone”) and cluster 11a was associated with ST68 (the “North American clone”) (1,22). This single-locus sequence-based method is rapid, standardized, and cost-effective, making it an ideal candidate for use in small-scale laboratories (22,23).

In a previous study, significant differences have been reported in the distribution of the 2 main dru clusters, 9a and 11a, between Canada, the US, and Europe (22). The report also inferred that varia-tion within a country may exist, as some dru types were detected in some US states, but not the others included in the study. To date, there has been only one study investigating dru types in Canada, which was limited to Ontario; therefore, genetic information on MRSP isolates from Canadian regions other than Ontario is needed. Thus, the primary objective of this current study was to explore the strain type diversity of MRSP isolates from dogs using the dru typ-ing method from submissions to a regional diagnostic laboratory in Atlantic Canada. A secondary objective was to describe the antimi-crobial resistance (AMR) pattern of these MRSP isolates.


Isolate screening and collectionIsolates were recovered from canine specimens submitted

to the Atlantic Veterinary College (AVC) Diagnostic Services Bacteriology Laboratory for routine culture and susceptibility test-ing. Staphylococci were identified by colony morphology, including hemolysis, a positive tube coagulase test and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spec-trometry. Antimicrobial susceptibilities to the following drugs were determined using the Kirby-Bauer disk diffusion method, following Clinical Laboratory Standards Institute (CLSI) standard M31-A3 (25): ampicillin (10 mg), amikacin (30 mg), amoxicillin-clavulanic acid (30 mg), cephalexin (30 mg), cefovecin (30 mg), chlorampheni-col (30 mg), clindamycin (30 mg), doxycycline (30 mg), enrofloxacin (5 mg), erythromycin (15 mg), fusidic acid (10 mg), gentamicin (10 mg),

penicillin (10 mg), and trimethoprim-sulfamethoxazole (SXT; 25 mg). Methicillin resistance was detected by oxacillin (1 mg) disk diffusion, and confirmed by mecA expression using the PBP29 latex agglutina-tion test (PBP29 Latex Agglutination Test; Oxoid Company, Nepean, Ontario). All isolates identified presumptively as MRSP were placed in a medium containing 15% glycerol and frozen at 280°C.

Patient province of residence, submitting clinic, anatomical source/site of specimen, and antimicrobial susceptibility data were extracted from the laboratory data management system and descrip-tive statistics were computed. Only 1 isolate from each patient was included for analysis in most patients. If multiple MRSP isolates on repeated lab submissions were recovered from a patient but had the same dru type, only the first isolate was included for analysis. If dif-ferent dru types were isolated from the same patient on different lab submissions, then both isolates were retained for analysis.

Molecular identification and typingGenomic material was extracted using a resin matrix (InstaGene

Matrix; Bio-Rad Laboratories, Montreal, Quebec). Manufacturer’s guidelines were followed except a large loopful of bacterial colo-nies were suspended in 1.0 mL of polymerase chain reaction (PCR) water, and the incubation time at 56°C was increased from 30 min to 1 h. Isolated DNA was frozen at 220°C until use. A multiplex PCR reaction was used to identify coagulase-positive staphylococci to the species level based on partial amplification of the nuc gene locus (26). Staphylococcus pseudintermedius isolates were character-ized using the dru typing method, as previously described (17). A 40-mL reaction was prepared containing 4 mL of template DNA, 0.8 U of DNA polymerase, buffer mixture containing 1.5 mM MgCl2 and 200 mM of each dNTP (KAPA2G Fast Hot Start DNA polymerase; KAPA Biosystems, Boston, Massachusetts, USA), addi-tional 0.3 mM MgCl2, and 0.8 mM of the forward primer druGF (59-GTTAGCATATTACCTCTCCTTGC-39) and the reverse primer druGR (59-GCCGATTGTGCTTGATGAG-39). Reaction mixtures were thermally cycled for an initial denaturation step of 94°C for 2 min followed by 30 cycles at 94°C for 1 min, 52°C for 1 min, and 72°C for 1 min (17). The PCR products were purified using a kit (QIAquick PCR Purification Kit; Qiagen, Mississauga, Ontario) prior to sequencing.

The dru repeats and types were assigned using a website (www.dru-typing.org) following the previously described nomen-clature (17). Cluster analysis was used to compare the relatedness among dru types, and a minimum spanning tree (MST) was gener-ated. Distance intervals (or similarity values) were created using a bin distance of 1.0%, in which dru types separated by an MST distance of # 2 repeats (. 98.5% similarity) were considered closely related and assigned to the same cluster. Root nodes were assigned to the dru type with the greatest number of isolates. Cluster analysis was completed using the TRST plug-in tool (BioNumerics version 6.6; Applied Maths, Austin, Texas, USA), as described previously for MRSP dru typing (22–24,27).

Statistical analysisMulti-drug resistant (MDR) MRSP were defined as being resistant

to $ 2 antimicrobials classes in addition to b-lactams. Dru clusters were used for comparisons to decrease the number of tested groups.


Unconditional associations between dru cluster and patient data and dru cluster and resistance to the non-b-lactam antimicrobials were assessed using Chi-squared or Fisher’s exact tests, where appropri-ate, with significance set at P # 0.05. Subtables were explored for any significant association to determine where the differences were. All statistical computations were done using computer software (Stata/IC 13.1 for Mac; StataCorp, College Station, Texas, USA).

Re s u l t s

Isolate collectionThe diagnostic laboratory collected 129 isolates from 90 dogs

between January 2010 and December 2012. Of those, 98 isolates were retained for analysis. Twenty-three patients had multiple submis-sions. Isolates were collected from various specimens (n = 98): skin (n = 52, 53.1%), ears (n = 18, 18.4%), wounds (n = 8, 8.2%), surgical sites (n = 6, 6.1%), urine (n = 5, 5.1%), abscesses (n = 2, 2.0%), and other (n = 7, 7.1%). Most of the patients were seen by private vet-erinary clinics in the region (71.4%) compared to being seen by the AVC Veterinary Teaching Hospital (AVC-VTH) (28.6%).

Antimicrobial resistance patternsOnly 8.2% (n = 8) of isolates were susceptible to all tested drugs

with the exception of b-lactams (Table I). Another 20.4% (n = 20) of isolates were resistant to 1 non-b-lactam antimicrobial class, while 71.4% (n = 70) were resistant to $ 2 non-b-lactam antimicrobial classes. The most common resistance was to SXT (74.5%), followed by erythromycin (68.4%), clindamycin (55.1%), enrofloxacin (46.9%), gentamicin (34.7%), chloramphenicol (23.5%), doxycycline (15.3%), and fusidic acid (3.1%). In isolates that displayed multi-drug resis-tance (n = 70), 68.6% of isolates were resistant to SXT, erythromycin, and clindamycin, while 55.7% (n = 70) of those were also resistant to enrofloxacin. Amikacin resistance was not detected in any of the isolates.

Direct repeat unit typingThe dru types were determined for 94/98 isolates, because

4 isolates were not available for typing. From the 94 isolates, 18 dru types were recovered with 3 predominant dru types contributing more than 70% of the distribution: dt11a (30.9%), dt10h (24.5%), and dt9a (18.1%). The frequency of each dru type can be found in Table II. Nine of the dru types were previously unidentified at the time of analysis and are represented by one isolate each: dt5k, dt6t, dt8ag, dt9bd, dt10cc, dt10cj, dt11ca, and dt11cm.

Table I. Number (and percent) of antimicrobial resistant methicillin-resistant Staphylococcus pseudintermedius (MRSP) isolates overall and by dru cluster, following Clinical Laboratory Standards Institute (CLSI) guidelines. Multidrug resistance (MDR) in each cluster is also reported

Overall 9a 10h 11a No cluster Antimicrobial (n = 94) (n = 18) (n = 27) (n = 45) (n = 4)Amikacin 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0%)Chloramphenicol 23 (24.5%) 3 (16.7%) 0 (0%) 19 (42.2%) 1 (25%)Clindamycin 54 (57.5%) 16 (88.9%) 7 (25.9%) 29 (64.4%) 2 (50%)Doxycycline 14 (14.9%) 1 (5.6%) 7 (25.9%) 4 (8.9%) 2 (50%)Erythromycin 63 (67.0%) 17 (94.4%) 11 (40.7%) 32 (71.1%) 3 (75%)Enrofloxacin 46 (48.9%) 16 (88.9%) 0 (0%) 29 (64.4%) 1 (25%)Fusidic acid 2 (2.1%) 1 (5.6%) 1 (3.7%) 0 (0%) 0 (0%)Gentamicin 34 (36.2%) 10 (55.6%) 2 (7.4%) 20 (44.4%) 2 (50%)SXT 70 (74.5%) 18 (100%) 23 (85.2%) 27 (60.0%) 2 (50%)MDR 66 (70.2%) 17 (94.4%) 15 (55.6%) 31 (68.9%) 3 (75%)SXT — trimethoprim-sulfamethoxazole.

Table II. Frequency of methicillin-resistant Staphylococcus pseudintermedius dru types from 94 isolates collected at a regional diagnostic laboratory between 2010 and 2012

Frequency dru type (n = 94) Clusterdt11a 29 (30.9%) 11adt10h 23 (24.5%) 10hdt9a 17 (18.1%) 9adt11af 10 (10.6%) 11adt10as 3 (3.2%) 10hdt10cca 1 (1.1%) 10hdt10cja 1 (1.1%) 11adt11bn 1 (1.1%) 11adt11caa 1 (1.1%) 11adt11v 1 (1.1%) 11adt11y 1 (1.1%) 11adt5ka 1 (1.1%) —dt6ta 1 (1.1%) —dt8aga 1 (1.1%) —dt9baa 1 (1.1%) 9adt9bda 1 (1.1%) —dt11cma 1 (1.1%) 11aa Novel dru types identified in this study.


A minimum spanning tree (MST) was constructed to identify relatedness among dru types, and to determine the predominate clusters (Figure 1). The cluster analysis identified 3 main dru clusters that included 90/94 typed isolates (Figure 1). Cluster 11a contained the most diversity, comprising 9 dru types and 47.9% of the typed isolates. Cluster 10h contained 3 dru types and 28.7% of the isolates, while cluster 9a contained 2 dru types and 19.2% of the isolates. Table II contains the dru type and cluster association.

Associations among dru clusters, patient demographics, and AMR

Significant differences were determined among the distribu-tions of dru clusters in the 4 Canadian Atlantic provinces (P = 0.01; Table III). In Newfoundland and Labrador (NL), cluster 9a was over-represented with 5/7 strains belonging to dt9a. Cluster 10h was present in Nova Scotia (NS; 15/52) and New Brunswick (NB; 5/23), but not NL, and was the predominant cluster from Prince Edward Island (PEI; 7/12) isolates. Both mainland provinces, NB and NS, shared the same predominant cluster, 11a, which comprised over 50% of the isolates from each province.

The dru cluster was not significantly different among the AVC-VTH and private practice (P = 0.18) or specimen types (P = 0.91). Significant associations among AMR and dru clusters were detected for all antimicrobials except fusidic acid (P = 0.31; Table I). For clindamycin, erythromycin, enrofloxacin, and SXT, cluster 9a had a significantly higher proportion of resistant isolates (P # 0.01). Cluster 10h had significantly higher proportion of isolates resistant to doxycycline (P = 0.04), while cluster 11a had a significantly higher proportion of chloramphenicol resistance (P , 0.001). The propor-tion of MDR isolates was significantly different among dru clusters (P = 0.03), with cluster 9a having the highest proportion of MDR (94.4%), followed by 10h (55.6%) and 11a (68.8%).

D i s c u s s i o nThe majority of isolates (73.5%) collected as part of this con-

venience sampling at the AVC Diagnostic Services Bacteriology Laboratory were represented by dru types dt11a, dt10h, and dt9a, which is similar to the findings in a previous multi-national study (22). However, in the current study, clusters 11a and 10h comprised

Figure 1. Minimum spanning tree (MST) of methicillin-resistant Staphylococcus pseudintermedius in Atlantic Canada. Numerical values on the branches indicate the similarity (MST distance) among different dru types. The dru types separated by an MST distance of # 2 ($ 98.5% similar) were considered closely related and assigned to the same cluster.

dt6t

dt10cc

dt10as

dt10hdt9bd

dt11ca dt11adt11af

dt11bn

dt10cj

dt5k

dt8agdt9a

dt9ba

dt10v

dt11v dt11y

dt11cm

3

2

2

22

2


more than 75% of the isolates collected, whereas in the Ontario study, cluster 11a (including dt10h) and 9a were evenly distributed (22). The distribution of MRSP dru clusters in Atlantic Canada was more similar to what has been reported in California, Illinois, North Carolina, Tennessee, and Texas (22). Differences among geographic regions within the US were also noted in the previous study (22).

Significant differences in the diversity of strains were detected among provinces of Atlantic Canada (Table III). It should be noted that this study was not designed for prevalence estimation because of the convenience sampling approach. Although it may appear that the incidence of MRSP is higher in certain provinces, these numbers are reflective of the sample submission demographics from these regions (i.e., the laboratory receives approximately 45% of their canine samples from NS, but only 5% are from NL). These provin-cial differences in the distribution of dru types could be attributed to geographic separation, since differences among Newfoundland and Labrador and the Maritime Provinces (NB, NS, and PEI) exist, as well as population density, since provinces with larger populations have greater dru type diversity. The small sample size of isolates from Newfoundland and Labrador may bias its estimate. It is pos-sible that the true diversity of the isolates from some provinces is underestimated, since each province except PEI has its own animal health microbiology laboratory, decreasing submissions from these provinces to the AVC diagnostic laboratory.

This study detected 9 dru types that had not been previously iden-tified (Table II), and required entry into the dru database. These new dru types are results of a random single-nucleotide polymorphism in one of the 40bp dru repeat sequences, subsequently creating a new dru repeat and thus new dru type. Novel dru types have also been identified in a previous study (22). Although there were no significant differences in the distribution of the new dru types, most of the novel dru types were isolated from Nova Scotia (5/9), and from skin samples (5/9).

More than 70% of the isolates recovered in this study were resis-tant to $ 2 non-b-lactam antimicrobial classes, highlighting the clini-cal concerns regarding management of MRSP infections. Multi-drug resistance has been frequently reported in MRSP isolated from canine specimens (1,28). The isolates recovered in this study were from submissions to a regional diagnostic bacteriology laboratory that also is the diagnostic laboratory for the AVC-VTH, thus a selection bias is possible, based on submission of specimens from complicated referral cases from the AVC-VTH. However, most samples in this study were from primary care practices. Information about prior antimicrobial exposure was not always available, but submissions to a diagnostic laboratory for bacterial culture and susceptibility

testing can be from cases proving difficult to treat, with the primary reason being non-response to antimicrobial therapy. It is likely that diagnostic laboratory submissions are biased to be more resistant.

Interestingly, cluster 9a isolates had the highest proportion of MDR at 94.4%, and also had a significantly higher proportion of resistant isolates for each antimicrobial except fusidic acid, chloram-phenicol, and doxycycline. Strong associations between cluster 9a and ST71 have been shown (22), and reports have also shown ST71 to have an increased antimicrobial resistance compared with other STs (1,29). MLST was not done on the isolates in this study, but the clus-ter 9a isolates in this study likely belong to ST71. Although this study was not designed for prevalence estimation, the ST71 clone associ-ated dru cluster 9a seems to have not disseminated into Atlantic Canada (18.1%), with the possible exception of Newfoundland, to the degree that it has in Europe (90%), the US (66%), and Ontario, Canada (47%) (22).

Resistance of the MRSP isolates to the non-b-lactam antimicrobi-als in this study was lower than in previous reports of multi-drug resistance in MRSP, which could be explained by the low preva-lence of cluster 9a isolates. However, caution should be taken when comparing studies because of differences in testing methodology. Specifically, it was reported that 62% (n = 107) of study isolates were resistant to doxycycline in Ontario (24), while in our study doxycycline resistance of isolates was 15%. One study in Australia and another in the United Kingdom (UK) found similarly high tet-racycline resistance at over 50% and 35%, respectively (23,30). Since the time of this study, canine specific doxycycline breakpoints for S. pseudintermedius were proposed and implemented by CLSI (31,32). This change in zone diameter breakpoints would likely increase the number of doxycycline resistant isolates in both this study and the Ontario study. Similar trends can be observed where the proportion of resistant isolates in Atlantic Canada is much lower than those reported in Australia and the UK. A recent study completed at a Texas (USA) Veterinary Medical Teaching hospital reported ami-kacin resistance in 36% of their MRSP isolates from dogs, whereas in our study amikacin resistance was not detected (33). A system-atic literature review of antimicrobial resistance in MRSP isolates reported individual antimicrobial resistance ranging between 0% and 100%, with most studies reporting AMR estimates . 50% for most antimicrobials, except for chloramphenicol and amikacin (28). Thus, it can be inferred that the MRSP isolates in Atlantic Canada are typically less resistant, specifically to doxycycline and amikacin, than their counterparts in some other regions, even though multi-drug resistance is still common. A possible explanation for this low-level resistance could be the low population density of the region as a

Table III. Number (and percent) of methicillin-resistant Staphylococcus pseudintermedius (MRSP) isolates in each province overall and by dru cluster

dru cluster NB NL NS PEIOverall (n = 94) 23 (24.5%) 7 (7.4%) 52 (55.3%) 12 (12.8%)9a (n = 18) 6 (33.3%) 5 (27.8%) 6 (33.3%) 1 (5.6%)10h (n = 27) 5 (18.5%) 0 (0.0%) 15 (55.6%) 7 (25.9%)11a (n = 45) 12 (26.7%) 2 (4.4%) 28 (62.2%) 3 (6.7%)No cluster (n = 4) 0 (0.0%) 0 (0.0%) 3 (75.0%) 1 (25.0%)NB — New Brunswick; NL — Newfoundland and Labrador; NS — Nova Scotia; PEI — Prince Edward Island.


whole, when compared with larger, more densely populated regions, which could mean less exposure to AMR organisms and less total antimicrobial use.

The overall picture of MRSP in Atlantic Canada is similar to reports elsewhere in Canada and the world. The distribution of the dru types reported in this study is similar to other reports from North America; however, the distribution is more similar to what was observed in the US versus Ontario, Canada. The international MRSP clone, ST71, which is disseminated throughout Europe and made up almost half the isolates in Ontario, Canada, was less com-mon in Atlantic Canada. This confirms that dru type distributions can vary significantly across the same country, and a larger, more comprehensive study of the dru types in Canada could help further clarify the dissemination of this pathogen. Multi-drug resistance in these isolates is common, especially within cluster 9a isolates, but resistance to the non-b-lactam antimicrobials is still considerably lower than has been previously reported.

A c k n o w l e d g m e n t sThe authors thank AVC Diagnostic Services Bacteriology

Laboratory for their valuable work with sample and isolate collec-tion; Joyce Rousseau, Ontario Veterinary College, for assistance with molecular techniques; Spencer Greenwood, AVC Lobster Science Centre, for use of equipment; Henrik Stryhn, Atlantic Veterinary College, for assistance with statistical analyses; and Robert Page, University of Prince Edward Island, for laboratory data extraction. This study was financially supported by the Atlantic Veterinary College Companion Animal Trust Fund and the Sir James Dunn Animal Welfare Centre.

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Article


I n t r o d u c t i o nAn estimated 10 000 cases of canine osteosarcoma (OSA) are

diagnosed in pet dogs annually in the United States (1). It is a locally invasive and highly metastatic disease, with micrometastasis present in approximately 75% to 95% of dogs at the time of diagnosis, based on studies of survival following amputation (2,3). While surgery is indicated to remove the painful and functionally limiting primary tumor, amputation is considered palliative therapy and does not result in prolonged survival. Median survival times following ampu-tation alone range from 102 to 168 d, with 1- and 2-year survival rates of 11% to 21% and 2% to 4%, respectively. The administration of adjuvant chemotherapy in an effort to delay or prevent metas-tasis and improve the outcome, is the current standard of care in

veterinary medicine. Three drugs have been identified as having significant activity against osteosarcoma: cisplatin, carboplatin, and doxorubicin (2–4). Median survival times ranging from 290 to 324 d and 1-year survival rates of 45.5% have been reported with the administration of adjuvant cisplatin chemotherapy (3,4). Carboplatin chemotherapy is comparable, with a median survival of 321 d and 1-year survival of 35.4% (5). Similarly, the median survival time fol-lowing a course of doxorubicin chemotherapy is 366 d with 1- and 2-year survival rates estimated at 50.5% and 9.7%, respectively (6).

A variety of strategies have been attempted in an effort to improve outcomes with chemotherapy. These include altering the time at which chemotherapy is initiated and using cisplatin or carboplatin in combination with doxorubicin (7,8). Unfortunately, these efforts have failed to demonstrate significant improvements in median

The impact of carboplatin and toceranib phosphate on serum vascular endothelial growth factor (VEGF) and metalloproteinase-9 (MMP-9)

levels and survival in canine osteosarcomaTracy L. Gieger, Julie Nettifee-Osborne, Briana Hallman, Chad Johannes, Dawn Clarke,

Michael W. Nolan, Laurel E. Williams

A b s t r a c tIn this pilot study, 10 dogs with osteosarcoma (OSA) were treated with amputation and subsequent carboplatin chemotherapy (300 mg/m2 IV q3wk 3 4 doses) followed by toceranib phosphate (2.75 mg/kg PO q48h starting at day 14 post carboplatin). Monthly clinical monitoring and serum measurements of vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) were acquired. No dogs were removed from the study due to toxicity. Levels of VEGF and MMP-9 did not change over time. Seven dogs died related to local recurrence and/or pulmonary or bone metastasis and the remainder died of other causes. Median OSA-free survival was 238 d with 34% 1-year progression-free survival. Median overall survival was 253 d with 30% alive at 1.5 y and 10% alive at 2 y. Although this regimen was well-tolerated, survival times did not exceed previously published data from dogs treated with amputation plus chemotherapy alone.

R é s u m éDans cette étude pilote, 10 chiens avec un ostéosarcome (OSA) ont été traités par amputation et chimiothérapie subséquente avec du carboplatin (300 mg/m2 IV q3sem 3 4 doses) suivi de phosphate de toceranib (2,75 mg/kg PO q48h débutant au jour 14 suivant le carboplatin). Un suivi clinique mensuel et une mesure des taux sériques du facteur de croissance de l’endothélium vasculaire (FCEV) et de la matrice de la métalloprotéinase-9 (MMP-9) ont été obtenus. Aucun chien ne fut retiré de l’étude pour cause de toxicité. Les niveaux de FCEV et MMP-9 n’ont pas changé dans le temps. Sept chiens sont décédés dues à une rechute locale et/ou des métastases osseuses et les autres sont morts d’autres causes. La durée médiane de survie libre d’OSA était de 238 j avec 34 % de survie sans progression de la condition après 1 an. La durée de survie médiane était de 253 j avec 30 % en vie après 1,5 an et 10 % en vie après 2 ans. Bien que ce traitement ait été bien toléré, les temps de survie n’ont pas excédé les résultats publiés antérieurement pour des chiens traités par amputation et chimiothérapie uniquement.


Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, 1052 William Moore Drive, Raleigh, North Carolina 27607, USA (Gieger, Nettifee-Osborne, Hallman, Johannes, Clarke, Nolan, Williams); Iowa State University College of Veterinary Medicine, 1600 S 16th Street, 1569 LVMC, Ames, Iowa 50011, USA (Johannes); University of Georgia College of Veterinary Medicine, 2200 College Station Road, Athens, Georgia 30602, USA (Clarke); Veterinary Specialty Hospital of the Carolinas, 6405 Tryon Road, Cary, North Carolina 27518, USA (Williams); Comparative Medicine Institute, North Carolina State University, 1052 William Moore Drive, Raleigh, North Carolina 27607, USA (Gieger, Nettifee-Osborne, Nolan).

Address all correspondence to Dr. Tracy L. Gieger; telephone: (919) 513-6690; e-mail: [email protected]

Received September 4, 2016. Accepted December 20, 2016.


survival times over those seen with conventional single-agent treat-ment protocols. Novel therapeutic approaches are needed to control metastatic disease and improve survival.

Angiogenesis is the process by which tumors induce new blood vessel formation and is essential for continued tumor growth and metastasis. In the absence of angiogenesis, tumors are restricted to sizes ranging from several microns up to 1 to 2 mm (9). Endogenous angiogenesis inhibitors have been detected in the urine of OSA tumor-bearing dogs and these factors demonstrated potent inhibi-tion of endothelial cell proliferation (10). These endogenous factors were absent in urine collected from the same dogs 1 to 12 wk after surgical amputation of the limb bearing the primary tumor. The pro-angiogenic shift that occurs after primary tumor removal suggests that a strategy designed to pharmacologically control angiogenesis should be evaluated for its capacity to suppress the rapid progres-sion of microscopic metastases to life-threatening metastatic disease. The use of angiogenesis inhibitors has emerged as a clinically valid therapeutic approach in human oncology. This strategy may also have applicability to veterinary oncology, and is particularly appeal-ing for OSA where death is the result of progressive enlargement of metastatic foci.

Anti-angiogenic treatment strategies fall into 2 categories: i) those that are directly cytotoxic to endothelial cells, and ii) those acting indirectly by eliminating critical pro-angiogenic factors [e.g., vascular endothelial growth factor (VEGF)] and promoting anti-angiogenic factors. Vascular endothelial growth factor is a 45-kDa homodimer with pluripotent effects in angiogenesis — stimulating the prolifera-tion, invasion, and migration of endothelial cells. In addition, VEGF is a key target of several angiogenic factors and proteases such as matrix metalloproteinases (MMPs), with roles in tumor metastasis (11). Matrix metalloproteinases are enzymes that degrade structural elements outside of cells and play a critical role in tissue remodel-ing during tumor invasion, angiogenesis, and metastasis. Among MMPs, MMP-9 is of particular interest. Matrix metalloproteinase-9 is induced via VEGF receptor (VEGF-R) activation and levels of this downstream factor may be useful as a surrogate marker of VEGF-R activity (12); however, there have been no studies directly correlat-ing serum MMP-9 levels with VEGF-R activity in serum or tissues of OSA-bearing dogs.

Vascular endothelial growth factor is expressed by a variety of tumors, and therapies designed to decrease production or otherwise block VEGF activity are being explored as a means of inhibiting tumor proliferation. In an evaluation of 24 dogs with OSA, pretreat-ment platelet-corrected serum VEGF levels correlated significantly with disease-free interval (13). Results of this study and similar human studies support an important role for VEGF in the develop-ment and progression of metastatic disease in OSA. We hypothesize that the inhibition of VEGF activity will delay the progression of OSA micrometastases.

Toceranib phosphate [TP (Palladia; Zoetis, Durham, North Carolina, USA)], an FDA-approved drug for use in dogs, is a selec-tive inhibitor of several members of the receptor tyrosine kinase fam-ily, including vascular endothelial growth factor receptor (VEGF-R), KIT, FLT-3, and platelet-derived growth factor receptor (PDGF-R). c-KIT wild type receptor overexpression was identified in a study of 74 human pediatric high-grade OSA, indicating a potential role of the

inhibition of KIT signaling in OSA (14). While initially developed for its activity against KIT — a receptor mutated in approximately 30% to 50% of canine mast cell tumors (MCT) — the activity of TP against other members of this receptor tyrosine kinase family, such as VEGF-R, suggests a potentially wider spectrum of activity. Anti-tumor responses are reported in MCT lacking a KIT mutation, providing support for this theory and suggesting that other inhibited receptors, such as VEGF-R, play an important role in tumor progres-sion and regression (15). Toceranib phosphate exerts antiproliferative effects on endothelial cells in vitro, suggesting a role in angiogenesis, and produces clinically meaningful disease stabilization in vivo in dogs with osteosarcoma (16–18). As part of a larger study of dogs with solid tumors being treated with TP, 11/23 (48%) dogs with metastatic OSA had stabilization of their disease (18). The oral dos-ing of TP on an alternate-day schedule ideally lends itself to chronic inhibition of tumor-promoted angiogenesis to prevent the progres-sion of OSA micrometastases.

The goals of this study are: i) to evaluate the impact of mainte-nance antiangiogenic therapy using TP as a sole agent following amputation and carboplatin chemotherapy as a means of improving outcome for dogs with OSA, and ii) to measure circulating levels of endogenous pro-angiogenic factors before and during treatment with TP, which may serve as biomarkers for survival and disease progression. We hypothesize that the administration of TP following amputation and carboplatin chemotherapy will inhibit the progres-sion of metastasis through disruption of receptor tyrosine kinases with subsequent inhibition of angiogenesis. This will lead to pro-longed remission duration and survival in dogs with appendicular OSA compared to rates previously achieved in a group of dogs treated with amputation and carboplatin alone (5). We hypothesize that the administration of TP following amputation and carboplatin chemotherapy will decrease circulating levels of pro-angiogenic MMP-9, a downstream factor and potential surrogate marker of VEGF-R inhibition in dogs with appendicular OSA.

M a t e r i a l s a n d m e t h o d sTen dogs with histologically confirmed appendicular OSA with

no radiographic evidence of pulmonary metastasis at the time of enrollment and for whom owner consent was given, were prospec-tively enrolled. Institutional IACUC approval was obtained. All dogs were evaluated with a physical examination, complete blood (cell) count (CBC), serum biochemistry panel, urinalysis, 3-view thoracic radiographs, and a quality-of-life (QOL) survey completed by the owner prior to treatment. Two weeks following amputation, carboplatin (Paraplatin; Bristol Myers Squibb, New York, New York, USA) chemotherapy was administered intravenously at a dosage of 300 mg/m2 at 3-week intervals for a total of 4 treatments. A CBC was performed immediately before each carboplatin treatment and 7 d and 14 d following the first 2 treatment cycles to monitor for potential hematologic toxicity. A 25% dosage reduction was pre-scribed for any patient developing grade 3 or higher bone marrow or gastrointestinal toxicity as defined by the Veterinary Co-operative Oncology Group Common Terminology Criteria for Adverse Events (VCOG-CTCAE) v1.0 (19). Three-view thoracic radiographs were repeated prior to the third carboplatin chemotherapy treatment and


immediately before initiation of TP. Two weeks following the final carboplatin chemotherapy treatment, patients began TP therapy at a dosage of 2.75 mg/kg body weight (BW), PO, q48h. This drug was to be continued indefinitely unless evidence of toxicity, development of concurrent co-morbidities, death, or owner withdrawal of the pet from the study, occurred. Dogs that developed local recurrence or metastasis were censored from statistical analysis but were allowed to continue to receive TP if the owner elected to do so; owners were also counseled about all other potential treatment options outside of the study. To minimize the likelihood of toxicity, all dogs received omeprazole (Prilosec 0.5 to 1.0 mg/kg BW, PO, q24h; AstraZeneca Pharmaceuticals, Stockholm, Sweden) or famotidine (Pepcid 0.5 to 1.0 mg/kg BW, PO, q12-24h; McNeil Consumer Pharmaceuticals, Fort Washington, Pennsylvania, USA) and, if needed, metoclo-pramide (0.2 to 0.4 mg/kg BW, PO, q8h) and/or maropitant (Cerenia 2 mg/kg BW, PO, q24h 3 consecutive 5 d each week; Zoetis, Parsippany-Troy Hills, New Jersey, USA). To avoid confounding variables, NSAIDs were discontinued for the duration of the study period. Patients requiring ongoing analgesia received tramadol (1 to 4 mg/kg BW, PO, q6-12h), amantadine (3 to 5 mg/kg BW, PO, q24h), and/or gabapentin (5 to 10 mg/kg BW, PO, q8-12h).

Sample collectionSerum samples for enzyme-linked immunosorbent assay (ELISA)

evaluation of VEGF and MMP-9 were collected for analysis: i) imme-diately before initiation of carboplatin chemotherapy, ii) immediately prior to initiation of TP, and iii) 3 and 6 wk after starting TP, and iv) every 8 wk thereafter for 18 mo or until the time of disease pro-gression if this occurred at less than 18 mo.

Patient monitoringDisease status was monitored by a physical examination and

3-view thoracic radiographs at 8-week intervals until disease pro-gression was documented. To monitor for potential TP-related toxicity, CBC and body weight were evaluated weekly for the first 6 wk and a chemistry panel and urinalysis were also performed at weeks 1, 3, and 6. Subsequently, a CBC, body weight, chemistry panel, and urinalysis were performed at 8-week intervals. Quality-of-life surveys were also submitted by pet owners at each of these time points. Toxicity was recorded at each time point. A 1-week cessation of TP therapy followed by incremental dosage reductions (0.5 mg/kg BW increments to a minimum of 2.2 mg/kg BW) and/or decreased TP administration frequency (from q48h to Monday, Wednesday, and Friday schedule) was instituted for any dog experiencing . grade 2 adverse event(s).

Sample analysisApproximately 7 to 10 mL of whole blood was collected into

serum and plasma separator tubes by jugular venipuncture. Immediately after collection, blood samples were centrifuged and the serum frozen at 270°C until assayed. Commercially available ELISA test kits were used for VEGF (CAVEOO Canine VEGF Quantikine ELISA Kit; R&D Systems, Minneapolis, Minnesota, USA) and MMP-9 (SEA553CA ELISA Kit for MMP9 Organism species; Cedarlane Labs) and tests were performed according to the manufacturers’ directions. To minimize the impact that VEGF released from platelets contrib-

uted to serum VEGF levels, serum VEGF levels were corrected for platelet counts as follows: serum VEGF (pg/mL)/platelet count (3 106/mL) (20,21).

Statistical analysisA sample size of 10 dogs provides a power of 80% and a 50%

level of significance to detect a 47% increase in 1-year survival (i.e., increase the probability of survival at 1 y, from 34.5% to 50.7%) in dogs with appendicular OSA treated with carboplatin chemo-therapy followed by TP following amputation. The historical control consisted of a previously reported group of 48 dogs treated with amputation and 4 cycles of carboplatin chemotherapy with a median survival of 321 d and 1-year survival of 34.5% (5). Osteosarcoma-free survival time was defined as the date of amputation to the date of documentation of local recurrence or OSA metastasis. Overall sur-vival (OS) was defined as the date of amputation until death from any cause. A commercially available statistical program was used (GraphPad Prism 6; San Diego, California, USA). The Kaplan-Meier method was used to calculate OSA-free survival and overall sur-vival (OS). A Friedman’s test was used to compare VEGF and MMP values at baseline, pre-TP, and end of study. A Kruskal-Wallis test of repeated measures was used to compare platelet-corrected VEGF values at baseline, pre-TP, and end of study. P , 0.1 was considered significant. A P-value = 0.1 was selected due to the small sample size to try to avoid type II error.

Re s u l t sMedian age was 9.5 y (range: 5 to 11 y). Median body weight

was 31 kg (range: 19 to 48 kg). Seven were spayed females and 3 were castrated males. There were 3 golden retrievers, 1 mixed breed dog, and 1 of each of the following breeds: Belgian tervu-ren, great Dane, border collie, Rottweiler, Labrador retriever, and St. Bernard. Tumor locations included the distal radius (n = 5), distal femur (n = 2), distal tibia (n = 2), and proximal humerus (n = 1). No patients had CBC abnormalities prior to treatment. Serum bio-chemical abnormalities included increased alkaline phosphatase in 2 dogs [726 and 164 IU/L (normal , 140)] and in one dog, increased BUN [67 mg/dL (normal , 26)], creatinine [3 mg/dL (normal , 1.5 mg/dL)], phosphorous [6.1 mg/dL (normal , 5.6 mg/dL)], and potassium [5.8 mmol/L (normal , 5.6 mmol/L)] (in this dog, the urine specific gravity was 1.018). Seven dogs were treated with NSAIDs prior to being enrolled in the study for a variable duration of time; these drugs were discontinued on the first day of chemo-therapy in 3 dogs and 3 to 35 d prior to initiation of chemotherapy in the remaining dogs.

Chemotherapy administrationCarboplatin chemotherapy was started a median of 16 d post-

amputation (range: 11 to 30 d). Eight dogs received the first dose of carboplatin at 300 mg/m2; 1 dog received 239 mg/m2 (unknown reason; given at RDVM) and the dog with renal insufficiency received 150 mg/m2 (arbitrary dose reduction due to renal insuf-ficiency). Four dogs required dose reductions of carboplatin due to neutropenia; no dogs required hospitalization for supportive care. The carboplatin dose was reduced from 300 mg/m2 to 225 mg/m2


after the first dose in 3 dogs and after the second dose in 1 dog (who also had a 1-week treatment delay due to prolonged neutropenia).

Toceranib phosphate administrationEight dogs received TP at doses ranging from 2.2 to 2.9 mg/kg

(median dose: 2.7 mg/kg). After starting TP, 1 dog had two 1-week drug holidays related to grade I diarrhea and 1 dog had dose reductions and an extended dosing interval due to persistent, non-progressive grade I neutropenia.

Toxicity outcomesNo dogs were removed from the study due to toxicity or declining

QOL. Quality-of-life surveys were subjectively scored, and scores remained stable throughout the study (scores did not decline from baseline after starting carboplatin or TP) until the patient experi-enced a clinical decline typically related to disease progression.

Tumor-related outcomesSeven dogs developed metastasis as the first event. Pulmonary

metastasis was suspected based on thoracic radiographs in 6 dogs 60 to 240 d post-amputation (median 180 d) and osseous metasta-sis to the vertebrae and ribs was suspected in one dog based on radiographs at 575 d after amputation. One dog that developed pulmonary metastasis also concurrently developed an amputation-site stump recurrence 180 d post-amputation. Two of 10 (20%) dogs developed pulmonary metastasis while receiving carboplatin che-motherapy and did not continue on to the TP phase of the study. These 2 dogs were included in outcome analysis on an intent-to-treat basis. Of the 8 dogs that received TP, 3 died of metastasis (pulmo-nary in 2 dogs, osseous in 1 dog), 1 died of concurrent pulmonary metastasis and stump recurrence, 3 died of other causes [lymphoma that was diagnosed during the study while the dog was receiving TP (n = 1), metastatic hemangiosarcoma that developed while the dog was receiving TP (n = 1), and progressive spinal pain with no definitive diagnosis despite and extensive workup (n = 1)] and 1 dog was withdrawn from the study per the owner’s request with no evidence of OSA (n = 1). Toceranib phosphate was continued in

3 dogs at the owners’ request after metastasis was detected (dogs were censored from analysis on the date metastasis was diagnosed) and these dogs remained on the drug until death. An additional dog received treatment with doxorubicin after pulmonary metastasis was detected. All dogs were followed until the time of their death and all are deceased. Only 1 dog underwent necropsy.

Survival dataThe median OSA-free survival was 238 d (range: 50 to 575 d) with

34% progression-free survival at 1 y (Figure 1). Overall survival was 253 d with 30% alive at 1.5 y and 10% alive at 2 y (Figure 2).

VEGF and MMP-9 dataPre-treatment serum VEGF levels ranged from 0.8 to 20.4

(median: 6.5) and MMP-9 levels ranged from 8.9 to 25.2 (median: 12), with wide variations in intra- and inter-patient variability. There was no significant difference in MMP (Figures 3 and 5) or VEGF [absolute (Figures 4 and 6) or platelet-corrected] values from baseline and pre-TP, baseline and TP discontinuation, or pre-TP to TP discontinuation.

D i s c u s s i o nIn this study, continuous administration of TP was well-tolerated

in dogs with OSA treated with amputation and adjuvant carboplatin chemotherapy, although a survival benefit was not shown. In the present study, OS was 253 d with 30% alive at 1.5 y post-amputation, which is comparable to a historical group treated with amputation and adjuvant carboplatin chemotherapy in which OS was 321 d with 35% alive at 1 y (5). To date, 2 previously published studies of dogs with OSA treated with carboplatin chemotherapy and metro-nomic chemotherapy (MC) have failed to demonstrate improved survival times versus historical publications of dogs treated with carboplatin alone. In one publication, as part of a larger study of dogs with OSA treated with chemotherapy 1 MC [piroxicam and oral cyclophosphamide (10 to 12 mg/m2 per day)], 14 dogs were treated with carboplatin concurrent with MC (22). Three dogs were removed from the study due to toxicity (gastrointestinal). The overall

Figure 1. Median osteosarcoma-free survival time was 238 days with 34.3% event-free at 1 year.

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Figure 2. Median overall survival time was 253.5 days with 30% alive at 1.5 years and 10% alive at 2 years.

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disease-free interval was 192 d, with a median survival time of 217 d. The lack of improvement with post-chemotherapy MC (including TP in some dogs) was also confirmed in a recent, larger study (23). One hundred twenty-six dogs with appendicular OSA were treated with amputation followed by 4 doses of carboplatin chemotherapy and then randomized to receive MC [piroxicam/cyclophospha-mide (10 mg/m2 q48h)] with or without addition of TP [MC 1 TP (2.75 mg/kg PO q48h)]. Thirty-two dogs developed metastasis before the onset of MC or MC 1 TP, and a total of 35 received MC and 46 received MC 1 TP. Toxicities were greater in the MC 1 TP group, but most were mild and resolved with supportive care. The median DFI was 215 and 233 d and the OS was 242 and 318 d, respectively, for the MC versus MC 1 TP groups, which did not differ significantly. The 1-y survival rate was 35% and 38% for the MC versus MC 1 TP groups, respectively.

Although MC administration has not yet been shown to improve outcomes in dogs with OSA when used in combination with tradi-tional maximally tolerated dose (MTD) chemotherapy, its potential usefulness in highly metastatic diseases such as OSA, continues to be explored. In an attempt to further elucidate the tolerability and mechanism of action of MC, a recent study evaluated toxicity and lymphocyte profiles in tumor-bearing dogs treated with MTD doxorubicin (MTD-dox) only and those treated with MTD-dox alone and in combination with metronomic cyclophosphamide (MCTX) (24). A phase I study was performed and established that the standard doxorubicin dose of 30 mg/m2 (IV q21d) and a previ-ously published MC dose of cyclophosphamide of 15 mg/m2 per day was tolerable when administered concurrently. Dogs were then randomized to receive MTD-dox (n = 8) or MTD-dox 1 MCTX (n = 8). Both treatment groups showed declines in both total and

Figure 5. Individual patient metalloproteinase-9 concentrations at baseline, before toceranib phosphate, and at time of discontinuation of toceranib phosphate.

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Figure 6. Individual patient vascular endothelial growth factor concentra-tions at baseline, before toceranib phosphate, and at time of discontinu-ation of toceranib phosphate.

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Figure 4. Vascular endothelial growth factor concentrations at baseline, before toceranib phosphate, and at time of discontinuation of toceranib phosphate.

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T-regulatory lymphocytes (Tregs) by day 7 after MTD-dox, and there was no statistically significant difference between the groups in either total lymphocyte count or Tregs. In contrast to previous studies demonstrating that Tregs were selectively inhibited by CTX (25), there was no selective depletion of Tregs. The authors theo-rized that concurrent administration of MTD-dox and MC may not be beneficial if the MTD chemotherapy dampens the inhibition of the selective Treg depletion seen with MCTX alone. Although this study used doxorubicin and the current study as well as a previously published larger study used carboplatin, this theory could explain why MTD carboplatin 1 MC did not yield prolonged survival times in dogs with OSA. Other potential mechanisms of efficacy of MC include other immunomodulatory effects (e.g., restoring NK effec-tor function), anti-angiogenic effects [including decreased tumor microvessel density and decreased mobilization of circulating endothelial precursors (CEPs)], inhibition of normal stromal cells that support growing cancer cells, and possibly direct anti-tumor effects. The use of NSAIDs in MC can serve as a confounding factor when comparing studies since they inhibit COX-2, which may result in an inability of CEPs to survive and proliferate within the tumor microenvironment (24); therefore, the contribution of metronomic chemotherapy drugs such as cyclophosphamide to the outcome can be difficult to determine when NSAIDs are used concurrently. It is likely that the full potential of MC in addition to MTD chemotherapy for OSA will only be realized with clinical trials that compare use of standardized MTD chemotherapy and MC regimens both concur-rently and sequentially.

One aspect of the present study that is unique, is the attempt to quantify the degree of antiangiogenesis via measurement of serum MMP-9, VEGF, and platelet-corrected VEGF levels at baseline (post-amputation), pre-TP (after 4 cycles of carboplatin), over time during TP administration, and at the time when TP was discontinued. We anticipated changes in VEGF and MMP-9 levels concomitant with changes in disease status. If changes were repeatable and consistent among the study dogs, these serum markers may have served a sur-rogate markers for the remission status of OSA. In a study of dogs with OSA treated with amputation, pre-amputation platelet-corrected VEGF was significantly correlated with DFI (13). In a study of dogs treated with metronomic chemotherapy (NSAID 1 cyclophospha-mide) (26) for various metastatic tumors, VEGF levels were measured before (but not during) treatment, and dogs with lower VEGF levels had longer survival times and were more likely to respond to treat-ment than dogs with levels above the median. Unfortunately, in the current group of dogs, the MMP-9 and VEGF (absolute or platelet-corrected) values varied widely both inter- and intra-patient and there was no statistical correlation of the values at any time point. A limitation to this study is the lack of serum to test these factors before amputation because most dogs were referred for treatment and subsequent enrollment in the study after amputation had been performed. In addition to obtaining pre-treatment serum samples, ideally, all of the tumors would have been reviewed and graded by a single pathologist. This was a pilot study, and as a result, conclu-sions are inherently limited due to small sample size and lack of a contemporaneous control group. Failure to reliably assay angiogen-esis biomarkers in this study show that other measures are needed to determine subclinical implications of anti-angiogenic therapy in dogs.

To our knowledge, this is the first study evaluating the use of single-agent TP (without other concurrent MC drugs such as NSAIDs and/or cyclophosphamide) after amputation and carboplatin che-motherapy in dogs with OSA. Although there was no improvement in outcome compared to historical controls, further work evaluating the use of TP in dogs with OSA should be conducted. Since it has been shown that dogs with gross metastasis from OSA can have stable disease and a clinical benefit from treatment with TP (18), it is likely that using TP in a microscopic disease setting before the onset of visible metastasis may be beneficial.

In conclusion, the combination of carboplatin followed by TP was well-tolerated by dogs with appendicular OSA. There was inter- and intra-patient variability of VEGF and MMP-9 levels at all time points and survival times did not differ from previously published data from dogs treated with amputation and adjuvant chemotherapy. Further larger studies to devise more effective therapeutic options for this disease are warranted.

A c k n o w l e d g m e n t sThis study was supported, in part, by a grant from Bone Cancer

Dogs and provision of drugs and finances from Zoetis. Preliminary results were presented as an abstract at the American College of Veterinary Internal Medicine Annual Forum, Denver, Colorado in June 2016.

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16. Mendel DB, Laird AD, Xin X, et al. In vivo antitumor activity of SU11248, a novel tyrosine kinase inhibitor targeting vascular endothelial growth factor and platelet-derived growth factor receptors: Determination of a pharmacokinetic/pharmacody-namic relationship. Clin Cancer Res 2003;9:327–337.

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Article


I n t r o d u c t i o nOxidative stress is a consequence of the accumulation of reac-

tive oxygen species (ROS), reactive nitrogen species (RNS), such as hydroxyl radicals (OH-), superoxide anion radicals (O2), lipid per-oxyl radicals (LOO-), and nitrate radicals (NO-) (1). Factors involved in the production of free radicals and reactive metabolites include physical exercise, characterized by an increase in the body’s oxygen consumption and correlated to an increase in ROS production, which

causes oxidative stress (2–4). It has been shown that exercise-induced oxidative stress contributes to increased muscle fatigue, muscle fiber damage (5), and eventually leads to immune system impairment (6). Both muscles and blood are endowed with antioxidant enzymes, such as superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), and non-enzymatic antioxidants, such as vitamins A, C, and E, to counteract damage caused by free radicals (2,7,8).

Oxidative damage induced by physical exercise has been reported in horses (9–11) and dogs (12,13). One study investigated the

Oxidative stress and food supplementation with antioxidants in therapy dogs

Sara Sechi, Filippo Fiore, Francesca Chiavolelli, Corrado Dimauro, Anna Nudda, Raffaella Cocco

A b s t r a c tThe objective of this study was to evaluate the ability of a long-term antioxidant-supplemented diet to regulate the oxidative stress and general health status of dogs involved in animal-assisted intervention (AAI) programs. Oxidative stress is a consequence of the accumulation of reactive oxygen species (ROS). Exercise-induced oxidative stress can increase muscle fatigue and fiber damage and eventually leads to impairment of the immune system. A randomized, placebo-controlled, crossover clinical evaluation was conducted with 11 healthy therapy dogs: 6 females and 5 males of different breeds and with a mean age of 2.7 6 0.8 y (mean 6 SEM). The dogs were divided into 2 groups, 1 fed a high quality commercial diet without antioxidants (CD) and the other a high quality commercial diet supplemented with antioxidants (SD) for 18 wk. After the first 18 wk, metabolic parameters, reactive oxygen metabolite-derivatives (d-ROMs), and biological antioxidant potential (BAP) levels were monitored and showed a significant reduction of d-ROMs, triglycerides, and creatinine values in the SD group (P , 0.05) and a significant increase in amylase values in the CD group (P , 0.01). At the end of this period, groups were crossed over and fed for another 18 wk. A significant decrease in amylase and glutamate pyruvate transaminase (GPT) values was observed in the CD and SD group, respectively (P , 0.05). In conclusion, a controlled, balanced antioxidant diet may be a valid approach to restoring good cell metabolism and neutralizing excess free radicals in therapy dogs.

R é s u m éL’objectif de la présente étude était d’évaluer la capacité d’une diète long-terme supplémentée en antioxydant à réguler le stress oxydatif et l’état de santé général de chiens impliqués dans des programmes d’intervention avec assistance animale (IAA). Le stress oxydatif est une conséquence de l’accumulation d’espèces oxygène réactive (EOR). Le stress oxydatif induit par l’exercice peut augmenter la fatigue musculaire et les dommages aux fibres et éventuellement mener à un mauvais fonctionnement du système immunitaire. Une évaluation clinique croisée, randomisée, et avec groupe témoin-placebo a été menée avec 11 chiens d’assistance en santé : 6 femelles et 5 mâles de races différentes et d’un âge moyen de 2,7 6 0,8 ans (moyenne 6 écart-type). Les chiens ont été divisés en deux groupes, un premier groupe nourri avec une diète commerciale de haute qualité sans antioxydant (DC) et l’autre groupe avec une diète commerciale de haute qualité supplémentée avec des antioxydants (DS) pour 18 semaines. Après les premières 18 semaines, les paramètres métaboliques, les métabolites dérivés d’oxygène réactive (MDOR), et les niveaux de potentiel antioxydant biologique (PAB) ont été surveillés et ont montré une réduction significative des valeurs des MDOR, des triglycérides et de la créatinine dans le groupe DS (P , 0,05) et une augmentation significative des valeurs de l’amylase dans le groupe DC (P , 0,01). À la fin de cette période, les groupes ont été croisés et nourris pour 18 semaines supplémentaires. Une diminution significative des valeurs de l’amylase et de la glutamate pyruvate transaminase (GPT) a été obtenue dans les groupes DC et DS, respectivement (P , 0,05). En conclusion, une diète contrôlée, balancée en antioxydant pourrait être une approche valide pour restaurer un bon métabolisme cellulaire et neutraliser les radicaux libres excédentaires chez les chiens d’assistance.


Department of Veterinary Medicine, Pathology and Veterinary Clinic Section (Sechi, Fiore, Cocco) and Department of Agriculture, Livestock Sciences Section (Dimauro, Nudda), University of Sassari, Sassari, Italy; Department of Diagnostic Medicine, Clinical and Public Health, University of Modena and Reggio Emilia, Modena, Italy (Chiavolelli).

Address all correspondence to Dr. Raffaella Cocco; telephone: (139) 079 229520; fax: 139079228211; e-mail: [email protected]

The authors certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.

Received November 7, 2016. Accepted January 4, 2017.


oxidative status of 9 dogs after 20 min and 4 h of aerobic exercise, after 30 d of rest (14). Results showed a significant oxidative status due to the high level of reactive oxygen metabolite-derivatives (d-ROMs) and the low level of biological antioxidant potential (BAP) in relation to the values before starting the exercises (P , 0.01) (14).

Ethane exhaled in the breath may be considered a marker of oxi-dative stress as it is a product of free radical-mediated oxidation of cell membrane lipids (15). The authors evaluated oxidative stress in terms of exhaled ethane in 8 human, 12 dog, and 11 equine athletes after a physical performance (15). A significant increase in exhaled ethane was observed in all species after training and was associated with oxidative stress.

Evaluation of oxidative status with specific parameters could become an interesting and practical method of monitoring the activ-ity of working dogs. Indeed, it is difficult to quantify ROS in practice because of their very short half-life and it requires complex tech-niques over a long period of time. Due to their high reactivity, ROS react with practically every organic molecule they meet, producing reactive oxygen metabolites (ROMs), which are more stable than the ROS and are therefore easier to quantify. Conversely, BAP matches the total antioxidant capability of plasma and includes either exog-enous (ascorbate, tocopherols, carotenoids) or endogenous (protein, GPx, SOD, CAT) components that can oppose the oxidant action of reactive species (16).

In conditions of oxidative stress, a controlled, balanced antioxidant diet may be a valid approach to restoring good cell metabolism and neutralizing excess free radicals. Two studies in dogs and rabbits demonstrated an increase in tissue oxidative stability by supple-menting with a diet enriched with different antioxidants (17,13). The effect on exercise-induced oxidative stress of supplementing with oral vitamin E (11 to 14 mg) for 10 wk was studied in 8 dogs and induced a significant reduction in paroxonase-1 activity, eryth-rocyte membrane fluidity, and vitamin E and a significant increase in plasma malondialdehyde levels (18). After 10 d of vitamin E supplementation, these modifications were significant in the placebo group, but not in the supplemented group.

The objective of this study was to ascertain the ability of a long-term antioxidant-supplemented diet to regulate the oxidative stress and general health status of working dogs involved in animal-assisted intervention (AAI) programs.

M a t e r i a l s a n d m e t h o d sOperative procedures and animal care were carried out in compli-

ance with national and international regulations (Italian regulation D.L. vo 116/1992 and European Union regulation 86/609/EC). The recommendations of the Consolidated Standards of Reporting (CONSORT) 2010 statement on randomized controlled trials were also consulted and considered (19).

SubjectsEleven therapy dogs (6 females and 5 males) of different breeds

(1 Cirneco dell’Etna, 6 Labrador retrievers, 3 mixed breed, and 1 pit-bull), aged 2.7 6 0.8 y and with a body weight of 22.12 6 8.43 kg (mean 6 SEM), were enrolled in this clinical evaluation. Before the recruitment, laboratory examinations (hemogram, biochemical pro-

file, urine analysis, and serum protein electrophoresis) and serological tests (Ehrlichia canis, Rickettsia spp., Leishmania infantum, Anaplasma phagocytophilum, Bartonella spp., Toxoplasma gondii, Neospora caninum, and Borrelia burgdorferi) were conducted on all dogs to confirm their good health status. Owner consent was obtained for each dog.

All dogs were involved in 5 weekly (30 min each) animal-assisted intervention (AAI) sessions in the rehabilitation or social care of humans. All sessions were held in the same place and at similar tem-perature and humidity conditions. Patients involved in AAI sessions consisted of 55 children with pervasive developmental disorders. Each 30-minute session consisted of 15 min of active playing, such as ball throwing or running, 10 min of collaborative activities, such as the child and the dog looking for a hidden object together, and 5 min of dog-care activities, such as feeding and brushing the dog.

DietsThe dogs were fed 2 varieties of dry dog food: Natural Trainer

Adult Medium and Personal Trainer Long Life Adult Medium, which is supplemented with antioxidants (both from Nova Foods, Vicenza, Italy). The Natural Trainer Adult Medium diet consisted of fresh chicken and turkey meat, corn, rice, dehydrated chicken and turkey meat, lard, dehydrated pork meat, flax seed, beet, corn oil, dehy-drated fish flour, chicory extract, fructooligosaccharides, brewer’s yeast, pea fiber, Spirulina algae (Arthrospira platensis), ribonucleotides extract from yeast, green-lipped mussels (Perna canaliculus), apple dried extract, black raspberry extract, salt, chloride-choline, methio-nine, vitamin E, vitamin A (18 000 UI/kg), vitamin D3 (1.350 UI/kg), vitamin E (265 mg/kg), and copper (20 mg/kg).

The Personal Trainer Long Life Adult Medium dog food consisted of corn, dehydrated chicken and turkey meat, fresh chicken and tur-key meat, rice, corn oil, lard, hydrolyzed proteins of chicken, beet, corn gluten, flax seeds, dehydrated fish flour, brewer’s yeast (0.50%), sodium chloride, pea fiber, wood cellulose, calcium carbonate, fruit oligosaccharides, broccoli dried extract, green-lipped mussels (Perna canaliculus) (0.05%), Vitaberry Plus (grape seed extract, quercetin, blueberry dried extract, resveratrol, and strawberry and blackberry dried extracts), and silicon dioxide (0.025%).

Experimental designDogs were randomly divided into 2 groups using a dedicated

software (Research Randomizer) (20). The first group (n = 5) received a high quality commercial control diet (CD) without antioxidant (Natural Trainer Adult Medium, Nova Foods), while the second group (n = 6) received a high quality commercial diet (SD) sup-plemented with antioxidants (Personal Trainer Long Life Adult Medium, Nova Foods) for 18 wk. At the end of this period, a cross-over design of the same duration was conducted.

Sample collectionBlood samples were collected from each dog at the beginning of

the evaluation and at the end of the first experimental period after 18 wk. The same procedure was carried out before and at the end of the crossover period. Blood samples were collected from the cephalic vein and stored in 2 tubes, one with heparin and the other without anticoagulant. Heparinized plasma samples and serum samples were obtained by blood centrifugation at 6000 3 g for 1.5 min at 37°C.


Metabolic profile evaluationTen milliliters of blood were collected from the cephalic vein

for the oxidative stress assessment. After blood centrifugation in heparinized test tubes, the plasma was separated from the serum and analyzed. Oxidative stress was measured with a spectro-photometric point-of-care assay (Free Radical Analytical System FRAS 4; Evolvo, Langhirano, Italy). The dROMs test and the BAP test were completed. In the dROMs test, reactive oxygen metabo-lites (primarily hydroperoxides) of the sample generated alkoxyl and peroxyl radicals, in the presence of iron released from plasma proteins by an acidic buffer, according to the Fenton reaction. Such radicals then oxidized an alkyl-substituted aromatic amine (N,N-dietylparaphenylendiamine), thus producing a pink-colored deriva-tive, which is photometrically quantified at 505 nm. The concentra-tion of the dROMs is directly proportional to the color intensity and is expressed as U CARR (Carratelli units). One U CARR corresponds to 0.08 mg/dL hydrogen peroxide. In normal dogs, dROM values range from 50 to 90 U CARR.

In the BAP test, the plasma samples were mixed with a colored solution obtained by mixing a ferric chloride solution with a thio-cyanate derivative solution that causes a discoloration, the intensity of which is measured photometrically at 505 nm and is proportional to the ability of the plasma to reduce ferric ion (16). The results are expressed as mMol/L of reduced ferric ions. In normal dogs, BAP values range from 2400 to 2200 mMol/L. The reference values of dROMs and BAP test were validated for canine species (14,21) and are summarized in Table I.

A metabolic profile was also investigated for each dog, includ-ing albumin, alkaline phosphatase (ALP), total bilirubin, calcium, cholesterol, creatine phosphokinase (CPK), creatinine, phosphorus, gamma-glutamyl transferase (GGT), glucose, aspartate aminotrans-ferase (GOT), glutamate pyruvate transaminase (GPT), total proteins, triglycerides, and urea (Dimension RxL Max Integrated Chemistry System; Siemens Healthcare Diagnostics, Milan, Italy).

Statistical analysisData were analyzed using GraphPad Prism 6 software (GraphPad

Software, La Jolla, California, USA). All data are presented as the means 6 SEM and were first checked for normality using the D’Agostino-Pearson normality test. Differences in dROMs, BAP, and metabolic profile parameters between the 2 diets before (T0) and at the end (T1) of the first evaluation and crossover were analyzed using a 2-way analysis of variance (ANOVA), followed by Sidak’s multiple comparisons test.

Re s u l t sBoth diets were similar in chemical composition and fatty acid

profile. The predominant fatty acids of the lipid fraction in both diets were palmitic acid (C16:0) and oleic acid (C18:1 c9), which were present in similar proportions (22% for palmitic acid and 35% for oleic acid). The amount of linolenic acid (C18:3 n3) was higher (128%) in the experimental than in the control diet.

The metabolic profile trend of the CD and SD group during the evaluation period is shown in Figure 1. Amylase concentration sig-nificantly increased from a T0 value of 778 6 32.10 U/L to 1059 6 82.75 U/L at T1 in the CD group (P , 0.01) (Figure 1C). Conversely, creatinine and triglycerides decreased significantly in the SD group from a T0 value of 1.22 6 0.06 mg/dL to 1.00 6 0.06 mg/dL at T1 and from a T0 value of 164.6 6 55.73 mg/dL to 60.20 6 9.51 mg/dL at T1, respectively (P , 0.05) (Figures 1H to P).

The concentration of dROMs and BAP decreased significantly from a T0 value of 142.6 6 13.86 U CARR to 111.4 6 7.97 U CARR at T1 in the SD group and from a T0 value of 3332 6 177.5 mMol/L to 2954 6 58.14 mMol/L at T1 in the CD group, respectively (P , 0.05) (Figure 2).

At the end of this period, groups were crossed over and fed for another 18 wk and the metabolic profile was reassessed (Figure 3). Amylase and GPT concentration significantly decreased from a T0 value of 1059 6 82.75 U/L to 859.2 6 61.02 U/L at T1 in the CD group and from a T0 value of 70.20 6 10.83 U/L to 50.60 6 5.38 U/L at T1 in the SD group, respectively (P , 0.05) (Figures 3C to L). Although no significant variation of dROMs was observed, a decrease in the CD group and an increase in the SD group was observed at the end of the crossover period, probably due to the antioxidant activity of the diet (Figure 4A). Similar to what was observed in the first evaluation, a decrease in BAP concentration was observed in the CD group (Figure 4B).

D i s c u s s i o nThe objective of this study was to evaluate the potential ability

of a long-term antioxidant-supplemented diet to control the oxida-tive stress and general health status of therapy dogs involved in AAI programs. Environmental stress, such as temperature, work-ing, and dog competitions may induce psychological and physical stress, which could also influence the food intake of dogs, whether it is excessive or not enough.

An unbalanced diet lacking in essential nutrients weighs on the general health status of dogs and may represent a risk factor for

Table I. Reference values of dROMs (1 U Carr = 0.08 mg H2O2/dL) and BAP test

dROMs reference values BAP reference valuesNormal values 50 to 90 U CARR Optimal values 2400 to 2200 mmol/LThreshold borderline 92 to 95 U CARR Threshold borderline 2200 to 2000 mmol/LMild oxidative stress 100 to 120 U CARR Slight deficiency 2000 to 1800 mmol/LOxidative stress 140 to 200 U CARR Deficiency 1800 to 1600 mmol/LStrong oxidative stress 220 to 300 U CARR Strong deficiency 1600 to 1400 mmol/LStrong oxidative stress Over 300 U CARR Very strong deficiency , 1400 mmol/LdROMs — reactive oxygen metabolites-derivatives; U Carr — Carratelli units; BAP — biological antioxidant potential.


Figure 1. The metabolic profile trend of the commercial diet (CD) and supplemental diet (SD) groups during the evaluation period (continues on next page).


metabolic and degenerative diseases. Among the mechanisms that can influence health status, the balance of oxidative stress plays a relevant role. Metabolic oxidative reactions constantly take place in animals in order to balance the production of free radicals with antioxidant molecules.

The significant reduction (P , 0.05) of dROMs and BAP values observed in dogs fed the SD diet during the first experimental period clearly demonstrated the ability of the diet to reduce oxidative stress,

thus indicating a positive trend towards the normal values observed for healthy dogs (14). Conversely, the significant reduction observed in the CD-fed group confirmed the lack of any antioxidant effect related to the diet. It is worth noting that, while a long period of washout was not observed, the overall trend of BAP and dROMs improved after SD supplementation, although slightly influenced by the previous diet assumption.

On the other hand, the reduction of the amylase enzyme after the crossover could be related to the inhibitory activity of antioxidant molecules on key enzymes linked to type-2 diabetes (a-amylase and a-glycosidase) as previously observed in in-vitro studies by using flavonoid, anthocyanin, and other polyphenol compounds (22–25). The decrease in GPT level after the crossover suggests that the SD diet is endowed with liver-protective properties as reported in studies where different polyphenol extracts were tested in laboratory animals (26,27). As a result, the beneficial effects of antioxidant supplementation on liver and pancreatic metabolism were demonstrated.

The antioxidant formulation used in this study was based on grape seed extract, quercetin, blueberry, resveratrol, and strawberry and blackberry dried extracts (Vitaberry Plus). All these compounds contain anthocyanins and polyphenols, which have antioxidant effects. The total proanthocyanidin content of grape seed extract also included catechins and epicatechins and possesses antioxidant properties and protects the vascular system (28). The present study clearly showed a decrease in dROM species after the administration of a specific diet enriched with antioxidants. This scavenger activity was in agreement with other studies about the antioxidant effects of single active ingredients included in antioxidant supplements. For example, it has been demonstrated that supplementing with grape seed extract decreased the activity of the oxidative stress-responsive transcription factors NF-kB and Nrf2 in the duodenal mucosa of pigs (29).

In a similar study, grape seed extract significantly reduced (by 60%) the formation of azoxymethane-induced aberrant crypt foci, probably due to its activity in reducing the levels of cyclin D1, COX-2, and iNOS involved in the foci genesis (30). While grape seed and blueberry extract are endowed with antioxidant activity, quercetin supplementation is known to prevent age-related dis-eases in dogs (31). Moreover, the anthocyanins and polyphenols in blueberry extract have been shown to exert antioxidant effects in

Figure 2. Graphical representation of oxidative stress parameters concen-tration of dogs. A significant decrease in dROMs and BAP concentration was observed after 18 weeks of supplementation with SD and CD diet, respectively (*P , 0.05). A — dROMs and B — BAP values before (T0) and after 18 weeks (T1) of diet supplementation.

Figure 1. The metabolic profile trend of the commercial diet (CD) and supplemental diet (SD) groups during the evaluation period.


Figure 3. Graphical representation of metabolic profile of dogs. A significant decrease in GPT concentration was observed after SD diet supplementa-tion (3L, *P , 0.05). (A) albumin, (B) alkaline phosphatase, (E) calcium, (G) creatine phosphokinase, (I) Gamma-glutamyl transpeptidase, (J) glucose, (K) Glutamic-oxaloacetic transaminase, (L) Glutamic — pyruvic transaminase, (O) total protein (continues on next page).


Figure 3. Graphical representation of metabolic profile of dogs. A significant decrease in GPT concentration was observed after SD diet supplementa-tion (3L, *P , 0.05). (A) albumin, (B) alkaline phosphatase, (E) calcium, (G) creatine phosphokinase, (I) Gamma-glutamyl transpeptidase, (J) glucose, (K) Glutamic-oxaloacetic transaminase, (L) Glutamic — pyruvic transaminase, (O) total protein.

some pathological conditions, such as hypobaric hypoxia, due to the inhibition of lipid peroxidation by interfering with glutathione activity (32,33). The antioxidant levels of sled dogs supplemented with blueberries were studied and blood creatine kinase levels and oxidative status were evaluated 48 h after exercise (34). Although the exercise protocol did not influence concentrations of creatine kinase, supplementation with blueberries significantly increased the total antioxidant status after exercise. In addition to being an antioxidant,

blueberry extract has also demonstrated genoprotective and behav-ioral effects (35). Specifically, 30 mice received a supplementation of blueberry extract for 30 d. After that period, all treated animals had a higher significant memory retention in all test sessions after 7 and 30 d of supplementation, while in-vitro examination revealed significantly reduced DNA damage in hippocampal tissues than in the control group.

Resveratrol (3,5,49-trihydroxystilbene), a polyphenol naturally occurring in grapes, berries, peanuts, and other vegetables, has also been shown to have antioxidant, anti-inflammatory, and antiproliferative properties (36,37). Although there is a lack of scientific studies on resveratrol supplementation in pets, it has been demon-strated that resveratrol was able to protect from oxidative stress in hepatic steatosis conditions, significantly increasing GSH and GPx levels in liver tissues and significantly decreasing ROS levels in 30 mice (38).

Antioxidant and anti-inflammatory activity have been ascribed to strawberry extract due to its high content of flavonoids (39). For example, a daily intake of 500 g of strawberries for 9 d increased non-urate plasma 2,2-diphenyl-1-picrylhydrazyl radical decomposition, thus possibly decreasing the risk of systemic oxidants over activity (excessive activity) (40).

Antioxidant and anti-inflammatory activities were also investi-gated for a polyphenolic blackberry extract by means of the oxygen radical absorbance capacity assay (ORAC) (41). The ORAC value of the blackberry extract was higher than the ORAC of quercetin and ellagic acid. Moreover, the blackberry extract inhibited superoxide production by NADPH oxidase (nicotinamide adenine dinucleo-tide phosphate-oxidase) in THP-1 cells and nitrite production in J774A.1 cells after 4 h of incubation, showing a scavenger activity, while a reduction of nitrites was observed after 24 h of incubation.

A dog’s diet may be qualitatively and quantitatively appropriate to the type and intensity of the activity as well as to its age, breed, and gender. Balanced nutrition could therefore be crucial to restor-ing good overall health status. This study confirms that long-term supplementation with a specific diet enriched with antioxidants is able to regulate the antioxidant status and general health status of working dogs involved in animal-assisted intervention (AAI) programs and prevent damage due to oxidative stress before and after their work.

Figure 4. Graphical representation of oxidative stress parameters con-centration of dogs. A — dROMs values and B — BAP before (T0) and after 18 weeks (T1) of diets supplementation.


A c k n o w l e d g m e n tThis study was supported by a grant from Nova Foods in Vicenza,

Italy.

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22. Gao J, Xu P, Wang Y, Wang Y, Hochstetter D. Combined effects of green tea extracts, green tea polyphenols or epigallocatechin gallate with acarbose on inhibition against alpha-amylase and alpha-glucosidase in vitro. Molecules 2013;18:11614–11623.

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Article


Evaluation of transmission infrared spectroscopy and digital and optical refractometers to identify low immunoglobulin G

concentrations in alpaca serumIbrahim Elsohaby, Jennifer J. Burns, Christopher B. Riley, J. Trenton McClure

A b s t r a c tThis study aimed to evaluate the digital Brix and optical serum total protein (STP) refractometers for measuring concentrations of serum immunoglobulin G (IgG) in alpacas and compare them to IgG concentrations measured by the reference method of radial immunodiffusion (RID) assay. The appropriate cutoff point for Brix and STP refractometers and the transmission infrared (TIR) spectroscopy method was determined for low IgG concentrations (, 10 g/L). Serum samples were collected from alpacas (N = 169) and tested by both refractometers. The correlation between Brix % and STP was high [correlation coefficient (r) = 0.99]. However, the correlation coefficients between Brix % and STP with serum RID-IgG concentrations were only 0.56 and 0.55, respectively. Twenty-one (12.4%) of 169 alpaca serum samples had IgG concentrations of , 10 g/L. Using receiver operator characteristic curve (ROC) analysis, the optimal cutoff points for the TIR assay, digital Brix, and optical STP refractometers for assessing low IgG (RID , 10 g/L) were 13 g/L, 8.8%, and 50 g/L, respectively. The TIR assay showed higher sensitivity (Se = 95.2%) and specificity (Sp = 96.8%) than either the digital Brix (Se = 90.5% and Sp = 65.5%) or optical STP (Se = 81% and Sp = 73.7%) refractometers for assessing alpacas with low IgG. In conclusion, the Brix and STP refractometers lack accuracy in measuring alpaca IgG concentrations, but may be useful for screening animals for low serum IgG. However, the TIR assay with a cutoff point of 13 g/L was more appropriate for identifying low IgG than either refractometer. Another study that focuses on neonatal crias is recommended in order to evaluate the usefulness of these assays for field diagnosing of failure of transfer of passive immunity (FTPI).

R é s u m éCette étude visait à évaluer un réfractomètre Brix digital et un réfractomètre optique pour protéines sériques totales (PST) pour mesurer les concentrations sériques d’immunoglobulines G (IgG) chez les alpagas et de les comparer aux concentrations d’IgG mesurées par la méthode de référence d’immunodiffusion radiale (IDR). La valeur seuil appropriée pour les réfractomètres Brix et PST ainsi que pour la méthode de spectroscopie infrarouge à transmission (SIT) fut déterminée pour des faibles concentrations d’IgG (, 10 g/L). Des échantillons de sérum ont été prélevés d’alpagas (N = 169) et testés avec les deux réfractomètres. La corrélation entre le % Brix et PST était élevée [coefficient de corrélation (r) = 0,99]. Toutefois, les coefficients de corrélation entre % Brix et le PST et les concentrations d’IgG déterminées par IDR étaient seulement de 0,56 et 0,55, respectivement. Vingt-et-un (12,4 %) des 169 échantillons de sérum d’alpaga avaient des concentrations d’IgG , 10 g/L. En utilisant l’analyse de la courbe de la fonction d’efficacité du récepteur, les valeurs seuils optimales pour l’épreuve SIT, et les réfractomètres Brix digital, et PST optique pour évaluer un faible niveau d’IgG (IDR , 10 g/L) étaient de 13 g/L, 8,8 % et 50 g/L, respectivement. L’épreuve SIT a montré une sensibilité (Se = 95,2 %) et une spécificité (Sp = 96,8 %) plus élevée que le réfractomètre Brix digital (Se = 90,5 % et Sp = 65,5 %) ou le réfractomètre PST optique (Se = 81 % et Sp = 73,7) pour évaluer les alpagas avec de faibles concentrations d’IgG. En conclusion, les réfractomètres Brix et PST manquent de précision pour mesurer les concentrations d’IgG d’alpagas, mais peuvent être utiles pour vérifier les animaux pour des faibles concentrations d’IgG sériques. Toutefois, l’épreuve SIT avec une valeur seul de 13 g/L était plus appropriée que les deux réfractomètres pour identifier des valeurs faibles d’IgG. Une autre étude qui s’attardait aux crias nouveau-nés est recommandée afin d’évaluer l’utilité de ces épreuves pour le diagnostic des déficiences de transfert d’immunité passive.


Department of Health Management, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, Prince Edward Island C1A 4P3 (Elsohaby, Burns, Riley, McClure); Infectious Diseases, Department of Animal Medicine, Faculty of Veterinary Medicine, Zagazig University, Zagazig City 44511, Sharkia Province, Egypt (Elsohaby); Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Palmerston North 4442, New Zealand (Riley).

Address all correspondence to Dr. Ibrahim Elsohaby; telephone: (902) 566-6063; fax: (902) 566-0823; e-mail: [email protected]

Received November 24, 2016. Accepted January 11, 2017.


I n t r o d u c t i o nCamelids are born hypogammaglobulinemic as the diffuse epi-

theliochorial structure of their placentation does not allow immuno-globulins to pass from dam to fetus (1). Newborn camelids therefore rely on the transfer of maternal immunoglobulins through intake of the dam’s colostrum in order to acquire passive immunity (2). Failure of transfer of passive immunity (FTPI) occurs when newborn camelids fail to ingest or absorb a sufficient amount (, 10 g/L) of colostral IgG within 48 h of birth (1,3,4). There is a recognized asso-ciation between FTPI and increased infectious disease, including septicemia, diarrhea, pneumonia, and septic arthritis, in neonatal camelids (5–7). Early diagnosis of FTPI is therefore a vitally impor-tant part of neonatal camelid husbandry and is critical in decreasing neonatal morbidity and mortality rates (8).

Several assays are available to measure IgG concentrations directly or indirectly and to assist in assessing conditions characterized by low IgG, such as FTPI in camelids (1,9). Direct methods of assessment include the radial immunodiffusion (RID) assay (10), immuno turbidimetric (IT) assay (11), and transmission infrared (TIR) spectroscopy (12). Indirect methods include zinc sulfate turbidity (13), glutaraldehyde coagulation assay, serum gamma-glutamyl transferase (GGT) activity (14), and total serum protein and globulin concentration measurements (4,13). The RID assay is the historical gold standard method for direct quantification of IgG level in camelids (15). Radial immunodiffusion (RID) has significant disadvantages, however, including the time it takes to obtain results (18 to 24 h), higher costs than other methods, and a lack of agree-ment about which commercially available RID kits should be used to measure IgG concentration in camelids (10,11).

While infrared spectroscopy has recently been used to measure alpaca serum IgG (12), refractometry is the most common indirect method for estimating serum IgG and assessing FTPI on-farm (13). Previous studies have evaluated optical serum total protein (STP) refractometry for assessing FTPI in neonatal camelids (13,16). To the authors’ knowledge, however, no study has been published that describes the use of digital Brix refractometry for measuring serum IgG concentrations and assessing IgG of , 10 g/L, which is the cutoff point for FTPI in alpacas.

The objectives of this study were to i) evaluate the digital Brix and optical STP refractometers for measuring serum IgG concentrations in alpacas and compare values with those measured by the reference method of radial immunodiffusion (RID) assay; and ii) determine the optimal cutoff points for assessing low IgG concentrations (, 10 g/L) in alpacas using digital Brix and optical STP refractom-eters and the transmisssion infrared (TIR) spectroscopy method.


Study population and samplingBanked sera from a previous study stored at −80°C were used

for this study (12). The study was approved by the University of Prince Edward Island (UPEI) Animal Care Committee and the Animal Ethics Committee of the University of Adelaide. Alpacas (N = 175) enrolled in this study between 2009 and 2011 were from

6 farms in Ontario and New Brunswick, Canada (n = 82) and 3 farms in the Adelaide Hills region of South Australia (n = 93). All alpacas appeared clinically healthy and were not visibly dehydrated. Age and gender data were only available for 129 of the 175 alpacas. Of these 129 alpacas, 81 were female and 48 were male. Four of these alpacas were , 2 mo old, 5 were 2 to 3 mo old, 15 were 3 to 6 mo old, 12 were 6 mo to 1 y old, and 93 were . 1 y old.

In Canada, blood samples were collected from alpacas by jugular venipuncture into a sterile, plastic vacutainer tube without anti-coagulant, then centrifuged at 1500 3 g for 10 min at room tempera-ture. Serum was collected into a cryovial and frozen at −20°C until transport to the Atlantic Veterinary College (AVC). Blood samples collected from alpacas in Australia were taken for hematologic and biochemical analysis as part of a separate study. The remaining serum was stored at −80°C before being shipped frozen to AVC, UPEI, Canada where it was stored at −80°C for later batch analysis.

IgG direct measurement methodsResults of serum RID and TIR IgG (g/L) measurement for the

same samples from a related previous study were used (12). In the earlier study, a commercial alpaca RID IgG assay (Triple J Farms, Bellingham, Washington, USA) and transmission infrared (TIR) spectroscopy (Tensor 37; Bruker Optics, Coventry, UK) were used. Radial immunodiffusion (RID) IgG assay was used as a reference method to determine each alpaca’s serum IgG concentration. Each serum sample and assay standard was tested in replicates of 5 to generate an average IgG concentration value. For TIR spectroscopy, each sample was tested in replicates of 6 as described in a previous study (12). Sample spectra were collected over the wavenumber range of 4000 to 400 cm21, with a nominal resolution of 4 cm21, with 512 scans collected for data acquisition. Data for the measured TIR IgG values were used to calculate an optimal cutoff point to identify low IgG (, 10 g/L) for this study.

IgG indirect measurement methodsOnly 169 of the original 175 serum samples were available for

testing by refractometers, as there was not enough volume in the remaining 6 samples to allow for further testing. Samples were vor-texed for 10 s and serum Brix (%) and STP (g/L) levels were deter-mined using a digital Brix refractometer (PAL-1; Atago, Bellevue, Washington, USA) and an optical handheld refractometer (Westover RHC 200ATC handheld refractometer; Woodinville, Washington, USA), respectively. The same person conducted both refractometer

Table I. Descriptive statistics of immunoglobulin G (IgG) concentrations, Brix percentage (%) and serum total protein (STP) measurements in 169 alpaca serum samples

Parameters Method Median Minimum MaximumIgG (g/L) RID 24.8 3.9 52.9IgG (g/L) TIR 26.7 22.3 51.8Brix (%) Digital 9 7.2 12.5STP (g/L) Optical 54 38 84RID — radial immunodiffusion assay; TIR — transmission infrared spectroscopy; Digital — Brix refractometry; Optical — serum total protein (STP) refractometry.


measurements at room temperature. However, the optical refrac-tometry to determine STP was conducted first to avoid observer bias. Both refractometers were cleaned and calibrated with distilled water before each reading.

Statistical analysisStatistical analyses were carried out using statistical software

(Stata Version 14.0; Stata Corporation, College Station, Texas, USA and MedCalc for Windows Version 15.8.0; MedCalc Software, Mariakerke, Belgium). A value of P , 0.05 was considered statisti-cally significant. The data were evaluated for normality by applying the Shapiro-Wilk test. Descriptive statistics for IgG measurements were calculated by RID, TIR spectroscopy, digital Brix, and optical STP refractometers. Spearman rank coefficient of correlation was calculated to determine the relationship among measurements by RID, Brix, and STP refractometers.

Alpacas with serum RID IgG concentrations of , 10 g/L were considered to have low IgG, which is consistent with the definition of FTPI in camelid neonates (1,3,4). A receiver operating charac-teristic (ROC) analysis was conducted on data obtained with TIR spectroscopy and Brix and STP refractometers to determine the optimal cutoff point based on the Youden index. Sensitivity (Se), specificity (Sp), accuracy, Youden index (J), and area under the curve (AUC) were used to compare the diagnostic performance of each method. Accuracy, which was defined as the percentage of correctly classified samples, was calculated as follows: [Se 3 p] 1 [(Sp) 3 (1 2 p)], where p represents the prevalence of samples with low IgG (, 10 g/L) observed in the studied population. Finally, kappa statistic was used to assess the level of agreement among the results of TIR spectroscopy, Brix, and STP refractometers versus the reference RID assay, as well as between results of digital Brix and optical STP refractometers.

Results and discussionWhile the IgG values from RID, Brix %, and STP assays were not

normally distributed (P , 0.001, respectively), the values for IgG

from TIR were normally distributed (P = 0.321). Descriptive statistics (median, minimum, and maximum) of IgG concentrations in alpaca serum obtained by Brix and STP measurement are shown in Table I. The median IgG concentration measured by RID assay (24.8 g/L) in the present study was marginally higher than that previously published, which ranged from 17.2 g/L to 23.4 g/L (3,4,11,14). The range of IgG concentrations in these studies was more likely to be higher, however, because they were not limited to healthy alpacas.

The RID and TIR assays were conducted at an earlier time than the refractometers, with the serum stored in a freezer at 280°C between testing. Degradation of IgG between testing periods is unlikely as it has been previously reported that freezing at this temperature over a period of years has no significant effect on IgG concentra-tion (17,18). Only 21 of the 169 alpacas had an IgG concentration of , 10 g/L, the most common cutoff point used to determine FTPI (1), which resulted in a true prevalence of 12.4%. This value falls within the range of FTPI prevalence (9% to 20.5%) previously reported in the literature (4,15). The differences in the prevalence of low IgG concentrations (, 10 g/L) in the present and previous studies may be related to the age of alpacas enrolled in these earlier studies, as well as to management practices on the farms, including biosecurity measures, nutrition, vaccination programs, geographic location, and climate (19). Since alpacas in this study population were of various ages and included crias and adults, we are limited in assessing the clinical usefulness of these assays for diagnosing FTPI. A future study focusing on neonatal crias is therefore recommended in order to better evaluate the usefulness of TIR spectroscopy (12), Brix, and STP refractometers for diagnosing FTPI.

CorrelationsThe Spearman rank correlation coefficient between IgG levels

was 0.56, as estimated by digital Brix refractometry and RID assay (Figure 1). The STP-based serum IgG measurements were correlated with those assessed by RID assay (r = 0.55; Figure 2). These findings were lower than correlations reported between immunoturbidimet-ric (IT) (11) or infrared spectroscopy methods (12) and RID-based

Figure 1. Correlation plot between Brix percentage (%) determined by digital Brix refractometry and immunoglobulin G (IgG) determined by radial immunodiffusion (RID) assay for 169 alpaca serum samples.

IgG concentration from RID assay (g/L)

Brix

(%)

12

10

8

6

r = 0.56

0 10 20 30 40 50

Figure 2. Correlation plot between serum total protein (STP) determined by optical handheld refractometry and immunoglobulin G (IgG) deter-mined by radial immunodiffusion (RID) assay for 169 alpaca serum samples.

IgG concentration from RID assay (g/L)

Ser

um to

tal p

rote

in (g

/L)

80

70

60

50

40

r = 0.56

0 10 20 30 40 50


measurements of IgG concentrations. Furthermore, the correlations between the digital Brix or the optical STP refractometers and RID assay were lower than those reported for bovine (20–22) and lamb sera (23). There was a high correlation between digital Brix and opti-cal STP refractometers values (r = 0.99; Figure 3), which indicates that there was no advantage of one method over the other. Although a similarly high level of correlation between Brix and STP refractom-eters has previously been reported (24), correlation coefficients from 0.73 to 0.91 have been reported for other species (20–22).

Test characteristics for detection of low IgGTest characteristics of the TIR spectroscopy, digital Brix, and

optical STP refractometers for assessing alpacas with low IgG con-centration (, 10 g/L) were calculated using optimal cutoff points determined by ROCs (Table II). The optimal combination of Se

(95.2%; 95% CI: 90.9 to 98.2) and Sp (96.8%; 95% CI: 92.3 to 98.9) for the TIR spectroscopic diagnosis of low IgG was achieved at # 13 g/L (Figure 4). The calculated Se and Sp for the digital Brix refractometer using # 8.8% as the optimal cutoff point were 90.5% (95% CI: 69.6 to 98.8) and 65.5% (95% CI: 57.3 to 73.2), respectively (Figure 5). This compares to the best combination of Se (81%; 95% CI: 58.1 to 94.6) and Sp (73.7%; 95% CI: 65.8 to 80.5) for the optical STP refractometer at a cutoff point of # 50 g/L (Figure 6).

The optimal Se and Sp values for the TIR spectroscopic approach were higher than those of the Brix and STP refractometers. Also, the Se (95.2%) of TIR spectroscopy at a cutoff point of # 13 g/L was higher than the Se (81%) from the original study when a cutoff point of 10 g/L was used for the same samples (12). The objectives of the original study (12), however, were to develop an algorithm that best predicted serum IgG concentration values over the TIR spectrum and looked at how well the TIR spectroscopic method performed against the RID using the same cutoff value of , 10 g/L for each assay. In this study, we used ROC analysis to determine the TIR IgG value that best correlates with serum IgG of , 10 g/L as determined by the reference RID assay. The Se and Sp of the TIR spectroscopy using a cutoff IgG of # 13 g/L value was similar or higher when compared with the reported Se and Sp for other available methods to assess low serum IgG in alpacas (11,13,25). The digital Brix refrac-tometer showed higher Se and slightly lower Sp than the optical STP refractometer at a cutoff point of # 8.8%, which indicates that digital Brix may be preferred as a screening test compared to optical STP refractometer. The Se and Sp of optical STP refractometer at a cutoff point of # 50 g/L were close to the Se (71%) and Sp (80%) previ-ously reported with the same cutoff point (13). When a cutoff point of 51.5 g/L was used, however, a higher Se (87.5%) and Sp (87.9%) for assessment of low serum IgG were reported (25). The difference in the optimal cutoff points between studies may be due to varia-tions between instruments or differences in animal populations (22).

Agreement among testsThe agreement among results of TIR spectroscopy and Brix and

STP refractometers for assessing alpaca serum with low IgG concen-trations (, 10 g/L) compared to the reference RID assay is presented

Figure 3. Correlation plot between serum total protein (STP) determined by optical handheld refractometry and Brix % determined by digital Brix refractometry for 169 alpaca serum samples.

Brix (%)

Ser

um to

tal p

rote

in (g

/L)

80

70

60

50

40

r = 0.99

7 8 9 10 11 12 13

Table II. Test characteristics for detecting low serum IgG (, 10 g/L) in 169 alpaca serum samples by transmission infrared (TIR) spectroscopy and Brix and optical serum total protein (STP) refractometers as calculated using the radial immunodiffusion (RID) assay as the reference

RefractometersTest characteristics TIR Brix STPCutoff point # 13 g/L # 8.8% # 50 g/LSensitivity (Se) 95.2 90.5 81Specificity (Sp) 96.8 65.5 73.7Accuracy 96.6 68.5 74.6Youden index (J) 0.92 0.56 0.55Area under the curve (AUC) 0.99 0.81 0.81Kappa 0.82 0.30 0.33Cutoff point — optimal criteria determined by receiver operating characteristic curves (ROCs).

Figure 4. Receiver operating characteristic (ROC) curve, the best combina-tion of sensitivity and specificity for transmission infrared (TIR) spectros-copy obtained at a cutoff point of # 13 g/L in 169 alpaca serum samples.

100-Specificity

Sen

sitiv

ity

100

80

60

40

20

0

Se: 95.2Sp: 96.8

Criterion: # 13

AUC: 0.99

0 20 40 60 80 100


in Table II. The overall percentages of agreement among results of TIR spectroscopy, Brix, and STP refractometers to the reference RID assay, i.e., accuracy, with a corresponding kappa value are also listed in Table II. The level of agreement between the results of the 2 refractometers was 85.2%, however, with a kappa value of 0.66. This finding shows that TIR spectroscopy had a substantial agree-ment with the RID assay that was better than that observed for both refractometers. The analytic advantage of TIR is that the serum IgG content creates a unique molecular absorption (fingerprint) in the infrared spectrum, which can be used for qualitative and quantitative measurement (26). However, refractometers measure STP or Brix %, which indirectly related to serum IgG concentration as a portion of total solids (13). The level of agreement found between the 2 refrac-tometers (85.2%) was similar to the level of agreement (85%) reported between 2 refractometers for assessing FTPI in dairy calves (20).

In conclusion, this study found that the TIR spectroscopic method using the calculated optimal cutoff point of 13 g/L provided bet-ter sensitivity, specificity, and accuracy for diagnosing low IgG (, 10 g/L) than digital Brix or optical STP refractometers at their optimal cutoff points of 8.8% and 50 g/L, respectively. The digital Brix and optical STP refractometers were poor predictors of serum IgG concentrations, and although they may be useful for screening animals for low IgG (, 10 g/L), further confirmation testing would be required due to the limited specificity of the assays. Another study focusing on neonatal crias is recommended to further evaluate the specific usefulness of TIR spectroscopy for diagnosing FTPI.

A c k n o w l e d g m e n t sThe authors thank Cynthia Mitchell for technical assistance,

Professor Michael Reichel (University of Adelaide, Australia),

and Dr. Nicole Jewett (New Brunswick, Canada) for providing the samples used in this study. This study was partially supported by the Atlantic Canada Opportunities Agency.

Re f e r e n c e s 1. Wernery U. Camelid immunoglobulins and their importance

for the new-born — A review. J Vet Med B Infect Dis Vet Public Health 2001;48:561–568.

2. Walker P, Tibary A. Neonatal care of camelids: A review and case reports. J Camel Pract Res 1999;6:255–263.

3. Bravo P, Garnica J, Fowler M. Immunoglobulin G concentrations in periparturient llamas, alpacas and their crias. Small Rumin Res 1997;26:145–149.

4. Weaver DM, Tyler JW, Scott MA, Wallace LM, Marion RS, Holle JM. Passive transfer of colostral immunoglobulin G in neonatal llamas and alpacas. Am J Vet Res 2000;61:738–741.

5. Adams R, Garry F. Llama neonatology. Vet Clin North Am Food Anim Pract 1994;10:209–227.

6. Dolente BA, Lindborg S, Palmer JE, Wilkins PA. Culture-positive sepsis in neonatal camelids: 21 cases. J Vet Intern Med 2007;21:519–525.

7. Whitehead CE. Management of neonatal llamas and alpacas. Vet Clin North Am Food Anim Pract 2009;25:353–366.

8. Fowler ME, Bravo PW. Neonatology. In: Fowler ME, ed. Medicine and Surgery of Camelids, 2nd ed. Ames: Iowa State University Press, 1998:507–524.

9. Berne BH. Differing methodology and equations used in quan-titating immunoglobulins by radial immunodiffusion — A comparative evaluation of reported and commercial techniques. Clin Chem 1974;20:61–69.

Figure 5. Receiver operating characteristic (ROC) curve, the best combi-nation of sensitivity and specificity for digital Brix refractometry obtained at a cutoff point of # 8.8% in 169 alpaca serum samples.

100-Specificity

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sitiv

ity

100

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60

40

20

0

Se: 90.5Sp: 65.5

Criterion: # 8.8

AUC: 0.81

0 20 40 60 80 100

Figure 6. Receiver operating characteristic (ROC) curve, the best combi-nation of sensitivity and specificity for optical serum total protein (STP) refractometery obtained at a cutoff point of # 50 g/L in 169 alpaca serum samples.

100-Specificity

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100

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Se: 81Sp: 73.7

Criterion: # 50

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10. Hutchison JM, Salman MD, Garry FB, Johnson LW, Collins JK, Keefe TJ. Comparison of two commercially available single radial immunodiffusion kits for quantitation of llama immuno-globulin G. J Vet Diagn Invest 1995;7:515–519.

11. Pinn TL, Gagliardo LF, Purdy SR, Appleton JA, Stokol T. Comparison of three immunoglobulin G assays for the diagnosis of failure of passive transfer of immunity in neonatal alpacas. J Vet Diagn Invest 2013;25:91–98.

12. Burns J, Hou S, Riley C, Shaw R, Jewett N, McClure J. Use of Fourier-transform infrared spectroscopy to quantify immuno-globulin G concentrations in alpaca serum. J Vet Intern Med 2014;28:639–645.

13. Weaver DM, Tyler JW, Marion RS, Wallace LM, Nagy JK, Holle JM. Evaluation of assays for determination of passive transfer status in neonatal llamas and alpacas. J Am Vet Med Assoc 2000;216:559–563.

14. Johnston NA, Parish SM, Tyler JW, Tillman CB. Evaluation of serum gamma-glutamyltransferase activity as a predictor of passive transfer status in crias. J Am Vet Med Assoc 1997;211: 1165–1166.

15. Garmendia AE, Palmer GH, DeMartini JC, McGuire TC. Failure of passive immunoglobulin transfer: A major determinant of mortality in newborn alpacas (Llama pacos). Am J Vet Res 1987; 48:1472–1476.

16. Nagy DW, Chakwenya J, Tyler JW, Holle J. A note on colostral immunoglobulin G concentrations vs. subsequent serum con-centrations in naturally suckled llama (Llama glama) and alpaca (Llama pacos) crias. J Camel Pract Res 2002;9:171–172.

17. Proverbio D, Spada E, Baggiani L, et al. Effects of storage time on total protein and globulin concentrations in bovine fresh frozen plasma obtained for transfusion. Sci World J 2015; Article ID: 752724, 5p.

18. Reynolds B, Taillade B, Médaille C, Palenché F, Trumel C, Lefebvre HP. Effect of repeated freeze-thaw cycles on routine plasma biochemical constituents in canine plasma. Vet Clin Pathol 2006;35:339–340.

19. Cebra C, Anderson DE, Tibary A, Van Saun RJ, Johnson LW. Llama and Alpaca Care: Medicine, Surgery, Reproduction, Nutrition, and Herd Health. 1st ed. St. Louis, Missouri: Elsevier, 2014:288–291.

20. Elsohaby I, McClure JT, Keefe GP. Evaluation of digital and optical refractometers for assessing failure of transfer of passive immunity in dairy calves. J Vet Intern Med 2015;29:721–726.

21. Thornhill JB, Krebs GL, Petzel CE. Evaluation of the Brix refrac-tometer as an on-farm tool for the detection of passive transfer of immunity in dairy calves. Aust Vet J 2015;93:26–30.

22. Hernandez D, Nydam D, Godden S, et al. Brix refractometry in serum as a measure of failure of passive transfer compared to measured immunoglobulin G and total protein by refractometry in serum from dairy calves. Vet J 2016;211:82–87.

23. Massimini G, Britti D, Peli A, Cinotti S. Effect of passive transfer status on preweaning growth performance in dairy lambs. J Am Vet Med Assoc 2006;229:111–115.

24. Deelen SM, Ollivett TL, Haines DM, Leslie KE. Evaluation of a Brix refractometer to estimate serum immunoglobulin G concen-tration in neonatal dairy calves. J Dairy Sci 2014;97:3838–3844.

25. Drew ML, Fowler ME. Comparison of methods for measuring serum immunoglobulin concentrations in neonatal llamas. J Am Vet Med Assoc 1995;206:1374–1380.

26. Riley CB, McClure JT, Low-Ying S, Shaw RA. Use of fourier-transform infrared spectroscopy for the diagnosis of failure of transfer of passive immunity and measurement of immunoglob-ulin concentrations in horses. J Vet Intern Med 2007;21:828–834.

Article


Cystic duct pressures after ligation with a novel absorbable device in an ex vivo caprine cholecystectomy model

Andrea J. Sundholm Tepper, Odd V. Höglund, Bonnie G. Campbell, Chi-Ya Chen, Boel A. Fransson

A b s t r a c tLaparoscopic cholecystectomy is the standard of care in human medicine for gall bladder disease. Although infrequently reported in veterinary literature, laparoscopic cholecystectomy is an option for uncomplicated gall bladder disease in canine patients. Due to the risk of cystic duct ligature slippage or clip dislodgement, we wanted to explore the use of a LigaTie; a novel absorbable medical device modeled after a cable tie. Our object was to describe the use of the LigaTie in a caprine cadaveric study of cholecystectomies as a model for canine patients and demonstrate the leak pressure of the cystic duct compared with cholecystectomies performed with 2 large endoscopic hemoclips. Samples of caprine gall bladder, liver, and cystic duct were collected. The cystic duct was ligated with either 2 large endoscopic hemoclips or a LigaTie. Maximum cystic duct pressure was recorded. Results showed that there was no statistically significant difference in the maximum cystic duct pressure achieved for cystic ducts ligated with 2 large endoscopic hemoclips or the LigaTie (P = 0.865). No leakage was observed from the cystic duct, hemoclip, or LigaTie site in either group. Supraphysiologic pressures were achieved in both groups and high pressure occlusion of the infusion pump determined the maximum intraluminal pressure achieved. Based on these results, the LigaTie may provide advantages in minimally invasive surgery, especially when considering ligation of a friable or thickened cystic duct during laparoscopic cholecystectomy. Future in vivo studies are warranted to determine minimally invasive maneuverability, tissue interaction, complications, and outcomes.

R é s u m éLa cholécystectomie par laparoscopie est le standard de soin en médecine humaine pour les maladies de la vésicule biliaire. Bien que rapportée peu fréquemment dans la littérature vétérinaire, la cholécystectomie par laparoscopie est une option pour les maladies non-compliquées de la vésicule biliaire chez les patients canins. Due au risque de glissement de la ligature du canal cholédoque ou au déplacement de l’agrafe, nous avons voulu explorer l’utilisation de LigaTie; un nouveau dispositif médical résorbable modélisé d’après une attache de câble. Notre objectif était de décrire l’utilisation de LigaTie dans une étude sur des cadavres de chèvres de cholécystectomies comme modèle pour des patients canins et de démontrer la pression de fuite du canal cholédoque comparativement à des cholécystectomies réalisées avec deux larges agrafes hémostatiques endoscopiques. Des échantillons de vésicules biliaires, de foies et de canal cholédoques caprins ont été prélevés. Le canal cholédoque était ligaturé avec soit deux larges agrafes hémostatiques endoscopiques ou du LigaTie. La pression maximale dans le canal cholédoque fut enregistrée. Les résultats ont montré qu’il n’y avait pas de différence statistiquement significative dans la pression maximale atteinte dans le canal cholédoque par les deux méthodes de ligature (P = 0,865). Aucune fuite ne fut observée des sites de ligature du canal cholédoque que ce soit du groupe avec agrafes ou celui avec LigaTie. Des pressions supra-physiologiques ont été atteintes dans les deux groupes et l’occlusion de la pompe à infusion due à la haute pression a déterminé la pression intraluminale maximale atteinte. En fonction de ces résultats, le LigaTie est avantageux comme méthode chirurgicale minimalement invasive, surtout si l’on considère la ligature d’un canal cholédoque friable ou épaissi durant une cholécystectomie par laparoscopie. Des études in vivo ultérieures sont nécessaires afin de déterminer la manœuvrabilité invasive minimale, l’interaction tissulaire, les complications et les résultats.


Department of Veterinary Clinical Sciences (Sundholm Tepper, Campbell, Chen, Fransson), College of Veterinary Medicine, Washington State University, Pullman, Washington, USA; Department of Clinical Sciences, Swedish University of Agricultural Sciences, Uppsala, Sweden (Höglund).

Address all correspondence to Dr. Boel A. Fransson; telephone: (509) 335-5669; e-mail: [email protected]

Dr. Fransson’s current address is Washington State University, College of Veterinary Medicine, 205 Ott Road, Pullman, Washington, 99164, USA.

Dr. Chen’s current address is the Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Atlantic Veterinary College, 550 University Avenue, Charlottetown, Prince Edward Island, C1A 4P3, Canada.

Disclaimer. The LigaTies were provided by Dr. Odd Höglund, founder and partner of Resorbable Devices AB, which is the legal manufacturer of the device. The devices were donated for research purposes.

Received December 1, 2016. Accepted February 6, 2017.


I n t r o d u c t i o nThe laparoscopic revolution started in the late 1980s and early

1990s, with minimally invasive surgery (MIS) for cholecystectomy and adrenalectomy in humans (1). By the early 2000s, cholecys-tectomy via laparoscopic approach had become the procedure of choice, with more than 75% of cholecystectomies in North America being performed laparoscopically (2), a number rising to exceed 90% within the last decade (3). The procedure is typically conducted on an outpatient basis with conversion to open surgery occurring in only 5% to 10% of cases (2). Advantages of laparoscopic procedures are well-documented in human medicine and it is known that lapa-roscopic cholecystectomy results in decreased post-operative pain, more rapid return to normal activity, and improved cosmesis (4). Likewise, studies in small animal patients have shown reduced pain (5,6) and more rapid return to normal activity (7,8). Laparoscopic cholecystectomy in canine patients is infrequently reported in vet-erinary literature but is gaining popularity as a minimally invasive option. Mayhew et al (9) described laparoscopic cholecystectomy for uncomplicated mucoceles in 6 dogs. All dogs had successful lapa-roscopic cholecystectomy without conversion to an open approach and clinical signs improved or resolved in all cases. More recently, short-term outcome of 16 dogs with laparoscopic cholecystectomy was determined to be good, with 6/16 (38%) being converted due to necrotic or ruptured gall bladder (10). In addition, possible compli-cations of laparoscopic cholecystectomy in dogs include cystic duct rupture and bile spillage from cystic duct ligation (11).

Options for laparoscopic cystic duct ligation include extracorpore-ally or intracorporeally tied sutures, and hemoclips (9). Cystic duct ligation with a vessel sealant device has also been described in a cadaveric study with normal hepatobiliary systems (12). Concern for hemoclip slippage, especially in thickened or friable cystic duct tissue, and challenges encountered with intra/extracorporeally suture ligation have encouraged research into alternative methods for cystic duct ligation (13).

The LigaTie (Resorbable Devices AB, Uppsala, Sweden) is an absorbable self-locking device that was manufactured based on the principle of a cable tie. It is currently not commercially available in the USA. Initially, the device was made of polydioxanone (14); however, because of concerns of pliability, it is now a co-polymer of glycolide and trimethylene carbonate (15). The original intent of the LigaTie was vessel ligation due to the excellent tissue grip and ability to close to a loop end diameter of zero (14). Höglund et al (15) described its use for ligation of the spermatic cord in dogs, with specific interest in the feasibility as well as biocompatibility. The study showed that no complications were found related to the device and that the subcutaneous tissues were capable of resorbing the polyglycolic based co-polymer, indicating appropriate biocom-patibility. The same device was also used for ligation of the canine ovarian pedicle and its performance was compared to traditional suture ligation (16). This feasibility study showed that the ligation time of the mesovarium was significantly shortened by using the self-locking implant versus a single traditional ligature. A study by Aminlashgari et al (17) tested the mechanical performance of the device and its degradation over time in vitro as well as short-term tests in vivo in pigs. Results of this study suggested that sufficient

strength of the LigaTie was retained during healing time of the blood vessels. In contrast, non-absorbable cable ties, previously described in veterinary medicine for ovariohysterectomy, have resulted in adverse complications such as fistulous draining tracts, retroperitoneal abscesses, and granuloma formation (18–20); their use in surgery is generally considered not appropriate.

Due to the movement towards minimally invasive surgery and need for a secure and reliable device for cystic duct ligation, we wanted to explore the use of the LigaTie. The objective of this study was to describe the use of the LigaTie in an ex vivo caprine cadav-eric cholecystectomy model and demonstrate the leak pressure of the cystic duct compared with cholecystectomies performed with 2 large endoscopic hemoclips. Our null hypothesis was that the leak pressure of the cystic duct occluded with a LigaTie and 2 large endoscopic hemoclips in an ex vivo cadaveric model would not be different.

M a t e r i a l s a n d m e t h o d sThis study was approved by the Washington State Institutional

Animal Care and Use Committee. Twelve goats were used in the study. A caprine model was chosen due to anatomical similarities to dogs, and the limited availability of fresh canine cadavers. There were 4 males, 2 females, and 6 wethers. Breeds represented were Lamancha (n = 3), Boer cross (n = 3), alpine cross (n = 2), Saanen (n = 2), and pygmy cross (n = 2). Goats were quarantined for a minimum of 14 d prior to administration of general anesthesia, to ensure appropriate health status. A complete physical examination and packed-cell volume/total protein (PCV/TP) was carried out before induction of general anesthesia for a terminal surgery labo-ratory. Goats were euthanized for reasons unrelated to this study. Immediately after euthanasia, a right paracostal approach was conducted to access the duodenum, common bile duct, gall bladder, and right liver lobes. In the original study design, a duodenotomy was completed and the common bile duct was cannulated retro-grade from the intestinal lumen with a 4 Fr double lumen catheter (Milacath; Mila International, Florence, Kentucky, USA) and secured to the common bile duct with a circumferential suture. A chole-cystectomy was carried out through the right paracostal approach with the cystic duct being ligated with either 2 large stainless steel endoscopic hemoclips (Horizon Metal Ligation Systems; Teleflex, Morrisville, North Carolina, USA) or the LigaTie (Resorbable Devices AB, Uppsala, Sweden). A pilot sample elucidated multiple com-munications between the biliary system and the pancreatic duct in the goat, and infusion of fluid through a retrograde catheter (which resided in the common bile duct) resulted in marked pancreatic edema. Therefore, we were unable to maintain and determine the true pressure within the cystic duct as infused fluid exuded into the pancreas. In the final study design, after the paracostal approach was performed, the gall bladder, and approximately 4 cm of the cystic duct was removed with the surrounding parenchyma of the right medial and quadrate liver lobe by sharp transection. The gall bladder was examined for gross pathology and gently expressed to ensure a patent cystic duct. The cystic duct was dissected from the surrounding liver parenchyma with blunt dissection until a stalk large enough to fit 2 endoscopic hemoclips (Figure 1) or a LigaTie


(Figure 2) was exposed. The LigaTie was placed in a similar fashion as a cable tie and tension was placed on the LigaTie “tail” until no further closure of the device could be achieved. Ligation method was designated for the first sample based on a coin toss, and thereafter the method was alternating in the subsequent samples. Once either the hemoclips or LigaTie had been placed, the apex of the gall blad-der was cannulated using a 4 Fr double lumen catheter (Milacath; Mila International). One port of the double lumen catheter was con-nected to a 1-liter bag of sterile saline infused with fluorescein dye in order to augment identification of leakage within the system. The other port was connected to an extension set and pressure transducer (Hewlett Packard CMS 24 Omicare; HP, Palo Alto, California, USA) (Figure 3). The transducer was calibrated to atmospheric pressure before testing each gall bladder. No reinforcement or ligation was needed at the catheter-gall bladder interface due to the apparent water tight seal created when fluid infusion was initiated. Fluid was infused through the catheter at a rate of 999 mL/h while the system was observed for leakage. Leak pressure was recorded when fluid was seen extruding from the gall bladder wall, cystic duct, or at the

site of the clips or LigaTie. If no leakage was observed, the maximum pressure achieved with the fluid pump was recorded.

Data analysisStatistical analysis was performed with a commercially available

software program (GraphPad Prism 5; GraphPad Software, La Jolla, Californai, USA). Leak pressure between the 2 groups was normally distributed as determined with a D’Agostino-Pearson omnibus K2 test and the means were analyzed using a Student t-test. Mean weight of the goats was likewise normally distributed and the means were analyzed using a Student t-test. A P-value , 0.05 was considered significant.

Re s u l t sTwelve goats of various breeds were included in this study. There

were no statistically significant differences between the weight of goats between groups (P = 0.564). Abnormal physical examination findings included mucoid nasal discharge in 3, lice in 1, and stimu-lus induced myotonia which was clinically irrelevant in 1. Results of preoperative PCV, and total protein were within normal limits in all goats. Goats were treated by the attending clinician for abnormal preoperative findings and were considered healthy before anesthesia and euthanasia. Subjectively, all gall bladders and livers were free of gross disease, and when expressed, normal bile easily passed through the cystic duct, demonstrating patency. All cystic ducts were , 5 mm in diameter and visibly free of pathology.

There were 12 samples included in the study: 6 ligated by hemo-clips and 6 ligated by the LigaTie. The mean cystic duct pressure achieved in the hemoclip group was 310.5 mmHg [6 75.4 mmHg standard deviation (SD)]. The mean cystic duct pressure of the LigaTie group was 317.2 mmHg [6 55.9 mmHg]. There were no statistically significant differences between the maximum cys-tic duct pressures between groups (P = 0.865). No leakage was observed from the cystic duct, hemoclip site, or LigaTie site in either group. However, very small amounts of fluid were observed diffusely exuding from the gall bladder wall in 4 samples at

Figure 1. Gall bladder catheterized with double lumen catheter (black arrow). Cystic duct ligated with 2 large endoscopic hemoclips (white arrow).

Figure 2. Gall bladder with cystic duct ligated with the LigaTie (white arrow).

Figure 3. Gall bladder catheterized with double lumen catheter (large black arrow). One lumen is connected to a pressure transducer (small black arrow) and the other is connected to the fluid ingress.


pressures . 330 mmHg. Also, in 1 sample, a small amount of leak-age was observed around the catheter insertion site at the apex of the gall bladder, at a pressure of . 230 mmHg. Gentle digital pressure at the catheter entrance site was enough to allow continued filling of the gall bladder without loss of pressure. The final pressure mea-sures were obtained when the infusion pump stopped due to the high pressure within the system.

D i s c u s s i o nThe results of this study indicate that there was no difference

between the maximum cystic duct pressure occluded with 2 large endoscopic hemoclips or a LigaTie in an ex vivo caprine cadaveric model for cholecystectomies.

Both the large hemoclip and LigaTie group reached supraphysi-ologic pressures within the gall bladder and cystic duct. Small amounts of fluid that diffusely seeped through the gall bladder wall at pressures . 330 mmHg were observed in 4 gall bladder samples. This did not affect the overall pressure achieved within the gall bladder or cystic duct because the transducer would continue to show increases in pressure. This equivocal leakage was assumed to be secondary to the marked intraluminal increases in hydrostatic pressure and the infusion pump eventually stopped when the pres-sure became too high. Therefore, it is possible that higher pressures could have been achieved with manual pressure and leakage from the hemoclips or LigaTie site could have been observed. However, the pressures obtained are already supraphysiologic and testing to failure would not be clinically relevant. The normal common bile duct pressure in a dog is 12 mmHg (21). Although the normal caprine cystic duct or common bile duct pressure is unknown, it is unlikely to reach pressures close to those noted in this study, indicat-ing that both large hemoclips and the LigaTie may be appropriate for cystic duct ligation in dogs. Additionally, there are no known studies that measure cystic or common bile duct pressures in dogs with hepatobiliary disease. In humans, common bile duct pressures can increase up to 29.4 mmHg with gallstones and suppurative cholangitis (22).

The LigaTie was manufactured to display characteristics of hemo-stasis and excellent tissue grip making possible applications of the device quite diverse. Initial studies found that a single device placed on the renal artery of pigs showed complete hemostasis in 12 arteries for 5 min (14). To further document resistance to slippage, the ability of the device to withstand a ligature slip-off was tested by applying a force of 10 N (14). The LigaTie has successfully been used in vivo on ovarian pedicles (15) and the spermatic cord (23) with histological evidence of vascular occlusion and biocompatibility. Future applica-tions such as lung lobectomies are currently being investigated at the authors’ institutions. In humans, the use of a self-locking non-absorbable inert polymer clip (Hem-O-Lok clip) has been shown to be a secure option during basic laparoscopic procedures including cholecystectomy, appendectomy, splenectomy, and nephrectomy (24). These clips can ligate up to 10-mm of tissue through a 5-mm portal or up to 15-mm of tissue through a 10-mm portal (24). Due to the concern of migration or dislodgement of metallic clips, these locking clips have been preferred for various procedures and share common advantages to the LigaTie.

One main advantage with using the LigaTie on the cystic duct is its ability to occlude and compress tissue without major limitation on duct size or tissue pliability. During cholecystectomies, the cystic duct may be friable and not amendable to extensive dissection or manipulation. Additionally, if the tissue is fibrous or dilated, large hemoclips may not adequately occlude the duct or provide adequate hemostasis of the cystic artery; risking slippage and biliary leakage (9). The LigaTie is flexible and could be used on any size cystic duct with a single, secure ligation with no collateral thermal damage. Laparoscopic cholecystectomy is performed in other species, includ-ing wild animals (25), in which the LigaTie may be an alternative for wide cystic ducts with fibrous walls.

The ideal use for the LigaTie is implementing it in minimally inva-sive surgery, specifically laparoscopic cholecystectomy. However, there is hesitation for complete laparoscopic cholecystectomies in canine patients which is because of the inability to flush the com-mon bile duct to confirm patency. Recent reports have described open cholecystectomies without catheterization of the common bile duct (26). Malek et al (26) also found that 53.5% of common bile ducts were not catheterized before cholecystectomy. No significant association was found in patient outcome between the methods of catheterizing the common bile duct during cholecystectomy and whether or not the common bile duct was catheterized. However, recommendations on the necessity or technique for flushing the common bile duct during surgery could not be made based on their findings (26). A recent study by Scott et al (27) described periopera-tive complications and outcomes of laparoscopic cholecystectomy in 20 dogs. Six dogs (30%) required conversion to an open cholecys-tectomy; however, all dogs were discharged from the hospital and no significant difference was found between hospitalization time in laparoscopic versus conversion to an open procedure (27). Patients with evidence of extra-hepatic biliary obstruction were not included in the study; therefore, patency of the common bile duct was not confirmed intraoperatively. Based on that study, laparoscopic cho-lecystectomy can be performed successfully for uncomplicated gall bladder disease with careful case selection. The authors of the current study are not necessarily advocating for laparoscopic cho-lecystectomy without catheterizing of the common bile duct, rather, understanding that it may be an option and prospective studies are required to help further define the ideal patient.

Limitations of this study include small sample size, using caprine cadavers instead of live or cadaver canine models, and the inability to model a standard cholecystectomy. However, given that this study was intended to be a proof of concept, future studies can be more targeted in both the species and study design to better analyze the clinical uses of LigaTie in traditional open and laparoscopic chole-cystectomy. An analysis of the histologic differences of a caprine gall bladder compared to a canine gall bladder was not conducted. Given there were no differences between the groups in this study and the supraphysiologic pressures that were obtained, the differ-ence in histologic appearance would likely not be clinically signifi-cant. It would have been ideal to be able to model a clinical canine cholecystectomy in this study’s caprine model. However given the gross anatomic differences from a canine cadaver, this was not pos-sible. Instead, it was decided that the focus would be on mechanical testing, in a consistent and repeatable manner, although the design


set-up is not applicable to in vivo conditions. Additionally, no attempt was made to place the LigaTie in a laparoscopic model; therefore, we cannot comment on the ability to maneuver the device in that setting. Instrument design is underway at our institution to use the LigaTie in a minimally invasive fashion.

In conclusion, no statistically significant differences were found in the maximum cystic duct pressures between 2 large endoscopic hemoclips and a LigaTie in a caprine cholecystectomy model. Future in vivo studies are necessary to characterize its interaction with canine cystic ducts as well as to determine any short- and long-term complications associated with this novel device.

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7. Culp WT, Mayhew PD, Brown DC. The effect of laparoscopic versus open ovariectomy on postsurgical activity in small dogs. Vet Surg 2009;38:811–817.

8. Mayhew PD, Brown DC. Prospective evaluation of two intra-corporeally sutured prophylactic laparoscopic gastropexy tech-niques compared with laparoscopic-assisted gastropexy in dogs. Vet Surg 2009;38:738–746.

9. Mayhew PD, Mehler SJ, Radhakrishnan A. Laparoscopic cho-lecystectomy for management of uncomplicated gall bladder mucocele in six dogs. Vet Surg 2008;37:625–630.

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and Thoracoscopy. 1st ed., Ames, Iowa: Wiley-Blackwell, 2015: 156–166.

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Short Communication Communication brève


Heartworm disease (HWD) is one of the most life-threatening infectious heart diseases in dogs, and typically causes cardiac and pulmonary disorders including right-side congestive heart failure, endarteritis, and pulmonary hypertension with abnormal immune reactions (1). Clinical signs are generally related to worm burden (1) and azotemia is often seen in dogs with HWD (1,2).

Symmetric dimethylarginine (SDMA) has recently been evaluated as a renal biomarker that can efficiently detect early renal dysfunc-tion in dogs (3). The level of serum SDMA has shown a significant relationship with the glomerular filtration rate (GFR) because it is only excreted through renal clearance in dogs (3). Unlike serum blood urea and creatinine, SDMA concentration is not significantly affected by age, body mass, and systemic diseases (4). Furthermore, SDMA, compared to creatinine, tends to start increasing at an earlier stage of renal failure in dogs (3). Therefore, SDMA may have good potential for early diagnosis of renal impairment in dogs with HWD. The aim of this study was to evaluate the diagnostic value of serum SDMA in dogs with HWD-mediated renal disorders.

The study population consisted of 12 dogs infected with HWD (6.6 6 4.5 kg, mixed breed) and 6 HWD-negative clinically normal control dogs (8.7 6 6.5 kg, mixed breed). All dogs were acquired

from a private animal shelter. The owner of the shelter gave consent for the dogs to participate in this study. The ages of dogs enrolled in this study could not be determined, although all dogs were mature. Heartworm preventive drugs were not given to the dogs in this study population. The affected dogs received no medical therapy for HWD prior to renal testing. The dogs had no significant medical history, but had undergone deworming and vaccination. Heartworm infection in this study population was diagnosed using a commercial enzyme-linked immunosorbent assay (ELISA) test kit (SNAP 4Dx Plus Test, IDEXX Laboratory, Westbrook, Maine, USA) used accord-ing to the manufacturer’s directions. Dogs with HWD were further divided into 2 groups based on clinical signs using physical, radio-graphic, or echocardiographic evaluation. Briefly, dogs in group A [n = 6, 7.2 6 3.4 kg of body weight (BW)] had either no or mild clini-cal signs with/without abnormal findings on physical, radiographic, or echocardiographic examinations (i.e., occasional cough with or without exercise intolerance, mild pulmonary infiltration, and pul-monary arterial dilation, few visible worms in pulmonary arteries, and no evidence of pulmonary hypertension). While dogs in group B (n = 6, 5.4 6 3.1 kg BW) had moderate to severe clinical signs with abnormal findings on physical, radiographic, or echocardiographic

Evaluation of serum symmetric dimethylarginine in dogs with heartworm infection

Bom-Sul Choi, Hyeongsun Moon, Sang-IL Suh, Changbaig Hyun

A b s t r a c tThis study evaluated the circulating levels of serum symmetric dimethylarginine (SDMA) in 12 dogs with different severities of heartworm disease (HWD) and assessed the biochemical renal markers (blood urea nitrogen, creatinine). Dogs were classified into 2 groups based on the severity of clinical signs. Group A — asymptomatic to mild clinical signs, group B — moderate to severe clinical signs. The serum SDMA levels were higher in dogs in group B. Although the serum SDMA levels in dogs in group A were also higher than those of the control dogs, the difference was not statistically significant. There was a good correlation between renal markers and severity of clinical signs. This study demonstrated that the glomerular filtration rate was significantly decreased in dogs in group A; therefore, earlier detection of renal impairment is required for successful management of dogs with HWD.

R é s u m éLa présente étude visait à évaluer les niveaux sériques de diméthylarginine symétrique (DMAS) chez 12 chiens atteints de maladie du vers du cœur (MVC) de différentes sévérités et d’examiner les marqueurs biochimiques rénaux (azote uréique sanguin, créatinine). Les chiens ont été classés en deux groupes sur la base de la sévérité des signes cliniques. Le Groupe A — asymptomatique à signes cliniques légers, groupe B — signes cliniques modérés à sévères. Les niveaux de DMAS sériques étaient plus élevés chez les chiens du groupe B. Chez les chiens du groupe A, bien que les niveaux sériques de DMAS étaient également plus élevés que ceux des chiens témoins, la différence n’était pas statistiquement significative. Il y avait une bonne corrélation entre les marqueurs rénaux et la sévérité des signes cliniques. Cette étude a permis de démontrer que le taux de filtration glomérulaire était diminué de manière significative chez les chiens du groupe A; ainsi, une détection précoce de déficience rénale est nécessaire pour la gestion réussie des chiens avec MVC.


Department of Small Animal Internal Medicine, College of Veterinary Medicine, Kangwon National University, Chuncheon, Korea (Choi, Suh, Hyun); Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA (Moon).

Address all correspondence to Dr. Changbaig Hyun; fax: 82 33 244 2367; e-mail: [email protected]

Received August 3, 2016. Accepted September 23, 2016.


examinations (i.e., persistent coughing, severe exercise intoler-ance, signs related to right-sided congestive heart failure, marked pulmonary infiltration with pulmonary arterial dilation and right ventricular enlargement, echocardiographic evidence of pulmonary hypertension with . 2.8 m/s peak velocity of tricuspid regurgitation or . 2.2 m/s peak velocity of pulmonary regurgitation.

To minimize the influence of feeding, all dogs were fasted for 12 h before the collection of blood samples. Whole blood was withdrawn from either the cephalic or jugular veins for determination of serum level of SDMA. Blood samples were drawn directly into sterile tubes (Vacutainer tubes; BD, Franklin Lakes, New Jersey, USA) and then centrifuged at 1.500 3 g for 10 min at 4°C. The SDMA concentrations were determined by a reference laboratory (IDEXX Laboratories). Levels of blood urea nitrogen (BUN) and creatinine were determined using an automated biochemistry analyzer (VetScan VS2, Abaxis, Union City, California, USA).

Statistical analyses were done using commercially available statistical software (SPSS 15.0 for Windows; IBM, New York, USA). Descriptive statistics for quantitative variables between study groups were calculated using analysis of variance (ANOVA) with Tukey’s multiple comparison test. The correlation between renal markers (e.g., BUN, creatinine, and SDMA) was determined by Pearson’s coefficient of bivariate correlation analysis. A probability value less than 0.05 was considered statistically significant in our analysis.

Serum SDMA concentrations were 8 6 2 mg/dL in controls, 13 6 5 mg/dL in group A, and 21 6 4 mg/dL in group B. Serum BUN was 18 6 7 mg/dL in controls, 26 6 13 mg/dL in group A, and 41 6 25 mg/dL in group B. Serum creatinine was 0.7 6 0.2 mg/dL in controls, 1.0 6 0.4 mg/dL in group A, and 1.5 6 0.5 mg/dL in group B. Pair-wise comparisons showed significant differences in serum BUN, creatinine, and SDMA concentrations in control and disease groups (P , 0.05), as well as in HWD groups with different severities of clinical signs (P , 0.05).

Based on previous veterinary references (3,5), upper limits were set as 25 mg/dL for BUN, 1.4 mg/dL for creatinine, and 14 mg/dL for SDMA. The number of dogs with BUN higher than the upper limit was 0/6 in controls, 2/6 in group A, and 3/6 in group B; the number with creatinine higher than the upper limit was 0/6 in controls, 1/6 in group A, and 2/6 in group B. The number of dogs with SDMA above upper limit values was 0/6 in controls, 2/6 in group A, and 6/6 in group B.

Bivariate analysis revealed close correlations among renal mark-ers (i.e., SDMA, creatinine, and BUN) were found. Serum BUN, creatinine, and SDMA concentrations were not correlated with body weight but were closely correlated with the severity of clinical signs (Table I). Serum SDMA concentration was correlated with serum BUN and creatinine concentration (Table I).

Azotemia is one of the most common complications of advanced heart failure caused by HWD and other heart diseases (6,7). Unlike other heart diseases, HWD in dogs can induce azotemia through immune-mediated glomerulonephritis caused by microfilaria and debris of dead adult worms, while lower cardiac output-induced ischemic renal injury in decompensated heart failure is a major risk factor for azotemia (1). Therefore, early detection of renal impair-ment in HWD would enable clinicians to establish a safe but efficient management plan in dogs with HWD. Current diagnostic tests for

renal impairment are limited to dogs with apparent renal disease or dysfunction, although direct measurement of GFR with creatinine or iohexol can detect earlier stages of renal impairment (5). However, indirect methods using BUN and creatinine are less sensitive, and are influenced by age, body mass, and other systemic diseases (5). Recent veterinary studies found that the level of circulating SDMA was more accurate and useful for early detection of renal dysfunc-tion in dogs, and is not significantly affected by lean body mass and age (3,4). Our study also demonstrated gradual but significant elevation of serum SDMA with progression of HWD, implying that renal impairment might be more obvious with progression of clinical signs in HWD. Unlike creatinine, the serum level of SDMA started to increase in dogs with ~40% loss of nephrons (3). In this study, the serum level of SDMA was higher than the reference range in 2/6 dogs in group A and 6/6 dogs in group B, while the serum level of creatinine was higher in 1/6 dogs in group A and 2/6 dogs in group B. This finding suggests that, in dogs, circulating SDMA can detect renal impairment earlier than does creatinine. Further, one recent canine study of chronic mitral valve disease found that SDMA increased with the severity of mitral insufficiency and indi-cated renal impairment earlier than did creatinine in dogs (8). The increased serum levels of SDMA in dogs with advanced stages of HWD (i.e., group B) in this study suggested a higher risk of renal impairment in dogs with higher worm burden and severe clini-cal signs. Interestingly, 2/6 dogs in group A in this study showed serum SDMA levels higher than the reference range, suggesting that many dogs without clinical signs related to HWD could have reduced GFR via impaired renal perfusion or renal injury due to immunological causes, although they generally appeared clinically normal. Therefore, earlier intervention to prevent renal injuries might be beneficial for successful management of HWD in dogs. Routine SDMA testing in dogs with HWD will help clinicians to more properly decide the choice of HW therapeutic protocols (e.g., slow kill versus melarsomine), the timing of initiation of adulticidal therapy and management guideline during adulticidal treatment, based on the renal status of dogs.

There are several limitations in this study. First, the study popula-tion was small, which may not provide sufficient data to adequately correlate SDMA with the severity of HWD in dogs. Second, the SDMA levels of all affected dogs in this study population could not be monitored over time owing to refusal by the owners. We

Table I. Correlation of clinical severity of heartworm disease (HWD) and symmetric dimethylarginine (SDMA) concentrations with other variables

Clinical severity SDMA R P-value R P-valueBW 0.03 0.751 0.116 0.355Clinical severity — — 0.732 0.001BUN 0.434 0.002 0.743 0.001CRE 0.555 0.01 0.648 0.01SDMA 0.738 0.001 — —BW — bodyweight; BUN — blood urea nitrogen; CRE — creatinine: R-correlation coefficient.


obtained SDMA test results over time for only a few affected dogs (2/6 dogs in group B) after melarsomine therapy and found that the SDMA levels returned to the reference range 2 mo after treatment (unpublished data). Since melarsomine is eliminated by the kidney to an extent, further studies should be aimed at investigating the influence of melarsomine on renal function and SDMA levels in a larger study population. In addition, further study should also be aimed at identifying which therapeutic protocol (e.g., slow kill or melarsomine-based protocol) is more suitable for HWD infected dogs with prolonged kidney damage. Nevertheless, our study demon-strated that the GFR was significantly decreased in dogs with lower worm burden and milder clinical signs; therefore, earlier detection of renal impairment is required for successful management of dogs with HWD. Our study also implied that earlier recognition and stabi-lization of renal impairment prior to and during adulticidal therapy might be more important for clinicians to achieve therapeutic goals by slow kill or melarsomine.

Re f e r e n c e s1. Calvert CA, Rawlings CA. Canine heartworm disease. In:

Fox PR, ed. Canine and Feline Cardiology. New York, New York: Churchill Livingstone, 1988:519–549.

2. Atkins CE, Vaden SL, Arther RG, et al. Renal effects of Dirofilaria immitis in experimentally and naturally infected cats. Vet Parasitol 2011;176:317–323.

3. Nabity MB, Lees GE, Boggess MM, et al. SDMA assay valida-tion, stability, and evaluation as a marker for early detection of chronic kidney disease in dogs. J Vet Intern Med 2015;29: 1036–1044.

4. Hall JA, Yerramilli M, Obare M, Yerramilli M, Melendez LD, Jewel DE. Relationship between lean body mass and serum renal biomarkers in healthy dogs. J Vet Intern Med 2015;29: 808–814.

5. Braun JP, Lefebvre HP. Kidney function and damage. In: Kaneko JJ, Harvey JW, Bruss ML, eds. Clinical Biochemistry of Domestic Animals. 6th ed. London, UK: Elsevier, 2008: 485–528.

6. Ronco C, Haapio M, House AA, Anavekar N, Bellomo R. Cardio-renal syndrome. J Am Coll Cardiol 2008;52:1527–1539.

7. Tang WH, Mullens W. Cardiorenal syndrome in decompensated heart failure. Heart 2008;96:255–260.

8. Choi B-S, Moon H-S, Seo S-I, Hyun C. Evaluation of serum cystatin-C and symmetric dimethylarginine concentrations in dogs with heart failure from chronic mitral valvular insufficiency. J Vet Med Sci 2017;79:41–46.



Pregnant sheep are frequently used as an experimental model for the human maternal-fetal unit (1–4). During experimental research, maternal health is routinely assessed while fetal health is more difficult to assess and is not routinely performed. This approach has its limitations as fetal health is not always synonymous with maternal health (5). There is limited information in the literature regarding fetal blood gas results and their relationship with mater-nal blood gas results during maternal anesthesia in sheep. The aim of this study was to describe the differences between maternal and fetal blood gases in normotensive and normocapnic third trimester singleton ewes during general anesthesia. These data were collected opportunistically as the animals were part of a larger project requir-ing fetal intervention during maternal anesthesia, laparotomy, and hysterotomy.

The use of sheep in this study was approved by the Animal Ethics Committees of the University of Western Australia and Murdoch University in accordance with the Code of Practice for the Care and Use of Animals for Research and Teaching (6). All sheep were

housed in a purpose built biomedical research facility and cared for as previously described (7,8).

Sixteen singleton adult merino ewes weighing 60.1 6 5.1 kg at 125 d (124 to 126 d) gestation (term is 150 d) were anesthetized as previously described (7,8). In brief, the ewes were pre-medicated with a combination of acepromazine [0.03 mg/kg body weight (BW)] and buprenorphine (0.01 mg/kg BW) by intramuscular injection. Forty-five minutes later, anesthesia was induced with a combina-tion of midazolam (0.25 mg/kg BW) and ketamine (5 mg/kg BW) by intravenous injection. Anesthesia was maintained with isoflu-rane (vaporizer settings were 1% to 2%) in 100% oxygen delivered through a circle breathing system. Ewes were placed in dorsal recumbency in the Trendelenburg position with the table tilted at 25°C and were mechanically ventilated (Aestiva 5 with Smartvent 7900; Datex-Ohmeda, GE Healthcare, Bromma, Sweden), with a tidal volume of 7 to 10 mL/kg BW, I:E ratio 1:2 and a peak inspiratory pressure of 10 to 25 cmH2O. Intravenous fluid therapy was adminis-tered during anesthesia with a balanced isotonic electrolyte solution

Maternal and fetal arterial blood gas data in normotensive, singleton, isoflurane anesthetized sheep at 124–126 days of gestation

Claire M. Loughran, Matthew W. Kemp, Gabrielle C. Musk

A b s t r a c tThe aim of this case series was to describe the differences between maternal and fetal blood-gas results during anesthesia. Sixteen singleton adult merino ewes weighing 60.1 6 5.1 kg at 125.7 d (124 to 126 d) gestation were anesthetized. Maternal (radial) and fetal (umbilical) arterial blood gas samples were collected 79 6 6 min after the start of anesthesia if maternal mean arterial pressure (MAP) was stable and . 65 mmHg. Fetal pH, partial arterial pressure of oxygen (PaO2), glucose, arterial hemoglobin oxygen saturation (SaO2), sodium, and chloride were significantly lower and fetal partial arterial pressure of carbon dioxide (PaCO2), lactate, hematocrit, total hemoglobin, potassium, and calcium were significantly higher than maternal blood-gas values. Fetal pH, PaO2, and BE were lower and fetal lactate was higher than fetal umbilical arterial samples previously reported, which may indicate a non-reassuring fetal status. Further refinement of the ovine experimental model is warranted with fetal monitoring during maternal anesthesia.

R é s u m éL’objectif de l’étude était de décrire les différences dans les résultats d’analyse des gaz sanguins maternel et fœtal durant l’anesthésie. Seize brebis mérinos primipares pesant 60,1 6 5,1 kg à 125,7 j (124 à 126 j) de gestation ont été anesthésiées. Des échantillons de sang artériel maternel (radiale) et fœtal (ombilicale) ont été prélevés 79 6 6 min après le début de l’anesthésie si la pression artérielle moyenne (PAM) maternelle était stable et . 65 mmHg. Pour le sang fœtal, les valeurs de pH, de la pression artérielle partielle en oxygène (PaO2), du glucose, de la saturation en oxygène de l’hémoglobine artérielle (SaO2), du sodium, et du chlorure étaient significativement inférieures, et les valeurs de la pression artérielle partielle en dioxyde de carbone (PaCO2), du lactate, de l’hématocrite, de l’hémoglobine totale, du potassium, et du calcium étaient significativement supérieures que celles du sang maternel. Les valeurs fœtales du pH, de la PaO2, et de BE étaient plus basses et le lactate fœtal étaient plus élevées que les valeurs d’échantillons provenant du sang artériel ombilical fœtal rapportées précédemment, ce qui pourrait indiquer un statut fœtal non-rassurant. Des améliorations de ce modèle expérimental ovin sont souhaitées avec un suivi fœtal durant une anesthésie maternelle.


College of Veterinary Medicine, School of Veterinary and Life Sciences, Murdoch University, Murdoch, Western Australia 6150, Australia (Loughran); School of Women’s and Infants’ Health, The University of Western Australia, Perth, Western Australia 6009, Australia (Kemp); Animal Care Services, The University of Western Australia, Perth, Western Australia 6009, Australia (Musk).

Address all correspondence to Gabrielle Musk; e-mail: [email protected]

Received September 7, 2016. Accepted November 14, 2016.


(Compound Sodium Lactate solution; Baxter Healthcare, Toongabbie, New South Wales, Australia) at 10 to 20 mL/kg per hour and succi-nylated gelatin solution (Gelofusine 4%; Braun Australia, Bella Vista, New South Wales, Australia) at 2 to 10 mL/kg per hour as required to maintain normotension (MAP . 65 mmHg). The physiological changes associated with anesthesia were monitored continuously and displayed on a multivariable monitor (Advisor Vital Signs Surgivet Monitor V9212AR; Smiths Medical, Lincoln Park, New Jersey, USA). These parameters included heart rate, respiratory rate, electrocardiography, pulse oximetry, esophageal temperature, inva-sive blood pressure, and inspiratory and ETCO2. The experimental procedure involved laparotomy and hysterotomy to access the fetus. While remaining in utero the fetal trachea was intubated and the fetal lung was ventilated. At the end of the procedure the ewe and fetus were euthanized with intravenous pentobarbitone 160 mg/kg (Valabarb, Pentobarbitone 320 mg/mL; Jurox, Rutherford, New South Wales, Australia).

The paired maternal and fetal arterial blood gas samples were collected 70 to 90 min after the onset of anesthesia and analyzed provided maternal MAP had been stable for 15 min and was greater than 65 mmHg at the time of blood sample collection. Maternal arte-rial blood samples were collected from the radial arterial catheter and fetal arterial samples were collected from the umbilical artery. The samples were collected into heparinized syringes, any air bub-bles were expelled and a cap was placed on the syringe immediately. Samples were analyzed within 15 min of collection using a benchtop arterial blood gas analyzer (RapidLab 1265; Siemens Healthcare Diagnostics). The data collected from these analyses included pH, PaO2, PaCO2, BE, lactate, glucose, hematocrit (Hct), tHb, SaO2, sodium (Na1), potassium (K1), calcium (Ca21), and chloride (Cl2).

Maternal and fetal arterial oxygen content (CaO2; mL/L) was cal-culated as follows: CaO2 (mL/L) = [([Hb] (gdL21) 3 1.36 3 SaO2 (%)) 1 (0.0031 3 PaO2 (mmHg))] 3 10. Normally distributed data were compared with a Student’s t-test. Otherwise a Mann–Whitney Rank Sum test was used. All tests were performed with Prism 6, 2015 (Graphpad Software; La Jolla, California, USA); P , 0.05 was considered to be statistically significant. Normally distributed data are presented as mean 6 SD, otherwise data are expressed as median (interquartile range).

Nineteen pairs of maternal and fetal blood gas results were obtained, but 3 pairs of results were excluded from analysis due

to maternal hypotension (MAP , 65 mmHg) at the time of arterial blood gas collection. Sixteen sets of arterial blood gas results were subsequently analyzed. Maternal physiologic parameters, ETCO2, peripheral capillary oxygen saturation measured by the pulse oxim-eter (SpO2), and MAP at the time of blood gas collection were within normal limits (Table I). Ten of the sheep had a mean arterial blood pressure between 55 and 65 mmHg for 5 to 15 min at the beginning of anaesthesia. This hypotension was managed with fluid therapy (temporary increase in the rate of infusion up to the high end of the aforementioned range for both the crystalloid and the gelofusin) and alteration of the vaporizer setting. Fetal pH, PaO2, glucose, SaO2, Na1, and Cl2 were significantly lower than maternal values while fetal PaCO2, lactate, Hct, tHb, K1, and Ca21 were significantly higher than maternal values (Table II). Maternal CaO2 was significantly higher than fetal CaO2.

This study compared paired maternal and fetal arterial blood gas data at a single time point from dorsally recumbent, normo-tensive isoflurane-anesthetized singleton ewes and their fetuses at a gestational age of 125 d (124 to 126 d). Comparison of these data demonstrated a significantly higher fetal PaCO2, lactate, Hct, tHb, K1, and Ca21 relative to maternal results and a significantly lower fetal umbilical PaO2, glucose, SaO2, Na1, Cl2, and CaO2 relative to maternal arterial values.

Simultaneous fetal and maternal arterial blood gas samples in this study revealed maternal PaO2 within the expected range, but fetal PaO2 lower than values recorded in previous studies (8). Anesthetized pregnant sheep breathing up to 98% O2, almost 5 times the oxygen concentration of room air, are expected to have a PaO2 of approximately 484 mmHg 6 21 mmHg according to the alveolar gas equation (9). This value is similar to the maternal PaO2 reported in this study. Maternal circulating oxygen is transported across the placenta and into the fetal circulation by passive diffusion in response to a pressure gradient. Consequently, fetal PaO2 should be significantly lower than maternal values, as reported in this study. The fetal PaO2 from the singleton animals in this study was unexpectedly lower than those reported for twin fetuses from anes-thetized ewes (8). This discrepancy may indicate a greater degree of maternal-fetal placental perfusion mismatch in the current study and a reduction in the oxygen transfer between the maternal and fetal blood. Furthermore, and perhaps more importantly, the fetal PaO2 values in this study were taken at 79 min 6 6 min, whereas the fetal twin arterial samples were collected at 28 min 6 6 min and 56 6 15 min after the onset of anesthesia for the first and second twin, respectively, indicating that the duration of anesthesia may negatively impact fetal PaO2.

Despite the low PaO2 of fetal blood, fetal tissue oxygen require-ments may exceed those of the mother. Consequently, the fetus has adapted to lower PaO2 levels by having a higher Hct and hemoglo-bin concentration with a greater affinity for oxygen than maternal hemoglobin. Maternal ovine Hct decreases during anesthesia (10). The maternal Hct reported in the current study, however, was lower than values reported previously in anesthetized non-pregnant sheep. This finding is consistent with a previous report that pregnant sheep are anemic during anesthesia (8). The fetal Hct in this study was significantly higher than maternal values and when the CaO2 in fetal and in maternal blood is compared, it is apparent that although

Table I. Mean 6 SD of the measured indices from ewes (N = 16) over the duration of anesthesia prior to collection of arterial blood

Parameter ValuesHeart rate (beats/min) 86.7 6 17.9Respiratory rate (breaths/min) 10.1 6 1.9MAP (mmHg) 70.8 6 5.5ETCO2 (mmHg) 37.6 6 3.4Temperature (°C) 38.2 6 0.9Isoflurane vaporizer setting (%) 1.5 6 0.5Time of blood gas collection after onset of 78.8 6 5.9 anesthesia (min)MAP — mean arterial pressure; ETCO2 — end tidal carbon dioxide.


maternal CaO2 is significantly higher, the difference is smaller than might be expected. It is unknown whether anemia during anesthesia of pregnant sheep is acute or chronic and this finding warrants fur-ther investigation. Pre-operative blood samples were not collected in this study.

Simultaneous fetal and maternal arterial blood gas samples in this study revealed that maternal PaCO2 was within the target range, but fetal PaCO2 was higher than values recorded in the fetus of conscious pregnant sheep (73 mmHg versus 52 mmHg) (11). Ventilation in this study was controlled with the aim of maintaining an ETCO2 of 35 to 45 mmHg. Consequently the maternal PaCO2 was similar to values reported in conscious pregnant ewes breathing room air at 103 to 104 d of gestation and confirms findings of previous studies which failed to demonstrate a significant difference between the PaCO2 of conscious pregnant and non-pregnant sheep (12). Fetal PaCO2 in this study was significantly higher than maternal PaCO2. These figures are consistent with those in other studies; however, the gradient for the exchange of CO2 between maternal and fetal blood was higher than the gradient reported in twin pregnant anesthetized ewes (8). The increased CO2 gradient could indicate a greater fetal-maternal perfusion mismatch in this study. The fetal PaCO2 in this study was 40% higher than fetal values recorded in singleton conscious ewes, indicating fetal hypercapnia (11). Based on the fetal-maternal PaCO2 gradient observed in this and other studies it may be war-ranted to target mild maternal hypocapnia (PaCO2: 30 to 35 mmHg) during anesthesia to reduce fetal PaCO2 to a level comparable to fetal measurements in conscious ewes (11). Maternal hypocapnia is normally tolerated for short periods, with few apparent effects (13). Extreme hypocapnia can reduce placental blood flow in sheep, but

this physiological state occurs when maternal PaCO2 values are considerably lower than those being recommended here (14).

Maternal blood glucose concentrations in this study were within the expected range, but fetal glucose concentrations were lower than values recorded in previous studies (8). This discrepancy may relate to the timepoint at which the sample was collected, which was 79 min 6 6 min after anesthesia. The fetus is dependent on maternal circulation to supply glucose so low fetal blood glucose could indicate a failure of placental transfer due to the development of maternal-fetal placental perfusion mismatch over time. Maternal lactate values in this study were within the expected range, but fetal lactate was higher than values recorded in previous studies (8). Lactate production by the fetus is a direct end product of anerobic metabolism.

Consistent with previous reports, the maternal pH in this study was similar to values recorded in conscious sheep and fetal pH was significantly lower than maternal levels (8,10). However, the fetal pH values in this study were lower than those recorded in fetal samples taken from singleton conscious ewes (11). A specific defini-tion of acidemia in the fetus is difficult to describe as the fetus has adapted to withstand short periods of acidosis. Acute fetal acidosis is almost always respiratory in origin, but quickly progresses to a mixed respiratory metabolic acidosis as seen in this study by the increase in fetal BE compared to fetal samples from anesthetized and conscious sheep (8,10).

The electrolyte concentrations in this study varied between maternal and fetal circulations. Ewes were slightly hypocalcemic, hypokalemic, normonatremic, and normochloremic (15). Fetal elec-trolyte levels were significantly lower (CL2 and Na1) and higher

Table II. Mean 6 SD or median (interquartile range) of measured and calculated indices of arterial blood gas data from the ewe and fetus during isoflurane anesthesia in singleton sheep at 124 to 126 d of gestation

Difference between maternal and Maternal Fetal fetal samples P-valuepH 7.4 6 0.05 7.2 6 0.07 0.2 6 0.09 , 0.0001BE (mmol/L) 1.4 6 2.4 20.9 6 3.0 2.3 6 4.3 0.0231PaCO2 (mmHg) 42.1 6 5.1 73.0 6 9.1 230.9 6 8.5 , 0.0001PaO2 (mmHg) 486.3 6 51.0 20.1 6 4.2 461.2 6 51.0 , 0.0001Hct (%) 19.6 6 2.6 35.6 6 2.7 215.9 6 3.5 , 0.0001tHb (g/dL) 6.6 (6.1–7.2) 12.1 (11.3–12.5) 25.4 [26.5–(24.2)] , 0.0001SaO2 (%) 99.9 (99.8–99.9) 54.7 (44.7–60.7) 45.2 (39.2–55.2) , 0.0001Glucose 3.0 6 0.5 1.0 6 0.2 2.2 6 0.4 , 0.0001Lactate (mmol/L) 1.4 6 0.5 3.63 6 0.9 22.3 6 1.0 , 0.0001Ca21 (mmol/L) 1.1 (1.0–1.1) 1.7 (1.6–1.8) 20.6 [20.7–(20.5)] , 0.0001Na1 (mmol/L) 144.1 6 1.5 137.6 6 1.7 6.4 6 1.5 , 0.0001K1 (mmol/L) 3.0 (2.8–3.2) 4.0 (3.7–4.3) 21.0 [21.1–(20.7)] , 0.0001Cl2 (mmol/L) 111.2 6 3.7 100.1 6 2.0 11.06 6 3.9 , 0.0001CaO2 (mL/L) 104.9 6 12.2 83.2 6 18.8 20.6 6 19.6 0.0006BE — base excess; PaCO2 — partial arterial pressure of carbon dioxide; PaO2 — partial arterial pressure of oxygen; Hct — hematocrit; tHb — total hemoglobin; SaO2 — percentage hemoglobin saturation with oxygen; Ca21 — calcium; Na1 — sodium; K1 — potassium; Cl2 — chloride; CaO2 — arterial oxygen content of the blood.


(Ca21 and K1) than maternal levels and lower than ranges found in 1- to 3-month-old sheep (16). Reference ranges are not available for fetal lambs and there is no information in the literature regarding electrolyte levels during gestation. The fetal electrolyte levels mea-sured in this study were similar to values in twin fetuses, with the exception of K1, which was lower in this study (8).

Although the fetuses in this study underwent ventilation, the placental circulation was intact. This intervention does not alter fetal blood gas results (17) so based on the results of this study, ovine maternal health as assessed by arterial blood gas analysis and routine physiological monitoring during anesthesia, does not neces-sarily constitute fetal health. While this study highlights poorer fetal blood gas parameters in anesthetized sheep compared to fetal blood gas parameters from conscious sheep, this finding is not unexpected given the negative impact of anesthesia on the fetal and maternal cardiovascular system, and by extension, placental blood flow, and nutrient and waste product transfer. Improvements in fetal blood gas results (lower PaCO2 and lactate; higher pH, PaO2, and BE) in future studies may be possible with refinements of the anesthetic protocol: determination of the optimal approach to the management of hypotension (fluid therapy versus vasopressors or inotropes); sophisticated regional anesthesia; neuromuscular blockade; intra-operative analgesic drug infusion (e.g., fentanyl), and propofol based total intravenous anesthesia (18).

This case series presents the differences between maternal and fetal arterial blood gas samples in dorsally recumbent isoflurane anesthetized singleton pregnant ewes. Despite the ewes being normotensive and normocapnic, the fetus showed signs of acidosis signaling non-reassuring fetal status. Further work is required to determine both the significance of these findings on fetal health beyond delivery, and to optimize the anesthetic regime for pregnant ewes at different gestational ages in a biomedical research setting.

A c k n o w l e d g m e n t sThanks is also extended to Astrid Armitage, Andrew Wilson, and

the Animal Care Services staff at the Large Animal Facility, for their expert care and husbandry of the pregnant ewes.

Re f e r e n c e s 1. Collins JJP, Kuypers E, Nitsos I, et al. LPS-induced chorioam-

nionitis and antenatal corticosteroids modulate Shh signaling in the ovine fetal lung. Am J Physiol Lung Cell Mol Physiol 2012;303:L778–L787.

2. Gisslen T, Hillman NH, Musk GC, et al. Repeated exposure to intra-amniotic LPS partially protects against adverse effects of intravenous LPS in preterm lambs. Innate Immunity 2014;20: 214–224.

3. Kuypers E, Collins JJP, Kramer BW, et al. Intra-amniotic LPS and antenatal betamethasone: Inflammation and maturation in preterm lamb lungs. Am J Physiol Lung Cell Mol Physiol 2012;302:L380–L389.

4. Uemura K, Shimazutsu K, McClaine RJ, et al. Maternal and preterm fetal sheep responses to dexmedetomidine. Int J Obstet Anesth 2012;21:339–347.

5. Künzel W, Misselwitz B. Unexpected fetal death during preg-nancy — A problem of unrecognized fetal disorders during ante-natal care? Eur J of Obstet Gynecol Reprod Biol 2003;110:S86–S92.

6. National Health and Medical Research Council. Australian code for the care and use of animals for scientific purposes, 8th ed [guidelines on the Internet]. Canberra, Australia: National Health and Medical Research Council c2013. Available from: https://www.nhmrc.gov.au/_files_nhmrc/publications/attachments/ea28_code_care_use_animals_131209.pdf Last accessed February 21, 2017.

7. Davis J, Musk GC. Pressure and volume controlled mechani-cal ventilation in anaesthetized pregnant sheep. Lab Anim 2014;48:321–327.

8. Musk GC, Kemp MW. Maternal and fetal arterial blood gas data during general anaesthesia for caesarean delivery of preterm twin lambs. Lab Anim 2016;50:198–203.

9. Dugdale A. Blood gas analysis. In: Veterinary Anaesthesia: Principles to Practice. Oxford, England: Wiley-Blackwell, 2010: 182–197.

10. Onmaz AC, Gunes V, Atalan G, Gelfert CC, Atalan G. Compari-son of arterial and venous blood gas values in sheep before and during isoflurane anaesthesia. Revue Méd Vét 2009;160:356–361.

11. Fleming JA, Lambert TF, Walker AM. The effects of bupivacaine on the umbilical circulation and placental gas exchange in the fetal lamb. Anesth Analg 1987;66:1121–1126.

12. Mathai S, Booth LC, Davidson JO, et al. Acute on chronic expo-sure to endotoxin in preterm fetal sheep. Am J Physiol Regula Integr Comp Physiol 2013;304:R189–R197.

13. Laffey JG, Kavanagh BP. Hypocapnia. N Engl J Med 2002;347: 43–53.

14. Levinson G, Shnider SM, deLorimier AA, Steffenson JL. Effects of maternal hyperventilation on uterine blood flow and fetal oxy-genation and acid-base status. Anesthesiology 1974;40:340–347.

15. Seeler DC. Fluid, electrolyte, and blood component therapy. In: Tranquilli WJ, Thurmon JC, Grimm KA, eds. Lumb and Jones’ Veterinary Anaesthesia and Analgesia. 4th ed. Ames, Iowa: Blackwell Publishing, 2007:183–201.

16. Eshratkhah B, Sadaghian M, Nezhad MS, Sabri V, Geyglou BF. Evaluation of electrolytes normal values in blood of Moghani sheep breed. J Anim Vet Adv 2008;7:437–440.

17. Hillman NH, Moss TJ, Nitsos I, Jobe AH. Moderate tidal volumes and oxygen exposure during initiation of ventilation in preterm fetal sheep. Pediatr Res 2012;72:593–599.

18. Seehase M, Houthuizen P, Collins JJ, Zimmermann LJ, Kramer BW. Propofol administration to the fetal-maternal unit reduces cardiac oxidative stress in preterm lambs subjected to prenatal asphyxia and cardiac arrest. Pediatr Res 2016;79: 748–753.



Due to global public health concerns, antimicrobial use (AMU) in animals is of significant interest, including international comparisons of AMU. These comparisons have typically been based on total national estimates of antimicrobials sales standardized by the national animal biomass calculated as the population correction unit (PCU). This approach has been criticized in favor of daily defined dose animals metrics (DDDA) which account for drug potency and usage at a species level, if not by animal age or weight (1). However, current and future implementation of DDDA is hampered by its high resource demands (2), including antimicrobial use by species, if not by animal age or weight, and the lack of a global DDDA standard. A European Union standard that has been under development since 2012, has recently been released and addresses poultry, pigs, and cattle while recognizing the need for DDDAs for all food producing species including other ruminants, horses, fish, rabbits, and companion animals (2,3).

In contrast, national estimates of antimicrobial usage standardized using PCU are available for over 25 countries, including Canada, and encompass use in all food-producing species (4). Therefore, for the foreseeable future it is likely PCU will continue to be used in international comparisons of animal AMU, as well as in comparison of usage between species (5).

The purpose of PCU is to control for animal demographics, which can vary over time within a country, and between countries. The PCU is calculated by totalling the number of livestock or poultry in an animal category multiplied by a standardized theoretical weight of an animal in that category at the age it would most likely be treated with antimicrobials, which is called average weights at treatment (AWT) (6).

There are 2 potential problems with the PCU method for calcu-lating animal biomass. Firstly, it is not clear how AWT is estimated

Towards an improved estimate of antimicrobial use in animals: Adjusting the “population correction unit” calculation

Brian R. Radke

A b s t r a c tInternational comparisons of animal antimicrobial use (AMU) have typically been based on total national estimates of antimicrobials sales standardized by the national animal biomass calculated as the population correction unit (PCU). The objective of this paper was to compare the currently accepted PCU calculation with that of the adjusted population correction unit (APCU), which re-evaluates the standard animal weights used in the calculation and accounts for animal lifespan. The APCU calculation resulted in substantial changes to the 2009 national biomass estimates for cattle, pigs, and poultry in 8 European countries and Canada. The estimated national biomass for cattle increased 35% to 43%, while the estimated national biomass of pigs and poultry typically decreased by approximately 51% and 87%, respectively. Among the 9 countries, the total national APCU ranged from an increase of 1% to a decrease of 40% relative to PCU, and these differences were statistically significant. Adjusted population correction unit is preferred over PCU in comparing and contrasting AMU among animals with different lifespans because it is more transparently derived and is a reasonable approximation of the animal biomass at risk of antimicrobial treatment.

R é s u m éLes comparaisons internationales de l’utilisation d’antimicrobiens chez les animaux (UMA) ont typiquement été basées sur les totaux nationaux estimés de ventes d’antimicrobiens standardisés pour la biomasse animale nationale calculée comme l’unité de correction pour la population (UCP). Les objectifs de cet article étaient de comparer les calculs d’UCP présentement acceptés à ceux de l’unité de correction pour la population ajustée (UCPA), qui réévalue les poids animaux standards utilisés dans les calculs et tient compte de la durée de vie des animaux. Les calculs de l’UCPA ont entrainé des changements substantiels aux estimés nationaux de 2009 de la biomasse pour les bovins, porcs et volailles dans 8 pays européens et le Canada. La biomasse nationale estimée pour les bovins a diminué de 35 % à 43 %, alors que les biomasses nationales estimées pour les porcs et les volailles ont typiquement diminué d’environ 51 % et 87 %, respectivement. Parmi les neuf pays, l’UCPA nationale totale variait d’une augmentation de 1 % à une diminution de 40 % relativement à l’UCP, et ces différences étaient statistiquement significatives. L’UCPA est préférée par rapport à l’UCP pour la comparaison et la mise en contraste de l’UMA chez les animaux avec différentes durées de vie étant donné qu’elle est dérivée de manière plus transparente et qu’elle est une approximation raisonnable de la biomasse animale à risque d’un traitement antimicrobien.


Plant & Animal Health Branch, Ministry of Agriculture, British Columbia.

Address all correspondence to Dr. Brian Radke; telephone: (604) 556-3066; fax: (604) 556-3015; e-mail: [email protected]

Dr. Radke’s current address is 1767 Angus Campbell Road, Abbotsford, British Columbia V3G 2M3, Canada.

Received June 15, 2016. Accepted November 14, 2016.


or how it is related to antimicrobial use. Instead, PCU calculations typically reference Monforts (7) and the European Medicines Agency (8), that simply define AWT as the mean body weight for animals raised for slaughter, and the maximum body weight for other animal groups (e.g., breeding animals). Even based on these definitions, some of the currently used AWT values do not appear to accurately reflect animal weights. For example, the average mature cow weighs approximately 600 kg (9–12), while a weight of 425 kg is currently used for PCU calculations.

Antimicrobial use in an animal population is affected by the weight of the animals, and their length of life. The opportunity for antimicrobial use increases with increased length of life, and length of life of PCU’s livestock and poultry categories vary considerably. The PCU doesn’t take into account length of life (4) and this is the second concern with the PCU method of estimating animal biomass. The PCU’s failure to incorporate the variable lifespans of the animal categories has potential implications not only for AMU comparisons between species, but also for comparisons of total national usage. This failure potentially results in underestimation of AMU in coun-tries with a preponderance of short-lived animal categories, such as poultry, and overestimation in countries with disproportionately more longer-lived categories such as cattle.

It is important that PCU calculations accurately reflect animal biomass for the animal categories of interest because inaccurate PCU values may lead to erroneous conclusions when comparing and con-trasting AMU data. The objective of this paper is to compare the cur-rently accepted PCU biomass calculation with one that re-evaluates AWT (based on current data regarding production animal weights) and accounts for the lifespan of the animal categories in question, using data from 8 European countries and Canada.

Currently, a country’s PCU is calculated as follows:

PCU = S ncAWTc 2 S niAWTi 1 S neAWTe (Equation 1) c i e

where nj is the total number of animals in category j (i.e., for breed-ing animals, nj is the number of animals present in a year; if j are slaughter animals, nj is the total number of animals slaughtered annually); AWTj is the average weight at treatment of an animal in j (kg); c is the animal categories raised and slaughtered within the country in question; i is the animal categories imported to the country; and e is the animal categories exported from the country.

The proposed equation, adjusted PCU (APCU), is as follows:

APCU = S ncLAWc 2 S niLAWi 1 S neLAWe (Equation 2) c i e

where LAWj is the life adjusted weight of an animal in category j. Life adjusted weight (LAW) is calculated as:

LAWj = AWjLLj (Equation 3)

The AWj is the adjusted weight of an animal in j (kg) calculated using Monforts’ (7) and the European Medicines Agency’s (8) definitions of the animal weights (i.e., the mean weight for slaughter animal categories and the maximum weight for all other animal catego-ries). The LLj variable is the length of life for category j animals as measured in years.

For the animal categories most commonly included in PCU calcu-lations, established AWT values were obtained from the European Medicines Agency (6) (Table I).

Adjusted weights (AW) were arrived at in several different ways depending on the data available. For the cattle categories, the slaugh-ter (ending) weights were calculated by dividing the average carcass weight by a live-to-carcass weight conversion factor (13) to deter-mine the average weight of live animals. Average carcass weights for 28 EU countries were calculated by dividing the total animal weight at slaughter for a given animal category, by the number of animals slaughtered for that category (14). Subsequently, using Monforts’ definition, the mean body weight of the cattle slaughter categories was calculated by averaging a birth weight of 45 kg and the final weight at slaughter. The AW of imported and exported cattle for slaughter is the mean weight of slaughter heifers, bullocks, and bulls.

For the remaining animal categories, Eurostat data regarding car-cass weights and number of animals slaughtered were not available. For this reason, the international literature was reviewed to provide a contemporary estimate of animal weights in each category. It was determined that these AWT are generally consistent with the (aver-age) body weights defined elsewhere (9–12), including Canadian PCU calculations (Canadian Integrated Program for Antimicrobial Resistance personal communication, 2016). The AWT was therefore used as AW for the non-cattle categories with the exception of pigs imported or exported for fattening. The AWT of these pigs is 25 kg. However, Monforts (7) and the European Medicines Agency (8) include weight of piglets up to 25 kg in the sow weight so the AW for exported fattening pigs was set to zero (Table I). Similarly, the 25 kg AWT for imported fattening pigs was set to zero as this weight is already recognized as the beginning weight in the slaughter pig’s category (Table I).

An animal category’s length of life (LL) was calculated using the inverse of its number of cycles per year on an average farm. For example, if the typical broiler farm has 9 cycles per year, then the average LL for broiler chickens is 0.11 y (1/9). Data regarding the number of cycles per year for each animal category were obtained from Monforts (7) and the European Medicines Agency (8). Neither reference included the number of cycles for slaughter heifers, bull-ocks or bulls. A LL of 1.5 y is assigned to slaughter heifers, bullocks, and bulls based on knowledge of these industries.

Using the most recently published values of n for the 8 European countries (6) and Canadian data (Canadian Integrated Program for Antimicrobial Resistance personal communication, 2016), and AWT (Table I), the 2009 PCU for the 9 countries was reproduced using Equation 1. The APCU for each country was calculated using Equation 2, the same values of n, and LAW from Table I. A 2-tailed paired t-test was used to determine whether total PCU and total APCU were significantly different among countries using Stata v.13.1 (StatCorp, College Station, Texas, USA) and APCU as a percentage change from PCU [i.e., (APCU-PCU)/PCU 3 100%] was calculated.

The 2009 PCU for the 9 countries is reported in Table II, as is the APCU and the percentage change. For cattle, APCU was 35% to 43% greater than PCU for each of the 9 countries, while for pigs, poultry, sheep, and goats, the APCU was consistently less than the PCU. For example, using APCU, the national poultry biomass decreased by 81% to 89%. The estimated national biomass of horses and fish were


the same for APCU and PCU. The estimated national biomass for slaughtered rabbits decreased for France and Canada.

For each country as a whole, the difference between APCU and PCU was variable. For Finland and Norway, APCU was respectively 11% and 1% greater than PCU. For the remaining 7 countries, APCU was between 2% and 40% less than PCU. The APCU and PCU were

statistically significantly different (P = 0.02, t = 3.01, d.f. = 8) for the 9 countries.

Use of the 2 different animal biomass calculations (APCU versus PCU) resulted in substantially different national values for most animal categories included in this analysis, as well as for most of the countries as a whole. These differences could have substantial

Table I. Animal categories, population correction unit (PCU), average weight at treatment, and data used in calculating life adjusted weights

PCU average weight at Adjusted Length Life adjusted treatment weight of life (LL) weight (LAW)Animal category (AWT) (kg) (AW) (kg) (year) (kg year)Cattle Slaughter cows 425a 627 1 627 Slaughter heifers 200b 269 (45, 493)e 1.5 404 Slaughter bullocks and bulls 425a 329 (45, 612)e 1.5 494 Slaughter calves and young cattle 140c 169 (45, 293)e 0.56 94 Imported/exported cattle for slaughter 425a 299j 1.5 449 Imported/exported cattle for fattening 140c 169 (45, 293)e 0.56 94 Livestock dairy cows 425a 627 1 627

Pigs Slaughter pigs 65d (25, 105)e 65 0.33 22 Imported/exported pigs for slaughter 65 65 0.33 22 Imported/exported pigs for fattening 25f 0 Livestock sows 240g 240 1 240

Poultry Slaughter broilers 1 1 0.11 0.11 Slaughter turkeys 6.5 6.5 0.37 2.4 Imported/exported broilers for slaughter 1 1 0.11 0.11

Sheep and goats Slaughter sheep and goats 20 (NAh, 40–45)e 20 0.5 10 Imported/exported sheep and goats for slaughter 20 20 0.5 10 Livestock sheep 75 75 1 75

Horses Living horses 400i 400 1 400

Fish Slaughter fishk

Rabbits Slaughter rabbits 1.4 1.4 0.15 0.21a Adult cow weight.b 0-1-year-old bovine weight.c Veal calf weight.d Fattening pig (25 to 105 kg).e Beginning weight for animal category, ending weight for animal category.f Weaner pig (to 25 kg).g Weight for sow and piglets until 25 kg.h Not available.i Horses 600 kg, ponies 250 kg.j The mean of a slaughter heifer, bullock, and bull weight (i.e., 269 1 329/2).k Eurostat data available only as live-weight at slaughter; information on AWT is unavailable.


effects on international comparisons of AMU as well as national comparisons among animal categories. For example, using PCU as the denominator, 2009 AMU in UK cattle is over 3-fold greater than AMU in pigs and poultry (5). In contrast, 2009 AMU in UK cattle is less than the AMU in pigs and poultry when APCU is used as the denominator (calculations not shown). These differences are primarily attributable to including length of life in the calculation, although for cattle categories and traded fattening pigs, adjusting the weights used in the calculation also had an impact. Given that analyses of AMU rely on accurate estimation of animal biomass to enable comparisons among animal categories and between countries, these results have significant implications on how AMU is calculated.

Amending or replacing the conventional PCU biomass calcula-tions with the APCU calculation presented here should be considered for 2 reasons. First, the APCU uses weight values that are clearly defined and supported by current data regarding animal weights.

For example, cattle AW estimates using Canadian data yielded results similar to the Eurostat data. In the future, AW values could be further improved by collecting international contemporary weight data for all animal categories as is currently collected for cattle. The European Medicines Agency has revised the weight for beef and dairy cows upwards to 500 kg in their DDDA calculations (2), but these weights remain less than those suggested by the Eurostat data (Table I).

Second, although PCU is controlling for animal demographics which vary among countries and includes standardizing for dif-ferences in animal weights, it does not include controlling for differ-ences in animals’ lifespans (4). Bondt et al (1) objected, in principal, to the PCU approach of adding weights of breeding stock to those of animals slaughtered during the year without accounting for length of life because this approach does not accurately reflect the population at risk for antimicrobial treatment. The DDDAs used in

Table II. Calculation of population correction unit (PCU), adjusted population correction unit (APCU), and APCU as a percentage change (% D) from PCU for 8 European countries and Canada, 2009 (1000 tonnes)

Czech Republic Denmark Finland France NetherlandsAnimal category PCU APCU % D PCU APCU % D PCU APCU % D PCU APCU % D PCU APCU % DCattle 308 421 37% 403 566 41% 227 319 41% 3289 4437 35% 1009 1401 39%Slaughtered cows 52 76 80 119 36 53 756 1115 216 319Slaughtered heifers 5 10 9 18 7 14 85 172 3 6Slaughtered bullocks and bulls 47 54 47 55 62 72 500 581 26 30Slaughtered calves and 2 1 19 13 0 0 220 148 208 139 young cattleNet exported cattle for slaughter 30 32 0 0 0 0 18 19 1 1Net exported cattle for fattening 9 6 3 2 0 0 148 99 0 0Net imported cattle for slaughter 0 0 0 0 0 0 0 0 0 0Net imported cattle for fattening 0 0 0 0 0 0 0 0 109 73Livestock dairy cows 163 241 244 360 122 179 1561 2303 664 979Pigs 245 116 253% 1820 768 258% 190 88 254% 1941 836 257% 1484 668 255%Slaughtered pigs 211 70 1255 418 152 51 1619 540 898 299Net imported pigs for slaughter 3 1 0 0 0 0 0 0 0 0Net exported pigs for slaughter 0 0 80 27 0 0 37 12 315 105Net exported pigs for fattening 0 0 162 0 0 0 0 0 7 0Net imported pigs for fattening 9 0 0 0 0 0 0 0 0 0Livestock sows 47 47 323 323 37 37 284 284 264 264Poultry 154 17 289% 112 12 289% 57 8 286% 1179 229 281% 400 44 289%Slaughtered broilers 136 15 100 11 51 6 759 84 481 53Slaughtered turkeys 0 0 0 0 6 2 377 140 0 0Net exported broilers for 18 2 12 1 0 0 43 5 0 0 slaughterNet imported broilers for 0 0 0 0 0 0 0 0 81 9 slaughterSheep and goats 15 15 21% 9 8 211% 8 8 25% 677 621 28% 103 93 210%Slaughtered sheep and goats 0 0 2 1 1 0 105 52 15 8Net exported sheep for slaughter 0 0 0 0 0 0 8 4 6 3Livestock sheep 15 15 7 7 7 7 565 565 82 82Livestock horses 28 28 0% 70 70 0% 29 29 0% 168 168 0% 58 58 0%Live weight fish slaughtered 20 20 0% 34 34 0% 14 14 0% 234 234 0% 56 56 0%Slaughtered rabbits 0 0 0% 0 0 0% 0 0 0% 52 8 285% 0 0 0%Total 771 617 220% 2447 1458 240% 524 465 11% 7540 6533 213% 3109 2321 225%


the Netherlands (15) and Denmark (16) account for animal lifespan. The LL used in the APCU calculations are representative of Canadian production practices.

Population correction unit is a purely technical unit of measure-ment and not a real value for the animal population biomass that could potentially be treated with antimicrobial agents (6). By adjust-ing the animal weights and incorporating length of life, the APCU approach is an improved approximation of the actual animal biomass at risk of antimicrobial treatment.

Using a calculation that better reflects average weights and includes length of life for each animal category resulted in values for total annual animal biomass that were significantly different than those obtained using a traditional PCU calculation. As a result, APCU provides a reasonable approximation of the actual animal biomass at risk of antimicrobial treatment. Consideration should be given to replacing PCU with APCU in AMU calculations comparing

and contrasting AMU among animals with different lifespans. The methodology used to transparently derive APCU will increase the credibility of this measure of animal biomass, improve comparisons of AMU data among animal categories and countries, and foster increased acceptance and harmonization of AMU calculations.

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Table II. (continued)

Norway Sweden United Kingdom CanadaAnimal category PCU APCU % D PCU APCU % D PCU APCU % D PCU APCU % DCattle 231 315 36% 331 456 38% 1678 2395 43% 3925 5490 40%Slaughtered cows 51 75 65 95 204 301 276 407Slaughtered heifers 0 0 11 21 156 315 215 434Slaughtered bullocks and bulls 76 89 96 111 537 623 703 816Slaughtered calves and 2 1 10 6 6 4 41 28 young cattleNet exported cattle for slaughter 0 0 0 0 0 0 324 342Net exported cattle for fattening 0 0 0 0 0 0 40 27Net imported cattle for slaughter 0 0 0 0 14 15 0 0Net imported cattle for fattening 0 0 0 0 3 2 26 24Livestock dairy cows 102 150 150 222 792 1169 2332 3440Pigs 32 27 216% 231 103 256% 674 306 255% 1874 793 258%Slaughtered pigs 8 3 192 64 587 196 1352 451Net imported pigs for slaughter 0 0 0 0 32 11 0 0Net exported pigs for slaughter 0 0 1 0 0 0 78 26Net exported pigs for fattening 0 0 0 0 0 0 128 0Net imported pigs for fattening 0 0 0 0 2 0 0 0Livestock sows 24 24 38 38 121 121 316 316Poultry 71 8 289% 76 9 288% 942 131 286% 718 116 284%Slaughtered broilers 71 8 73 8 839 93 620 69Slaughtered turkeys 0 0 3 1 101 37 140 52Net exported broilers for 0 0 0 0 2 0 12 1 slaughterNet imported broilers for 0 0 0 0 0 0 254 26 slaughterSheep and goats 92 80 213% 46 43 26% 1915 1758 28% 56 49 213%Slaughtered sheep and goats 24 12 5 3 308 154 16 8Net exported sheep for slaughter 0 0 0 0 6 3 21 0Livestock sheep 68 68 41 41 1601 1601 41 41Livestock horses 14 14 0% 141 141 0% 520 520 0% 417 417 0%Live weight fish slaughtered 0 0 0% 0 0 0% 197 197 0% 142 142 0%Slaughtered rabbits 0 0 0% 0 0 0% 0 0 0% 1 0 2100%Total 440 443 1% 825 752 29% 5925 5307 210% 7133 7007 22%


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11. DANMAP 2011 — Use of antimicrobial agents and occurrence of antimicrobial resistance in bacteria from food animals, food and humans in Denmark. ISSN 1600-2032. Available from: http://www.danmap.org/Downloads/~/media/Projekt%20sites/Danmap/DANMAP%20reports/Danmap_2011.ashx Last accessed February 16, 2017.

12. Radke BR. Use of Over-the-Counter Antibiotics in BC Livestock and Poultry, 2002–2012. 2014. Available from: http://www.agf.gov.bc.ca/lhmr/pubs/otcu_amu.pdf Last accessed February 16, 2017.

13. Agriculture and Agri-Food Canada 2013. Red meat sector con-version factors. Available from: http://www.agr.gc.ca/eng/industry-markets-and-trade/statistics-and-market-information/by-product-sector/red-meat-and-livestock/red-meat-market-information-canadian-industry/carcass-weight/conversion-factors/?id=1415860000020 Last accessed February 16, 2017.

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