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Rapid Detection of Mycoplasma synoviae and Avian Reovirus in Clinical Samplesof Poultry Using Multiplex PCRAuthor(s): Carolina Reck, Álvaro Menin, Marina Feltrin Canever, and Luiz Claudio MilettiSource: Avian Diseases, 57(2):220-224. 2013.Published By: American Association of Avian PathologistsDOI: http://dx.doi.org/10.1637/10425-101712-Reg.1URL: http://www.bioone.org/doi/full/10.1637/10425-101712-Reg.1

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Rapid Detection of Mycoplasma synoviae and Avian Reovirus in Clinical Samples ofPoultry Using Multiplex PCR

Carolina Reck,AB Alvaro Menin,BC Marina Feltrin Canever,A and Luiz Claudio MilettiAD

ADepartamento Producao Animal e Alimentos, Universidade do Estado de Santa Catarina-CAV/UDESC, Lages, SC, Brazil, 88520-000BInstituto de Pesquisa e Diagnostico Veterinario–IPDVET, Curitibanos, SC, Brazil, 89520-200

CDepartamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de Santa Catarina, Florianopolis, SC, Brazil, 88040-970

Received 21 October 2012; Accepted 23 January 2013; Published ahead of print 25 January 2013

SUMMARY. Mycoplasma synoviae and avian reovirus (ARV) are associated with several disease syndromes in poultry and causenotable global economic losses in the poultry industry. Rapid and efficient diagnostics for these avian pathogens are important notonly for disease control but also for prevention of clinical disease progression. However, current diagnostic methods used forsurveillance of these diseases in poultry flocks are laborious and time-consuming, and they have low sensitivity. The multiplex PCR(mPCR) developed in this study has been proven to be both sensitive and specific for simultaneous M. synoviae and ARV detectionand identification in clinical samples. To evaluate the mPCR assay, the diagnostic test was applied to different clinical samples fromnatural and experimental M. synoviae and ARV-infected poultry. Results were compared with serologic, single PCR, andimmunofluorescence analyses. Tibiotarsal articulation could be the best target for simultaneous detection of M. synoviae and ARVinfection. The detection limit by visualization of mPCR-amplified products was 100 pg for both pathogens. Overall, the mPCRdeveloped and standardized in this research is a useful tool for diagnosis and screening and for surveillance and control of M.synoviae and ARV infection in poultry flocks.

RESUMEN. Deteccion rapida de Mycoplasma synoviae y reovirus aviar mediante PCR multiple en muestras clınicas avıcolas.Mycoplasma synoviae y el reovirus aviar (ARV) estan asociados con varios sındromes de enfermedades en las aves y causan

importantes perdidas economicas mundiales en la industria avıcola. El diagnostico rapido y eficaz para estos patogenos aviares esimportante no solo para el control de las enfermedades, sino tambien para la prevencion de la progresion de la enfermedad clınica.Sin embargo, los metodos actuales de diagnostico utilizados para la vigilancia de estas enfermedades avıcolas son laboriosos,consumen mucho tiempo, y tienen baja sensibilidad. El PCR multiple (mPCR) desarrollado en este estudio ha demostrado ser massensible y especıfico para la deteccion e identificacion simultanea de M. synoviae y reovirus en muestras clınicas. Para evaluar elensayo de PCR multiple, la prueba de diagnostico se aplico en diferentes muestras clınicas de aves comerciales infectadas de maneranatural y experimental con M. synoviae y con reovirus. Los resultados se compararon con analisis serologicos, con PCR simples, ycon analisis de inmunofluorescencia. La articulacion tibiotarsal podrıa ser la mejor muestra para la deteccion simultanea de lainfeccion por M. synoviae y reovirus. El lımite de deteccion por visualizacion de los productos amplificados por PCR multiple fue de100 pg para ambos patogenos. En general, el metodo de PCR multiple desarrollado y estandarizado en esta investigacion es unaherramienta util para el diagnostico, la deteccion, la vigilancia y el control de la infeccion por M. synoviae y reovirus en las avescomerciales.

Key words: multiplex polymerase chain reaction, Mycoplasma synoviae, avian reovirus, poultry disease

Abbreviations: ARV 5 avian reovirus; IBDV 5 infectious bursal disease virus; IBV 5 infectious bronchitis virus; IF 5 immuno-fluorescence; Ig 5 immunoglobulin; mPCR 5 multiplex PCR

Mycoplasma synoviae and avian reovirus (ARV) are importantavian pathogens for poultry and present several disease syndromesthat cause large economic losses for the poultry industry (25,30).Mycoplasma synoviae is responsible for infectious synovitis, arthritis,airsacculitis, bursitis, and septicemia in chickens (18,19,30). ARV isan RNA virus in the family Reoviridae, and it is associated with avariety of diseases and conditions in chickens and turkeys, includingenteric diseases, chronic respiratory diseases, myocarditis, hepatitis,arthritis/tenosynovitis, and malabsorption syndrome (4,23,25).Mixed infections with M. synoviae and ARV have been known tooccur in poultry flocks worldwide, and they also have been associatedwith severe immunosuppression (27), depression, retarded growth,weight loss, decrease in egg production, and particularly theelimination of lesioned carcasses at the slaughterhouse (15,21,22).These findings suggest simultaneous M. synoviae and ARV infec-tion has a synergistic effect upon clinical symptoms and pathologicchanges (14). In this context, efficient diagnostic testing is essential

for the surveillance and control of these avian pathogens in poultryflocks.

Rapid and early diagnostic detection of M. synoviae and ARV isimportant to prevent spread of infection and to limit economiclosses to the poultry industry. Serologic testing and isolation aremethods used for the diagnosis of M. synoviae and ARV (5,29);however, these methods are laborious and time-consuming, and theyhave low sensitivity. Moreover, serologic analysis is often plaguedby nonspecific reactions and problems with reagent cross-reaction(5,29,31). Although, single PCR also has been used to detect theseavian pathogens, the technique only allows for the detection ofnucleic acids from one specific pathogen per reaction (8,9,31). Theselimitations can be overcome by using a multiplex PCR (mPCR)assay that incorporates multiple primers to amplify molecular targetsfrom different avian pathogens simultaneously in one reaction(7,20). The benefits of mPCR include cost effectiveness, timeefficiency, and potential for use in screening and surveillance ofpathogens in commercial poultry flocks (2,10). In this context, themain objective of this research was to develop and optimize anmPCR assay that would allow simultaneous detection andDCorresponding author. E-mail: miletti@cav.udesc.br

AVIAN DISEASES 57:220–224, 2013

220

differentiation of M. synoviae and ARV in one reaction. Our resultsprovide insight on the use of mPCR for detection as well assurveillance of two important avian pathogens in commercial poultryflocks.

MATERIALS AND METHODS

Field samples and experimental infection. For this study, 212 (42-day-old) broiler chickens with lesions of infectious arthritis in tibiotarsalarticulations were obtained from 21 commercial flocks from the state ofSanta Catarina, Brazil. During slaughter, 10–12 tibiotarsal joints witharthritic injury were collected from each flock. Broiler chickens were notvaccinated for M. synoviae, ARV, or both and were not previouslyidentified for infection by these pathogens. Tissue samples includingsynovial membrane and digital flexor tendons were collected in theslaughterhouse and conserved in liquid nitrogen (2196 C) for nucleicacid extraction. Samples were embedded in Tissue-TekH OCTTM

compound (Sakura Finetek USA, Torrance, CA) for immunofluores-cence (IF) analysis and formalin fixed for histopathologic examination.

For experimental infection with M. synoviae and ARV, we used 16female broiler chicks with 10-day-old (lineage Cobb). The animalswere maintained in the experimental station of the Centro de CienciasAgroveterinarias/Universidade do Estado de Santa Catarina (CAV/UDESC), Lages, Brazil, under standard conditions and in accordancewith guidelines established by the current laws of animal protection inBrazil (3). All procedures were evaluated previously and approved by theEthics Committee on Animal Experiments, CAV/UDESC, underprotocol 1.31.11.

At 15 days old, chicks were simultaneously inoculated with 8 3 105

colony-forming units/ml M. synoviae, MS-H strain (Merial, Campinas,Brazil) through aerosol route and footpad injection, and a suspension ofARV S1133 strain (Biovet, Vargem Grande Paulista, Brazil) containing104.8 median tissue culture infective dose/0.05 ml, through oral routeand footpad injection. Thirty days after infection, euthanasia andnecropsy of the poultry were performed. Trachea, lung, air sacs, liver,spleen, tibiotarsal articulation, and blood samples were collected andconserved in liquid nitrogen (2196 C) for nucleic acid extraction,embedded in Tissue-Tek OCT compound (Sakura Finetek USA) for IFanalysis, and formalin fixed for histopathologic examination. Experi-mentally infected chickens were tested before and after inoculation.Serologic analyses to detect antibodies against M. synoviae and ARV wereperformed by ELISA using the IDEXX MS Ab TestH and IDEXX REOAb TestH (IDEXX Laboratories, Westbrook, ME), following manufac-turer’s instructions.

Nucleic acid extraction. The nucleic acid extraction protocol wasadopted for the simultaneous extraction of total DNA (M. synoviae) andRNA (ARV). The procedures were carried out according to the TRIzolH(Invitrogen, Carlsbad, CA) manufacturer’s protocol and as describedpreviously (11,28). In brief, after homogenization with TRIzol andaddition of chloroform, the tissue sample was centrifuged to separate thephases. The mixture separated into a lower, red, phenol-chloroformphase (DNA); an interphase; and a colorless, upper aqueous phase(RNA). RNA was precipitated by adding isopropyl alcohol, and thenDNA was precipitated by the addition of ethanol. Positive controls usedwere pure cultures of M. synoviae MS-H strain (Merial) and ARV S1133strain (Biovet).

Reverse transcription reaction and cDNA synthesis. The totalRNA (ARV) extracted was used for the reverse transcription-PCR andpreparation of the first-strand complementary DNA synthesis using

ProtoScriptH M-MuLV First Strand cDNA Synthesis kit (New EnglandBiolabs, Ipswich, MA), following the manufacturer’s instructions. Inbrief, the first-strand cDNA synthesis was initially performed in avolume of 8 ml, in which the reaction mixture contained 6 ml of viralRNA (100 ng) and 2 ml of random primer mix. RNA was denatured for5 min at 70 C. Next, 10 ml of Moloney murine leukemia virus (M-MuLV) reaction mix and 2 ml of M-MuLV enzyme mix were added tothe solution, for a final volume of 20 ml. Reverse transcription wascarried out in the thermal cycler for one cycle at 25 C for 5 min and at42 C for 60 min. The enzyme was then inactivated at 80 C for 5 min.Reaction was diluted to 50 ml with 30 ml of sterile deionized water. ThecDNA product was stored at 220 C.

Single PCR for M. synoviae and ARV detection. Single PCR for M.synoviae and ARV was performed to detect single avian pathogen in eachreaction. Oligonucleotide primer sets that specifically amplify thetarget sequence of the S1 gene from ARV (31) and MS-16S rRNAsequence from M. synoviae (17) are described in Table 1. All sets ofoligonucleotide primers were synthesized by Invitrogen. The addition ofboth extracted DNA and synthesized cDNA provided templates for theamplification of M. synoviae and ARV, respectively. The conditions forsingle PCR for M. synoviae and ARV detection were performed asdescribed previously (17,31).

mPCR for simultaneous detections M. synoviae and ARV. In themPCR assay, M. synoviae and ARV were detected using oligonucleotideprimers (Table 1) described previously (17,31). Detection involvedoptimizing several PCR parameters, including testing different primersconcentrations, concentrations of MgCl2, and annealing temperatures toachieve optimum conditions for the simultaneous amplification of targetgenomic sequences from M. synoviae and ARV.

The mPCR reactions were performed in a 20-ml volume. ExtractedDNA and synthesized cDNA were mixed in equal proportions of each(100 ng), thereby providing the template for M. synoviae and ARVdetection, respectively.

The mPCR reactions were carried out using optimized MgCl2, 103PCR buffer II (500 mM KCl and 100 mM Tris HCl, pH 8.3;Invitrogen), 200 mM of each dNTP (Invitrogen), 20 pmol of ARVprimers MK87R and MK88F, 20 pmol of M. synoviae primers MSR andMSF (Invitrogen), and 5 units of Taq DNA polymerase (Invitrogen).Various concentrations of MgCl2 (2.5, 3.0, 3.5, and 4.0 mM) weretested as well. All reactions were carried out using a gradient thermalcycler (Axygen, Inc., Union City, CA). The cycling protocol consistedof an initial denaturation at 94 C for 5 min and then 35 cyclesof denaturation at 94 C for 1 min, annealing for 1 min; varioustemperatures were tested (48.5, 49.8, 51.5, 52.6, and 54.0 C) andextension was at 72 C for 1 min. The sample was then heated at 72 C for10 min for a final extension. Each mPCR run included positive andnegative controls for PCR reagents and sample extractions. The negativecontrol did not contain template DNA and consisted of PCR mastermix, all four sets of primers, and deionized water.

The mPCR amplification products were analyzed by electrophoresisin 1.5% (w/v) agarose gel and TBE buffer (89 mM Tris, 89 mM borate,and 2 mM EDTA). Ten-microliter volumes of single PCR and mPCRproducts were separated in agarose horizontal gel by electrophoresisat 80 V. A 100-bp DNA ladder was used as the molecular-size marker.Gels were stained with ethidium bromide solution (0.5 mg/ml) for10 min, visualized by ultraviolet light, and photographed on a MiniBISProH digital transilluminator (BioAmerica Biotech, Miami, FL).

mPCR sensitivity and specificity. Sensitivity of the mPCR wasdetermined by performing 10-fold serial dilutions (100 ng) of the DNAextracted from M. synoviae (MS-H strain) and RNA extracted from ARV

Table 1. Oligonucleotide primer sequence used in single PCR and multiplex polymerase chain reactions.

Avian pathogen Primer Sequence(59-39) Amplicon size (bp) Reference

ARV MK87F 59-GGTGCGACTGCTGTATTTGGTAAC-39 (location 55–77) 532 25MK88R 59-AATGGAACGATAGCGTGTGGG-39 (location 568–587)

M. synoviae MSF 59-GAAGCAAAATAGTGATATCA-39 (location 1194–1213) 207 12MSR 59-GTCGTCTCCGAAGTTAACAA-39 (location 1381–1400)

Mycoplasma synoviae and avian reovirus using multiplex PCR 221

(S1133 strain). The different extracted ARV RNA dilutions were usedfor cDNA synthesis as described previously (2,12). Extracted DNA andcDNA of each dilution were used as the template for mPCR. The DNAand RNA were quantified spectrophotometrically (BioPhotometerH,Eppendorf AG, Hamburg, Germany).

Determination of the mPCR specificity was carried out by examiningthe ability of the test to detect and differentiate only M. synoviae andARV. The mPCR was tested against other agents found in broilers, suchas Mycoplasma gallisepticum, Escherichia coli, Pasteurella multocida,Staphylococcus aureus, Enterococcus faecalis, infectious bursal disease virus(IBDV), and infectious bronchitis virus (IBV). In addition, this assaywas used to test articulations of 15 uninfected chickens to examine thespecificity. The age of the healthy chickens varied from 35 to 47 days.

For further confirmation, amplified DNA bands were excised andpurified using QIAquick Gel Extraction (QIAGEN, Valencia, CA).Sequencing of mPCR amplicons that amplified 207- and 532-bpproducts of the 16S rRNA M. synoviae gene and ARV S1 gene,respectively, were cloned in a pGEMH-T Easy vector (Promega,Madison, WI) following the manufacturer’s instructions. CompetentDH5a E. coli cells were used for chemical transformation. Transformedclones were selected in Luria-Bertani medium supplemented withampicillin (100 mg/ml). Confirmation of the insert presence and size wasperformed by direct PCR amplification from the bacterial colony usingprimers specific for the pGEM-T Easy vector (pGEM-F and EXCEL-R). Colonies positive by PCR for insert presence were selected for

sequencing. Sequencing of clones was carried out using MegaBaceH1000 DNA Analysis System (GE Healthcare, Little Chalfont, U.K.).The sequencing reactions were prepared using the DYEnamic ET DyeTerminatorH kit (GE Healthcare), following the manufacturer’sguidelines. The sequences were analyzed and compared with thosefrom the international gene bank at the National Center forBiotechnology Information (Bethesda, MD) using the BLAST program.

Immunofluorescence assay and histopathologic analysis. IF assayswere performed on tissues (articular capsule/synovial membrane,tendon, lung, and liver) embedded in Tissue-Tek OCT compound.Four-micrometer sections were fixed with cold acetone in slidespretreated with poly-L-lysine (QIAGEN). The blocking of tissuesections was performed using skim milk solution (1%) for 60 min.Then, the sections were rinsed with phosphate-buffered saline (PBS) andincubated overnight at 4 C with the primary antibodies mouse anti-ARVantibody (Universidade Federal de Santa Catarina, Florianopolis, SC,Brazil) or rabbit anti-M. synoviae antibody (Bcell, RS, Brazil), diluted1:200 in PBS. Sections were rinsed with PBS and then incubated for60 min with the secondary antibody AlexaFluorH 488 anti-rabbitimmunoglobulin (Ig)G (Invitrogen) or fluorescein isothiocyanate anti-mouse IgG (2 mg/ml; Invitrogen), diluted 1:400 in PBS. Aftermounting, slides were analyzed in a fluorescence microscope (Axioplan;Carl Zeiss, Jena, Germany) and photographed with aDP72H/chassrge-coupled device system (Olympus America, Center Valley, PA).

For the histopathologic analysis, tissues samples were fixed in 10%neutral buffered formalin and dehydrated in ethanol solutions. Afterdehydration, samples were paraffin embedded, sectioned (4 mm), andstained by hematoxylin and eosin. The histologic lesions suggestive ofM. synoviae and ARV were defined as described previously (1,19,23).

RESULTS

The optimization of mPCR parameters, including annealingtemperature, extension time, cycle quantity, and primer concentra-tions, was performed to achieve efficient conditions for simultaneousM. synoviae and ARV detection. Optimal mPCR conditions fordetection of these two important avian pathogens were observedwhen using the concentration of 3.5 mM MgCl2 and a primerannealing temperature of 52.6 C (Fig. 1).

Detection by visualization of PCR-amplified DNA products was100 pg for M. synoviae and 100 pg of RNA used for cDNA synthesisfor ARV (Fig. 2). The primer specificities were confirmed bysequencing the mPCR products. Sequencing analysis demonstratedthe sequences of mPCR amplicons were identical to the sequences ofrespective ARV (MK87R/MK88F; 100%) and M. synoviae (MSR/MSF; 100%) templates.

The mPCR was found to be specific assay for M. synoviae andARV, with no amplification of nucleic acids from M. gallisepticum,

Fig. 1. Optimization of mPCR assay developed for the detectionof M. synoviae and ARV. (A) Agarose gel showing simultaneousmPCR amplification of target M. synoviae/ARV with different annealingtemperatures. Lane M, 100-bp DNA ladder; lane 1, only ARV; lane 2,only M. synoviae; lanes 3–7, different annealing temperatures mPCR:lane 3, 51.5 C; lane 4, 49.8 C; lane 5, 52.6 C; lane 6, 48.5 C; lane 7,54 C. (B) mPCR assay optimized with 52.6 C temperature of annealing.Lane M, 100-bp DNA ladder; lane 1, only M. synoviae; lane 2, onlyARV; lanes 3 and 4, M. synoviae + ARV.

Fig. 2. Sensitivity of mPCR. Detection limit of mPCR for M.synoviae and ARV to different concentrations of DNA and RNA (usedfor cDNA synthesis). Lane M, 100-bp DNA ladder; lane 1, only M.synoviae (100 ng); lane 2, only ARV (100 ng); lane 3, 100 ng; lane 4,50 ng; lane 5, 10 ng; lane 6, 1 ng; lane 7, 0.5 ng; lane 8, 100 pg; lane 9,10 pg; lane 10, negative control (agarose gel).

222 C. Reck et al.

E. coli, P. multocida, S. aureus, E. faecalis, IBDV, and IBV. Inaddition, mPCR analysis of 212 lesions of arthritis from broilerchickens (Fig. 3) showed positive amplification for M. synoviae in58.01% (131/212) of samples, ARV in 25.47% (54/212) of samples,and both M. synoviae and ARV in 18.86% (40/212) of samples(Table 2).

Histopathologic analyses of broilers field samples showedsuggestive lesions of M. synoviae, ARV infection, or both in76.88% (163/212) of samples. The broiler chickens presentedprimarily diffuse lymphohistiocytic inflammatory infiltrate withaccumulation of heterophils in the synovial capsule and digital flexortendon. Furthermore, we observed hyperplasia and hypertrophy ofsynovial cells.

DISCUSSION

The mPCR developed in this research was efficient in thesimultaneous detection of M. synoviae and ARV infection in tissuesamples from naturally and experimentally infected broiler chickens.These important avian pathogens cause chronic infectious diseaseand are transmitted rapidly among broiler chickens or poultry flocks(4,8,13). Despite the importance of these avian pathogens,systematic data on infection in Brazilian commercial flocks arescarce. Therefore, rapid diagnosis of these pathogens is an importanttool for surveillance programs and for control of the spread ofinfection.

Molecular biology has been used widely for the detection ofvarious infectious agents (9), including several avian pathogens(2,10). The optimization of mPCR involves important steps such astesting the best annealing temperature, determining the optimalconcentration of MgCl2 and Taq DNA polymerases, establishingefficient methods for DNA and RNA extraction, calibratingequipment (20). Low annealing temperature cycles can lead tononspecific amplification, and high annealing temperatures canreduce primer pairing and the PCR amplification yield (6,26).

Diagnosis of ARV is dependent on virus detection; however, thepresence of the virus cannot necessarily be confirmed as the cause ofthe disease, except when ARV is detected in the affected joint tissue(13). In addition, in arthritis/synovitis cases caused by M. synoviaeinfection, high bacterial load can be observed within the joint tissueaffected (22). Similarly, these studies have shown tibiotarsalarticulation can be a major target for simultaneous detection bymPCR of M. synoviae and ARV infection in broiler chickens. Thedifference between the M. synoviae/ARV (mPCR/IF) detection data

Fig. 3. Clinical application of the mPCR for the simultaneousdetection of M. synoviae and ARV. Lane M, 100-bp DNA ladder; lanes1–4, field samples positive for M. synoviae and ARV; lanes 5, 7, and 8,field samples positive for M. synoviae; lane 6, field samples positive forARV (50-ng concentration of DNA and cDNA template, agarose gel).

Tab

le2.

Det

ecti

onfr

equ

ency

ofM

.sy

novi

aean

dA

RV

bysi

ngl

eP

CR

,m

PC

R,

IF,

EL

ISA

,an

dh

isto

pat

hol

ogy

inex

per

imen

tally

and

nat

ura

llyin

fect

edcl

inic

alsa

mp

les.

Cli

nic

alsa

mp

le

Sin

gle

PC

Rp

osit

ive,

%(n

o./t

otal

)m

PC

Rp

osit

ive,

%(n

o./t

otal

)IF

pos

itiv

e,%

(no.

/tot

al)

Sero

logy

(EL

ISA

)H

isto

pat

hol

ogic

anal

ysis

AR

VM

SAA

RV

MS

AR

VM

SA

RV

MS

Exp

erim

enta

llyin

fect

edbr

oile

rsch

icke

ns

(n5

16)

Tib

iota

rsal

arti

cula

tion

100

(16/

16)

100

(16/

16)

100

(16/

16)

100

(16/

16)

100

(16/

16)

100

(16/

16)

NA

BN

A10

0(1

6/16

)L

un

g/ai

rsac

s0

100

(16/

16)

010

0(1

6/16

)0

100

(16/

16)

NA

NA

100

(16/

16)

Liv

er10

0(1

6/16

)0

100

(16/

16)

010

0(1

6/16

)0

NA

NA

100

(16/

16)

Seru

mN

TC

NT

NT

NT

NT

NT

100

(16/

16)

100

(16/

16)

NT

Fie

ldsa

mp

les

(n5

212)

Tib

iota

rsal

arti

cula

tion

25.4

7(5

4/21

2)58

.01

(131

/212

)25

.47

(54/

212)

58.0

1(1

31/2

12)

19.3

3(4

1/21

2)51

.41

(109

/212

)N

AN

A76

.88

(163

/212

)Se

rum

NT

NT

NT

NT

NT

NT

NT

NT

NT

AM

S5

M.

syno

viae

.BN

A5

not

app

lica

ble.

CN

T5

not

test

ed.

Mycoplasma synoviae and avian reovirus using multiplex PCR 223

and histopathologic analysis possibly is due to infection by M.synoviae, ARV, or both not having a lesional pathognomonicstandard (13,24), and lesions also can be associated with otherinfectious agents (13,16).

The mPCR developed in this research has been proven to besensitive and specific for detection and able to identify M. synoviaeand ARV simultaneously in one reaction. Nevertheless, thesensitivity of mPCR assay depends on reaction condition optimi-zation, purity of DNA and RNA, DNase and RNase activity, andthe presence of inhibitors (20). This mPCR is useful for diagnosis,screening, and epidemiologic studies that require fast processing withlarge numbers of samples, as well as for surveillance of poultry flocksfor early detection of pathogens and avoidance of economic losses.

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ACKNOWLEDGMENTS

The authors are indebted to Dr. Edmundo C. Grisard Lab ofUniversidade Federal de Santa Catarina for sequencing. This study wasfunded by Fundacao de Amparo a Pesquisa e Inovacao do Estado deSanta Catarina (FAPESC) and Coordenacao de Aperfeicoamento dePessoal de Nıvel Superior (CAPES).

224 C. Reck et al.