research article relationship between leishmania...

Research ArticleRelationship between Leishmania IFAT Titer andClinicopathological Manifestations (Clinical Score) in Dogs

Daniela Proverbio Eva Spada Giada Bagnagatti de GiorgiRoberta Perego and Emanuela Valena

Dipartimento di Scienze Veterinarie per la Salute la Produzione Animale e la Sicurezza Alimentare (VESPA)Universita degli Studi di Milano Via Celoria 10 20133 Milano Italy

Correspondence should be addressed to Daniela Proverbio danielaproverbiounimiit

Received 28 February 2014 Revised 12 May 2014 Accepted 12 May 2014 Published 3 June 2014

Academic Editor Xin Yao

Copyright copy 2014 Daniela Proverbio et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

During canine leishmaniasis (CanL) due to Leishmania infantum high levels of antibodies production are associated withthe presence of various clinical signs because of the deposition of soluble immune complexes in organs and tissues Theimmunofluorescence antibody test (IFAT) is one of the most commonly used techniques for detection of anti-Leishmaniaantibodies The purpose of this study was to assess whether there is a correlation between clinical signs and IFAT titers in dogsnaturally infected with Leishmania A retrospective study was performed on medical records of 49 dogs diagnosed with CanLInformation extracted from the medical records of each dog with CanL was clinical score IFAT titer serum total protein (TP)gamma globulin (IgG) and creatinine concentration and protein creatinine ratio in urine sample (UPUC) at each follow-upexamination Results show that dogs with highest IFAT titers recorded had higher mean clinical scores indicating a positiverelationship (119875 lt 00001) between anti-Leishmania antibodies (IgG) and clinical manifestations which becomes more evident insevere clinical forms of canine leishmaniasis Higher TP and IgG serum concentrations were recorded in dogs with higher clinicalscores Significant association was observed between UPUC and the IFAT titer (119875 = 0004)

1 Introduction

Leishmaniasis due to Leishmania spp infection has a widedistribution in four continents and affects mainly dogshumans and rodents Dogs are the main reservoir forzoonotic human visceral infection caused by L infantumparasite and play a pivotal role in the transmission of thedisease via phlebotomine sandflies (Phlebotomus spp andLutzomyia spp in the Old and New World) to people [1]Canine leishmaniasis (CanL) due to L infantum is a life-threatening disease which may be fatal before treatmentcan be instigated [1] Clinical presentations of CanL rangefrom subclinicalasymptomatic to full-blown disease withvariable laboratory findings depending on the hostrsquos immuneresponse [2] When the immune response is mediated byTh2 lymphocytes IL-4 secretors antibody production is highand this is associated with severe clinical manifestations [3]

Many studies [4 5] report that high levels of antibodiesproduction are associated with the presence of clinical signssuch as epistaxis proteinuria polyuria polydipsia uveitisand skin ulcers because of the deposition of soluble immunecomplexes in organs and tissues [3 6] Several authors [57 8] evaluated the profile of anti-Leishmania antibodies indogs with different clinical forms of visceral leishmaniasisusing methods not easily obtainable in clinical practiceThe immunofluorescence antibody test (IFAT) is one of themost commonly used techniques [9] for detection of anti-Leishmania antibodies and is recommended by World Orga-nization for Animal Health (OIE) as the reference serologicalmethod [10] High antibody levels are associated with highlevels of parasitism [2] and provide a definitive diagnosis ofCanL [9]

The purpose of this study was to assess whether there isa correlation between clinical signs and IFAT titers in dogs

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 412808 5 pageshttpdxdoiorg1011552014412808

2 BioMed Research International

naturally infected with L infantum to determine whetherIFAT titers could be used as a predictor of the severity ofclinical disease

2 Materials and Methods

A retrospective study was performed The medical recordsof 131 dogs diagnosed with CanL that were presented to theTeaching Animal Hospital of the Department of Health Ani-mal Science and Food Safety of University of Milan between2008 and 2013 were reviewedThe following inclusion criteriawere used clinical diagnosis of CanL confirmed by positiveserology for Leishmania infantumusing IFAT and cytologicalidentification of Leishmania amastigotes or detection of para-site DNA using polymerase chain reaction (PCR) IgG anti-Linfantum antibodies were measured by IFAT according to therecommendations of OIE [10] using MHOMIT80IPT1 as awhole-parasite antigen fixed onmultispot slides (BioMerieuxSpa Florence Italy) and fluorescently labeled anticaninegamma globulin (Sigma Aldrich Milan Italy) as conjugatePositive sera were serially diluted and tested to establish themaximumreaction titer starting at a dilution of 1 40 Positiveand negative controls were included on each slide

The real-time PCR analysis of whole blood or samplesfrom lymph node aspiration was performed using the IllustraBlood genomicPrep Mini Spin kit (GE Healthcare MilanItaly) following the manufacturerrsquos instructions The targetfor amplification was a 116-bp fragment in the constantregion of the kDNA minicircle of L infantum This is oneof the kDNA minicircle families that are used to identifythe Leishmania genus The primers used were QLK2-UP51015840-GGCGTTCTGCGAAAACCG-31015840 and QLK2-DOWN 51015840-AAAATGGCATTTTCGGGCC-31015840 the TaqMan probes wereQ Leish Probe 2 and 51015840-FAM TGGGTGCAGAAATCCCGT-TCA-31015840-Black Hole

At the moment of the diagnosis and at each follow-up a complete physical examination was performed onall dogs after which clinical assessment of the severity ofsigns attributable to Leishmania infection (scored on a scalefrom 0 to 3) was made (Table 1) During each follow-up acomplete blood count hematological and serum biochemicalexaminations (including the determination of total proteinand serum electrophoretic pattern) and urine examinationwere performed

All dogs in which concomitant infectious diseases (egbabesiosis ehrlichiosis and dirofilariasis) were diagnosedby parasitological orand serological examinations wereexcluded

21 Medical Records Review Information extracted from themedical records of each dog at each follow-up examinationwith CanL was clinical score IFAT titer serum total protein(TP) gamma globulin (IgG) and creatinine concentrationand protein creatinine ratio in urine sample (UPUC)

A total of 49131 dogs (374)met the criteria for inclusionin the study Ages of the dogs ranged from 07 to 14 yearswith a median age of 6 years Twenty-eight were intact malesand 21 were females (10 neutered) 21 were X-breeds and 28

were purebreds A mean of 139 (min 2-max 18) follow-upexaminations was done for each dog

To assess the relationship between the IFAT titer andthe parameters considered that is clinical score serumtotal protein IgG and creatinine concentration and UPUCthe recorded values from each follow-up examination weredivided into 9 groups according to the IFATvalue (1 40 1 801 160 1 320 1 640 1 1240 1 2560 1 5120 and 1 10240)regardless of the subject to which they belonged

22 Statistical Analysis Mean standard deviation medianof clinical score value (CS) TP IgG creatinine (Cr) andUPUC were calculated after calculating normal distributionof parametric data using the DrsquoAgostino-Pearson test Mann-Whitney was used for independent samples tests to assessany statistically significant difference in the mean of clinicalscores and PT IgG creatinine and UPUC of dogs withdifferent IFAT titers (40 versus 80 80 versus 160 160 versus320 etc)

Spearmanrsquos coefficient of rank correlation (rho) was usedto evaluate the degree of association between themean valuesof variables CS PT IgG Cr and UPUC and IFAT titer

For all tests significance was set as 119875 lt 005 Statisticalanalyses were performed using commercial software (Med-Calc Software v13000 Mariakerke Belgium)

3 Results

Comparison of mean values and standard deviation of clini-cal score total protein IgG creatinine and UPUC in the 9groups based on the IFAT titer are reported in Table 2

The significance and the degree of association betweenthe mean values of all variables IFAT titer clinical score TPIgG creatinine and UPUC are reported in Table 3

4 Discussion

Detection of specific serum antibodies is widely used inthe diagnosis of CanL [11] Treatment of sick dogs is oftenaccompanied by a decrease in the specific antibody levels[9 11 12] However in other cases clinical improvement hasnot been associated with a decrease in the titer of specificantibodies [13]

This study was conducted to investigate whether therewas a correlation between the IFAT titer and the clinical scorein dogs with CanL

Higher clinical score values were detected in dogs withhigher IFAT titers and a significant level of associationbetween IFAT titer and clinical score (119875 lt 00001) was foundThe dogwith themaximum clinical score (score = 30) had thehighest IFAT titer (1 10240)

There was a similar trend in serum total protein andIgG concentrations higher TP and IgG concentrations wererecorded in dogs with higher clinical scores As expectedthe IFAT titer was also related to TP and IgG (119875 lt 00001)Similar results were found by Corona et al [3] who reportedthat the changes in quantitative determination of specificantibodies paralleled those of total protein and IgG fractions

BioMed Research International 3

Table 1 Score for clinical parameters (on a scale from 0 to 3 maximum total score 87) in dogs with CanL

Clinical sign 0 1 2 3Appetite Normal Slight decrease Moderate decrease AnorexiaMentation Normal Slight depression Depression ProstrationExercise intolerance No Slight Moderate Refusal to moveWeight loss No Slight Moderate SeverePolyuria No Slight Moderate SeverePolydipsia No Slight Moderate SevereUPUC No lt1 gt1 lt 2 gt2Localized muscleatrophy (temporalmuscles)

No Slight Moderate Severe

Generalized muscleatrophy No Slight Moderate Severe

Lymphadenomegaly No 1-2 nodes gt2 lt 4 nodes GeneralizedSplenomegaly No YesConjunctivitis andorblepharitis No Unilateral and slight Bilateral or unilateral severe Bilateral and severe

Uveitis andorkeratitis No Unilateral and slight Bilateral or unilateral severe Bilateral and severe

Pale mucousmembranes No Slight Moderate Severe

Epistaxis Neverpresented Sporadic Frequent Persistent

Mouth ulcers ornodules No 1 or 2 small ulcers or

nodules gt2 small ulcers or nodules gt14 of oral cavity coveredby ulcers or nodules

Vomiting No Sporadic Frequent Frequent with bloodDiarrhea No Sporadic Frequent PersistentLameness No Sporadic Frequent Constant

Erythema No lt10 body surface or slightgeneralized erythema

10ndash25 body surface ormoderate generalizederythema

gt25 body surface

Dry exfoliativedermatitis No lt10 body surface or slight

generalized erythema


gt25 body surface

Ulcerative dermatitis No 1-2 ulcers 3ndash5 ulcers gt5 ulcersNodular dermatitis No 1-2 nodules 3ndash5 nodules gt5 nodulesSterile pustulardermatitis No 1-2 pustules 3ndash5 pustules gt5 pustules

Alopecia No lt10 body surface 10ndash25 body surfaceerythema gt25 body surface

Altered pigmentation No Localized Multifocal GeneralizedHyperkeratosis truffleand pads No Slight Moderate Severe

Generalizedhyperkeratosis No Slight Moderate Severe

Onychogryphosis No Slight Moderate Severe

It is well known that the clinical manifestations of CanLare the consequence of the host immune response and areassociated with deposition of soluble immune complexesin different tissues [9] In infected dogs the defective cell-mediated immunity results in uncontrolled multiplicationof the parasite and subsequent polyclonal and sometimesmonoclonal activation of B cells with overproduction ofimmunoglobulins These antibodies do not provide protec-tion for the host but result in the formation of immunecomplexes that can damage a variety of tissues and organs

Several studies have described the levels of specific Leish-mania IgG subclasses (IgG1 IgG2 IgM IgA and IgE)in sick asymptomatic and treated dogs sometimes withconflicting results [4 5 7 8] Solano-Gallego et al [14]determined the level of Leishmania-specific total IgG (IgG)IgG1 and IgG2 antibody responses in the sera of a widerange of canine populations symptomatic and asymptomaticdogs from endemic areas and naturally and experimentallyinfected dogs showing that clinical signs are directly relatedto IgG1 and IgG2 and that IgG2 concentrations are more


Table 2 Mean value and standard deviation of clinical score total protein IgG creatinine and UPUC in the 9 groups of dogs categorizedaccording to IFAT titer

119873∘ dogs IFAT titer Mean clinical

score (plusmnSD)

Mean totalprotein

(plusmnSD) gdL

Mean IgG(plusmnSD)

Mean creatinine(plusmnSD) mgdL

Mean UPUC(plusmnSD)

19 1 40 094 plusmn 097 672 plusmn 069 1071 plusmn 323 115 plusmn 035 029 plusmn 06846 1 80 098 plusmn 12 682 plusmn 066 1167 plusmn 35 11 plusmn 038 084 plusmn 14854 1 160 148 plusmn 202 697 plusmn 07 1344 plusmn 381 094 plusmn 04 058 plusmn 15648 1 320 265 plusmn 289 727 plusmn 088 1785 plusmn 74 094 plusmn 031 016 plusmn 02737 1 640 432 plusmn 348 794 plusmn 128 2579 plusmn 1218 105 plusmn 07 085 plusmn 219 1 1280 510 plusmn 471 852 plusmn 143 3169 plusmn 1303 126 plusmn 1 134 plusmn 2279 1 2560 478 plusmn 3 938 plusmn 139 4437 plusmn 941 162 plusmn 142 017 plusmn 0062 1 5120 16 plusmn 99 1075 plusmn 007 6465 plusmn 17 095 plusmn 035 mdash1 1 10240 30 107 532 07 mdash

Table 3 119875 and rho values of IFAT titer and clinical score totalprotein gamma globulin creatinine and UPUC 119875 value showsthe level of significance of the association and rho the degree ofassociation between the mean values of variables

119875 RhoIFAT

lt00001 0454Clinical scoreIFAT

lt00001 0532Total proteinIFAT

lt00001 0728Gamma globulinIFAT 00786 minus0117CreatinineIFAT 0004 0222UPUCTotal Protein

lt00001 0436Clinical scoreTotal protein

lt00001 0678Gamma globulinTotal protein 00037 minus0193CreatinineTotal Protein 00307 0168UPUCGammaglobulin

lt00001 0391Clinical scoreGammaglobulin 095 00042CreatinineGammaglobulin 00243 0175UPUCCreatinine 00148 minus0162Clinical scoreCreatinine 08171 00181UPUCUPUC 00035 0225Clinical score

strongly correlated with clinical illness than IgG1 De Freitaset al [5] investigated the profile of anti-Leishmania antibodiesin different clinical forms ofCanL andobserved that IgG2 and

IgM were positively correlated with clinical signs while totalIgG IgG1 and IgA were negatively correlated The varyingimmune response of infected animals may account for theconflicting results reported by various authors [4] The IFATtest is one of the recommendedmethods for the evaluation ofanti-Leishmania antibodies [15] and is widely used in clinicalpractice while the antibody assay of each class of antibodiesis not routinely practiced Our results are in accordancewith the findings of Almeida et al [6] and Vercammen etal [16] who both reported that IgG anti-Leishmania titersincreased significantly in symptomatic dogs when comparedto an asymptomatic control group

During CanL overproduction of IgG IgM and IgAconsequent to B cell activation causes the formation ofimmuno-complexes [17]

Immunomediated mechanisms have a pivotal role inthe development of renal pathology [18] and immunocom-plex deposition in glomeruli is the cause of four princi-pal types of glomerulonephritis focal or diffuse mesangialglomerulonephritismembranous glomerulonephritismem-branoproliferative glomerulonephritis and focal segmentalglomerulosclerosis [19] There is strong evidence that pro-teinuria is a risk factor for the development and progressionof renal failure and that renal disease is the most severecomplication of CanL and is a sign of poor prognosis [20]For these reasons we have correlated the value of creatinineand UPUC with the IFAT titer to assess whether there wasan association between renal function analyses and the anti-Leishmania antibodies (IgG)

Higher UPUC mean value has been reported in dogswith IFAT titers of 1 1280 and significant association betweenUPUC and IFAT (119875 = 0004) but not between IFAT titerand creatinine was observed (Table 3) Our results are inaccordance with Poli et al [21] who in a study of glomerularlesions in dog with CanL found no correlation betweenrenal involvement and anti-Leishmania titer and creatininebut found correlation between glomerular lesions and theamount of proteinuria Glomerulopathy that characterizedCanL causes proteinuria due to deposition of circulating anti-genantibody complexes this factmay explain the correlationbetweenUPUC and IgG value found in our study [18] Renal


damage with less than 75 percent of nonfunctional nephronsmay not cause increase of serum creatinine concentration[22] therefore elevation of this parameter during CanL mayshow up later than the presence of protein in the urine Thismay justify the lack of correlation between IFAT titer andcreatinine serum concentration found in our study

5 Conclusions

In conclusion our results show that dogs with the high-est IFAT titers recorded had higher mean clinical scoresindicating a positive relationship (119875 lt 00001) betweenanti-Leishmania antibodies (IgG) and clinical manifestationsthat becomes more evident in polisymptomatic subjectsSignificant correlation was found between IFAT titers andurinary UPUC

The results of this study may help to clarify whether IFATtiters could be usefully included in the short-term followupof dogs with CanL Results from the present study supportthe idea that high IFAT titers are related with the presence ofmultiple clinical symptoms suggesting a positive relationshipbetween IFAT titer and clinical manifestations

Conflict of Interests

None of the authors declare any conflict of interests

References

[1] L Manna S Reale F Vitale and A E Gravino ldquoEvidence fora relationship between Leishmania load and clinical manifesta-tionsrdquo Research in Veterinary Science vol 87 no 1 pp 76ndash782009

[2] A B Reis O A Martins-Filho A Teixeira-Carvalho etal ldquoSystemic and compartmentalized immune response incanine visceral leishmaniasisrdquo Veterinary Immunology andImmunopathology vol 128 no 1ndash3 pp 87ndash95 2009

[3] M Corona P Ciaramella A Pelagalli et al ldquoHaemostaticdisorders in dogs naturally infected by Leishmania infantumrdquoVeterinary Research Communications vol 28 no 1 pp 331ndash3342004

[4] L Solano-Gallego P Morell M Arboix J Alberola and LFerrer ldquoPrevalence of Leishmania infantum infection in dogsliving in an area of canine leishmaniasis endemicity using PCRon several tissues and serologyrdquo Journal of ClinicalMicrobiologyvol 39 no 2 pp 560ndash563 2001

[5] J C C De Freitas B E Lopes-Neto C R A De Abreu et alldquoProfile of anti-leishmania antibodies related to clinical picturein canine visceral leishmaniasisrdquoResearch in Veterinary Sciencevol 93 no 2 pp 705ndash709 2012

[6] M A O Almeida E E V JesusM L B Sousa-Atta L C AlvesM E A Berne and A M Atta ldquoAntileishmanial antibodyprofile in dogs naturally infected with Leishmania chagasirdquoVeterinary Immunology and Immunopathology vol 106 no 1-2 pp 151ndash158 2005

[7] A Rodrıguez L Solano-Gallego A Ojeda et al ldquoDynamicsof Leishmania-specific immunoglobulin isotypes in dogs withclinical leishmaniasis before and after treatmentrdquo Journal ofVeterinary Internal Medicine vol 20 no 3 pp 495ndash498 2006

[8] R G T Neto R C Giunchetti C M Carneiro et al ldquoRela-tionship of leishmania-specific IgG levels and IgG avidity withparasite density and clinical signs in canine leishmaniasisrdquoVeterinary Parasitology vol 169 no 3-4 pp 248ndash257 2010

[9] L Solano-Gallego A Koutinas G Miro et al ldquoDirections forthe diagnosis clinical staging treatment and prevention ofcanine leishmaniosisrdquo Veterinary Parasitology vol 165 no 1-2pp 1ndash18 2009

[10] World Organization for Animal Health (OIE) ldquoManual ofDiagnostic Tests and Vaccines for Terrestrial Animalsrdquo CAP218 section B paragraph 2 2008 httpwwwoieintfilead-minHomeengHealth standardstahm20108 LEISHMAN-IOSISpdf

[11] C Riera J E Valladares M Gallego et al ldquoSerological andparasitological follow-up in dogs experimentally infected withLeishmania infantum and treated with meglumine antimoni-aterdquo Veterinary Parasitology vol 84 no 1-2 pp 33ndash47 1999

[12] F Mancianti and N Meciani ldquoSpecific serodiagnosis of canineleishmaniasis by indirect immunofluorescence indirect hemag-glutination and counterimmunoelectrophoresisrdquo AmericanJournal of Veterinary Research vol 49 no 8 pp 1409ndash1411 1988

[13] L Ferrer M J Aisa X Roura and M Portus ldquoSerologicaldiagnosis and treatment of canine leishmaniasisrdquo VeterinaryRecord vol 136 no 20 pp 514ndash516 1995

[14] L Solano-Gallego C Riera X Roura et al ldquoLeishmaniainfantum specific IgG IgG1 and IgG2 antibody responses inhealthy and ill dogs from endemic areas evolution in the courseof infection and after treatmentrdquo Veterinary Parasitology vol96 no 4 pp 265ndash276 2001

[15] D Proverbio E Spada L Baggiani G Bagnagatti De Giorgiand R Perego ldquoComparison of a clinic-based ELISA testkit with the immunofluorescence antibody test for assayingLeishmania infantum antibodies in dogsrdquo BioMed ResearchInternational vol 2013 Article ID 249010 6 pages 2013

[16] F Vercammen F J Fernandez-Perez C Del Amo and J MAlunda ldquoFollow-up of Leishmania infantum naturally infecteddogs treated with allopurinol immunofluorescence antibodytest ELISA and Western blotrdquo Acta Tropica vol 84 no 3 pp175ndash181 2002

[17] M N Saridomichelakis ldquoAdvances in the pathogenesis ofcanine leishmaniosis epidemiologic and diagnostic implica-tionsrdquo Veterinary Dermatology vol 20 no 5-6 pp 471ndash4892009

[18] A F Koutinas and C K Koutinas ldquoPathologic mechanismsunderlying the clinical findings in canine leishmaniosis due toLeishmania infantumchagasirdquoVeterinary Pathology vol 51 no2 pp 525ndash539 2014

[19] A Zatelli M Borgarelli R Santilli et al ldquoGlomerular lesions indogs infected with Leishmania organismsrdquo American Journal ofVeterinary Research vol 64 no 5 pp 558ndash561 2003

[20] M Pierantozzi X Roura S Paltrinieri M Poggi and A ZatellildquoVariation of proteinuria in dogs with leishmaniasis treatedwith meglumine antimoniate and allopurinol a retrospectivestudyrdquo Journal of the American Animal Hospital Association vol49 no 4 pp 231ndash236 2013

[21] A Poli F Abramo F Mancianti M Nigro S Pieri and ABionda ldquoRenal involvement in canine leishmaniasis A light-microscopic immunohistochemical and electron-microscopicstudyrdquo Nephron vol 57 no 4 pp 444ndash452 1991

[22] J Palacio F Liste and M Gascon ldquoUrinary proteincreatinineratio in the evaluation of renal failure in canine leishmaniasisrdquoThe Veterinary Record vol 137 no 22 pp 567ndash568 1995

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

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Zoology


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GenomicsInternational Journal of


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BioinformaticsAdvances in

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Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



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Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


naturally infected with L infantum to determine whetherIFAT titers could be used as a predictor of the severity ofclinical disease

2 Materials and Methods

A retrospective study was performed The medical recordsof 131 dogs diagnosed with CanL that were presented to theTeaching Animal Hospital of the Department of Health Ani-mal Science and Food Safety of University of Milan between2008 and 2013 were reviewedThe following inclusion criteriawere used clinical diagnosis of CanL confirmed by positiveserology for Leishmania infantumusing IFAT and cytologicalidentification of Leishmania amastigotes or detection of para-site DNA using polymerase chain reaction (PCR) IgG anti-Linfantum antibodies were measured by IFAT according to therecommendations of OIE [10] using MHOMIT80IPT1 as awhole-parasite antigen fixed onmultispot slides (BioMerieuxSpa Florence Italy) and fluorescently labeled anticaninegamma globulin (Sigma Aldrich Milan Italy) as conjugatePositive sera were serially diluted and tested to establish themaximumreaction titer starting at a dilution of 1 40 Positiveand negative controls were included on each slide

The real-time PCR analysis of whole blood or samplesfrom lymph node aspiration was performed using the IllustraBlood genomicPrep Mini Spin kit (GE Healthcare MilanItaly) following the manufacturerrsquos instructions The targetfor amplification was a 116-bp fragment in the constantregion of the kDNA minicircle of L infantum This is oneof the kDNA minicircle families that are used to identifythe Leishmania genus The primers used were QLK2-UP51015840-GGCGTTCTGCGAAAACCG-31015840 and QLK2-DOWN 51015840-AAAATGGCATTTTCGGGCC-31015840 the TaqMan probes wereQ Leish Probe 2 and 51015840-FAM TGGGTGCAGAAATCCCGT-TCA-31015840-Black Hole

At the moment of the diagnosis and at each follow-up a complete physical examination was performed onall dogs after which clinical assessment of the severity ofsigns attributable to Leishmania infection (scored on a scalefrom 0 to 3) was made (Table 1) During each follow-up acomplete blood count hematological and serum biochemicalexaminations (including the determination of total proteinand serum electrophoretic pattern) and urine examinationwere performed

All dogs in which concomitant infectious diseases (egbabesiosis ehrlichiosis and dirofilariasis) were diagnosedby parasitological orand serological examinations wereexcluded

21 Medical Records Review Information extracted from themedical records of each dog at each follow-up examinationwith CanL was clinical score IFAT titer serum total protein(TP) gamma globulin (IgG) and creatinine concentrationand protein creatinine ratio in urine sample (UPUC)

A total of 49131 dogs (374)met the criteria for inclusionin the study Ages of the dogs ranged from 07 to 14 yearswith a median age of 6 years Twenty-eight were intact malesand 21 were females (10 neutered) 21 were X-breeds and 28

were purebreds A mean of 139 (min 2-max 18) follow-upexaminations was done for each dog

To assess the relationship between the IFAT titer andthe parameters considered that is clinical score serumtotal protein IgG and creatinine concentration and UPUCthe recorded values from each follow-up examination weredivided into 9 groups according to the IFATvalue (1 40 1 801 160 1 320 1 640 1 1240 1 2560 1 5120 and 1 10240)regardless of the subject to which they belonged

22 Statistical Analysis Mean standard deviation medianof clinical score value (CS) TP IgG creatinine (Cr) andUPUC were calculated after calculating normal distributionof parametric data using the DrsquoAgostino-Pearson test Mann-Whitney was used for independent samples tests to assessany statistically significant difference in the mean of clinicalscores and PT IgG creatinine and UPUC of dogs withdifferent IFAT titers (40 versus 80 80 versus 160 160 versus320 etc)

Spearmanrsquos coefficient of rank correlation (rho) was usedto evaluate the degree of association between themean valuesof variables CS PT IgG Cr and UPUC and IFAT titer

For all tests significance was set as 119875 lt 005 Statisticalanalyses were performed using commercial software (Med-Calc Software v13000 Mariakerke Belgium)

3 Results

Comparison of mean values and standard deviation of clini-cal score total protein IgG creatinine and UPUC in the 9groups based on the IFAT titer are reported in Table 2

The significance and the degree of association betweenthe mean values of all variables IFAT titer clinical score TPIgG creatinine and UPUC are reported in Table 3

4 Discussion

Detection of specific serum antibodies is widely used inthe diagnosis of CanL [11] Treatment of sick dogs is oftenaccompanied by a decrease in the specific antibody levels[9 11 12] However in other cases clinical improvement hasnot been associated with a decrease in the titer of specificantibodies [13]

This study was conducted to investigate whether therewas a correlation between the IFAT titer and the clinical scorein dogs with CanL

Higher clinical score values were detected in dogs withhigher IFAT titers and a significant level of associationbetween IFAT titer and clinical score (119875 lt 00001) was foundThe dogwith themaximum clinical score (score = 30) had thehighest IFAT titer (1 10240)

There was a similar trend in serum total protein andIgG concentrations higher TP and IgG concentrations wererecorded in dogs with higher clinical scores As expectedthe IFAT titer was also related to TP and IgG (119875 lt 00001)Similar results were found by Corona et al [3] who reportedthat the changes in quantitative determination of specificantibodies paralleled those of total protein and IgG fractions















gt25 body surface




gt25 body surface











score (plusmnSD)

Mean totalprotein

(plusmnSD) gdL

Mean IgG(plusmnSD)


Mean UPUC(plusmnSD)



119875 RhoIFAT














5 Conclusions





References






























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















gt25 body surface




gt25 body surface











score (plusmnSD)

Mean totalprotein

(plusmnSD) gdL

Mean IgG(plusmnSD)


Mean UPUC(plusmnSD)



119875 RhoIFAT














5 Conclusions





References






























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




score (plusmnSD)

Mean totalprotein

(plusmnSD) gdL

Mean IgG(plusmnSD)


Mean UPUC(plusmnSD)



119875 RhoIFAT














5 Conclusions





References






























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



5 Conclusions





References






























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article relationship between leishmania...

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