Research Article Serological and Molecular Evaluation of …downloads.hindawi.com/journals/isrn/2013/916376.pdf · ISRN Parasitology.. Data Collection. e following data were recorded:

Hindawi Publishing CorporationISRN ParasitologyVolume 2013 Article ID 916376 6 pageshttpdxdoiorg1054022013916376

Research ArticleSerological and Molecular Evaluation of Leishmania infantumInfection in Stray Cats in a Nonendemic Area in Northern Italy

Eva Spada1 Daniela Proverbio1 Antonella Migliazzo2 Alessandra Della Pepa1

Roberta Perego1 and Giada Bagnagatti De Giorgi1

1 Dipartimento di Scienze Veterinarie per la Salute la Produzione Animale e la Sicurezza AlimentareUniversita degli Studi di Milano Via G Celoria 10 20133 Milano Italy

2 Centro di Referenza Nazionale per le Leishmaniosi Istituto Zooprofilattico Sperimentale della SiciliaVia R Dicillo 4 90129 Palermo Italy

Correspondence should be addressed to Eva Spada evaspadaunimiit

Received 8 May 2013 Accepted 5 June 2013

Academic Editors M Mahieu P Somboon K R Trenholme J Venegas Hermosilla and R Zufferey

Copyright copy 2013 Eva Spada et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Infection by Leishmania species is increasing worldwide It was hypothesized recently that cats act as a secondary reservoir forLeishmania infection The aim of the present study was to assess the prevalence of Leishmania infantum antibodies and DNA inblood samples collected in a sample of stray cats in metropolitan area of Milan in northern Italy which is a nonendemic area forleishmaniasis An indirect immunofluorescence antibody test for L infantum showed that 59 of 233 cats (253) were seroreactive38 samples (163) had antibody titers of 1 40 15 (64) had antibody titers of 1 80 and 6 (26) had antibody titers of 1 160Feline immunodeficiency virus (FIV) seropositive status was statistically associated with seroreactivity to L infantum (119875 = 001) asshown by univariate andmultivariate logistic regression (119875 = 00098 OR= 734) All blood samples that were tested using real-timePCRwere negative for parasite DNAThese results were surprising since no autochthonous human or canine cases of leishmaniasishave ever been reported in this region of northern Italy It is possible that this high seroreactivity to L infantum could be due tocross-reactionwith antigens fromother parasites Additional studies that include parasite isolation are needed to clarify our findingson feline leishmaniasis in this region

1 Introduction

Leishmaniasis in the Old World is caused by the protozoaLeishmania infantum It is prevalent in countries in theMediterranean basin and dogs are the main reservoir of theparasite in that region [1] In recent years autochthonouscases of human and canine disease have been recorded athigher latitudes namely in Germany [2 3] The Netherlands[4] and North America [5] Infections have also beenreported in species other than dogs and humans includinghorses [6] and cows [7] There have been numerous reportsof feline leishmaniasis (FeL) mostly in cats living in knownendemic areas [8ndash10] some of the cats had concurrentimmunosuppressive infections [9ndash11] In countries in south-ern Europe where canine leishmaniasis (CanL) is endemicserological investigations of feline populations have revealedseroprevalence rates ranging from less than 1 to more than

60 [9ndash21] Given the diffusion of Leishmania infection andthe lack of information regarding infection rates in cats inthe Milan metropolitan area in northern Italy the aim of thepresent study was to assess the prevalence of leishmaniasisin a large representative sample of stray cats from thisnonendemic area A secondary aim was to analyze the resultsaccording to clinical laboratory and infectious data

2 Materials and Methods

21 Feline Population During a 2-year period (January 2008to January 2010) blood samples were collected from 233European shorthair stray cats from urban colonies in Milannorthern Italy during a trap-neuter-release (TNR) programthat was approved by the local authority of the city councilThe program was conducted as described previously [22]

2 ISRN Parasitology

22 Data Collection The following data were recorded sex(119899 = 233) age (119899 = 233) body condition score (BCS)(119899 = 215) area of colony of provenance that is one ofthe seven municipalities of Milan (119899 = 233) health statusbased on physical examination (119899 = 233) and dermatologicalevaluation (119899 = 121) Cats were classified as healthy orunhealthy depending on the clinical findings (Table 1)

23 Sample Collection Whole blood samples were collectedby cephalic or jugular venipuncture into tubes with EDTAanticoagulant for complete blood cell (CBC) count andpolymerase chain reaction (PCR) testing and into emptytubes for serology All samples used for serology and PCRwere stored at minus20∘C until use

24 Hematological and Serological Examination Within 24hours of sample collection a CBC count was performed onwhole blood (119899 = 127) using an ADVIA 120 System (SiemensHealthcare Diagnostics Milan Italy) Cats were categorizedas having alterations in the CBC as shown in Table 1

Serological assessment was performed to determine thepresence of the following antibodies to the feline immuno-deficiency virus (FIV) relative to the gp40 and p24 FIVantigens the feline leukemia virus (FeLV) p27 antigen (SnapFeLVFIV Combo Plus Test Idexx Laboratories HoofddorpThe Netherlands) (119899 = 137) and Toxoplasma gondii IgGantibodies (IFAT Fuller Laboratories Fullerton CA USA)(119899 = 79) The results of these serological tests have beenalready published [22] and were reanalyzed with the presentresults

For various technical reasons not all data were availablefor all 233 cats

25 Indirect Immunofluorescence Antibody Test The pres-ence of anti-L infantum antibodies was measured by anindirect immunofluorescence antibody test (IFAT) per-formed according to the recommendations of OIE [23]using MHOMIT80IPT1 as a whole-parasite antigen fixedon multispot slides (Bio Merieux Spa Florence Italy)and fluorescently-labeled antifeline gamma globulin (SigmaAldrich Milan Italy) as conjugate Positive sera were dilutedserially and tested to establish the maximum reaction titerstarting at a dilution of 1 40 Positive and negative controlswere included on each slide

26 PCR L infantum DNA was amplified from 200120583Lof whole blood by real-time PCR using the Illustra BloodgenomicPrep Mini Spin kit (GE Healthcare Milan Italy)following the manufacturerrsquos instructions The target foramplification was a 116-bp fragment in the constant region ofthe kDNAminicircle of L infantum This is one of the kDNAminicircle families that is used to identify the Leishmaniagenus The primers used were QLK2-UP 51015840-GGCGTTCTG-CGAAAACCG-31015840 and QLK2-DOWN 51015840-AAAATGGCA-TTTTCGGGCC-31015840 the TaqManprobes were Q Leish Probe2 and 51015840-FAM TGGGTGCAGAAATCCCGTTCA-31015840-BlackHole

27 Statistical Analysis Univariate analysis of the categoricaldata was performed using the chi-square test or Fisherrsquos exacttest Any parameters statistically linked to IFAT seroreactivityfor L infantum or to the presence of L infantum DNA asdetected by PCR were used in a logistic regression model totest for independent risk factors associated with the L infan-tum positivity Associations were considered statistically sig-nificant when 119875 lt 005 both the 119875 value and odds ratio (OR)are reported Data were analyzed using MedCalc Software(version 1230 Mariakerke Belgium)

3 Results

The characteristics of the feline study population are summa-rized in Table 1The serology test for L infantum showed that253 (59233) of the cats had L infantum seroreactivity 38(163) had antibody titers of 1 40 15 (64) had titers of1 80 and 6 (26) had antibody titers of 1 160 All bloodsamples tested using real-time PCR were negative for thepresence of L infantum DNA Standard curve and amplifi-cation curve of real-time PCR were reported in Figures 1 and2 respectively

No statistical association was found between seroreactiv-ity to L infantum and age sex BCS municipality of prove-nance clinical finding dermatological findings or FeLV andT gondii serology In contrast in terms of CBC neutrophiliawas statistically associated with seroreactivity to L infantum(119875 = 001) in univariate analysis but this association wasnot confirmed using multivariate logistic regression (119875 =057) In terms of serology for the retrovirus FIV seropositivestatus was statistically associated with seroreactivity to Linfantum (119875 = 001) This association was confirmed bymultivariate logistic regression 119875 = 00098 and OR = 734(95CI = 196 to 2759) The distribution of the parametersthat were evaluated and compared in L infantum seropositiveand seronegative cats is shown in Table 1

4 Discussion

This study is the first epidemiological investigation of felineLeishmania infection in the metropolitan area of Milanwhich is a nonendemic area for leishmaniasis We foundseroreactivity to L infantum by IFAT in 59 of the 233(253) stray cats that we examined These results weresurprising since no autochthonous human or canine casesof leishmaniasis have ever been reported in this region innorthern Italy In countries in southern Europe where leish-maniasis is endemic serological investigations performed infeline populations using different techniques have revealedprevalence rates that range from less than 1 to more than60 [9ndash21] In particular the seroprevalence in Italy rangesfrom 09 to 68 [9ndash11 13] in Spain from 37 to 60[14ndash16] and in Portugal from 06 to 28 [18ndash20] InGreece the seroprevalence is 39 [17] and in France it is124 [12]These results in L infantum endemic geographicalregions may reflect differences in the serological techniquesused in the cut-off values or positive thresholds and inthe populations of cats that were tested As here previous

ISRN Parasitology 3

Table 1 Characteristics of a population of stray cats in northern Italy and a comparison of characteristics in Leishmania infantum seropositiveversus seronegative cats as determined using an indirect immunofluorescence antibody test

Factor Category Totalpopulation

Seropositive

Seronegative Univariate 119875-value

Age Young (le6 months) 106 (455) 24 (407) 82 (471) 04788Adult (gt6 months) 127 (545) 35 (593) 92 (529)

Sex Female 153 (657) 38 (644) 115 (661) 09387Male 80 (343) 21 (356) 59 (339)

BCS Scarce (le39) 19 (88) 4 (74) 15 (93) 08802Good (ge49) 196 (912) 50 (926) 146 (907)

Colony of origin

Zone 2 11 (47) 2 (34) 9 (52)

00825

Zone 4 95 (408) 18 (305) 77 (442)Zone 5 9 (39) 0 (00) 9 (52)Zone 6 23 (99) 8 (136) 15 (86)Zone 7 53 (227) 17 (288) 36 (207)Zone 8 21 (90) 5 (85) 16 (92)Zone 9 21 (90) 9 (152) 12 (69)

Clinical examination

Healthy 49 (210) 12 (203) 37 (213) 09728Unhealthy 184 (790) 47 (797) 137 (787)Stomatitis 92 (395) 17 (288) 75 (431) 00740

Ocular discharge 35 (150) 10 (169) 25 (144) 07881Nasal discharge 21 (90) 5 (85) 16 (92) 09236

Pale mucous membranes 12 (52) 4 (68) 8 (46) 07532Lymphadenomegaly 117 (502) 30 (508) 87 (50) 09696

Dermatological examination

Absence of lesions 83 (686) 17 (548) 66 (733) 00912Presence of lesions 38 (314) 14 (452) 24 (267)Crusted dermatitis 22 (182) 7 (226) 15 (167) 06410

Scaling 5 (41) 1 (32) 4 (44) 08188Nodular dermatitis 3 (25) 2 (65) 1 (11) 03273

Alopecia 18 (149) 8 (258) 10 (111) 00910Ectoparasites 27 (223) 6 (222) 21 (778) 08346

Dermatophytosis 9 (74) 1 (32) 8 (89) 05225

CBC results

Absence of anemia 29 (228) 5 (167) 24 (247) 05015Presence of anemia 98 (772) 25 (833) 73 (753)

Decreased Ht 97 (764) 25 (833) 72 (742) 04352Decreased Hb 23 (181) 7 (233) 16 (165) 05627Decreased RBC 41 (323) 11 (367) 30 (309) 07158

Thrombocytopenia 10 (79) 2 (77) 8 (82) 09149Leukocytosis 5 (39) 2 (67) 3 (31) 07319Leukopenia 15 (118) 3 (100) 12 (124) 09776Neutrophilia 15 (118) 8 (267) 7 (72) 001 (057)lowast

Neutropenia 2 (16) 0 (00) 2 (21) 09631Lymphocytosis 2 (16) 0 (00) 2 (21) 09631Lymphopenia 33 (260) 12 (400) 21 (216) 00776Eosinophilia 12 (94) 4 (133) 8 (82) 06346Eosinopenia 33 (260) 9 (300) 24 (247) 07371

FIV status Positive 12 (88) 7 (212) 5 (48) 001 (00098)lowast

Negative 125 (912) 26 (788) 99 (952) OR = 734lowast (95 CI 196ndash2759)

4 ISRN Parasitology

Table 1 Continued

Factor Category Totalpopulation

Seropositive

Seronegative Univariate 119875-value

FeLV status Positive 5 (36) 0 (00) 5 (48) 0453Negative 132 (964) 33 (1000) 99 (952)

T gondii status Positive 26 (329) 9 (360) 17 (315) 08886Negative 53 (671) 16 (640) 37 (685)

BCS body condition score CBC complete blood count Ht hematocrit Hb hemoglobin RBC red blood cells FIV Feline immunodeficiency virus FeLVFeline Leukemia virus OR odds ratio CI confidence interval119875-values in bold are statistically significant (119875 lt 005)lowastResults from multivariate logistic regression analysis

epidemiological studies have used IFAT to detect antibodiesto Leishmania spp in cats An important concern is that thereis no standardized IFAT method for serological evaluationof antibodies to Leishmania spp in cats accordingly thereis no universally accepted antibody titer cut-off value thatcorresponds to active infection Cut-off titers validated indogs are used frequently for cats but the immune responsecould be different in cats than in dogs

None of the peripheral blood samples we examined usingreal-time PCR were positive for parasite DNA PCR has beenused previously by others either alone or in combinationwith serology as in our study to assess the prevalenceof feline Leishmania infection [9 11 14 24 25] Blood isnot the best specimen for PCR diagnosis of leishmaniasisSpecifically PCR performed on canine blood has lowersensitivity specificity and positive and negative predictivevalues compared to PCR performed on canine lymph nodeaspirates [26] and this may be true for samples from catsas well However blood sampling is less invasive and is easyto perform particularly for epidemiological studies involvingnumerous subjects as in our survey

Although dogs have been universally regarded as thedomestic reservoir hosts of zoonotic visceral leishmaniasiscaused by L infantum some researchers have hypothesizedthat cats may also act as a secondary reservoir host ofleishmaniasis rather than simply as an accidental host [9 1415] Differences in immune response vector host preferenceor innate resistance in cats to vector-borne diseases couldaccount for the observed differences in the prevalence ofinfection in canine versus feline populations in endemicareas Immunosuppressive agents such as FIV or FeLV ordisease and stress can induce immunological dysfunctionand impair the cellular immune response This allows activemultiplication of the parasite and widespread visceral dis-semination of the protozoa [27] In our survey FIV infectionwas statistically associated with seroreactivity to L infantumby IFAT and FIV-positive cats were 73 times more likelyto be L infantum seroreactive than FIV-negative cats (119875 =00098) This association has also been found in previousstudies performed in endemic area of Southern Italy [9 11]

Based on results from a recent survey continental north-ern Italy is now focally endemic for leishmaniasis but nosand-flies (vector) or autochthonous cases of human andcanine leishmaniasis have been identified in Milan or itssuburbs [28] Cases of CanL are commonly diagnosed in the

area where we performed our study but the histories of theaffected dogs always reveal that they have lived or travelled inareas that are endemic for CanL [29 30] A canine epidemio-logical survey of 313 dogs in a public animal shelter that weretested for L infantum by IFAT more than 10 years ago (2002-2003) in the urban area of Milan found a seroprevalence of34 [31] Although the history of dogs in animal shelters isoften unknown some of these dogs may have come fromareas that are endemic for L infantum infection In contrastit is unlikely that all of the Leishmania seropositive catsfound in our study population were infected in endemicareas In the present study the feline seroprevalence for Linfantum was much higher than the canine seroprevalencefound 10 years previously in a canine population in ananimal shelter in the same area Notably this area is stillconsidered nonendemic for leishmaniasis We speculate thatthe serology results for leishmaniasis in our survey maybe an overestimation due to the possibility of IFAT cross-reactivity between L infantum and other pathogens Cross-reactivity with other pathogens is possible on some serologictests especially those that use a whole-parasite antigen aswe did here There was no significant correlation betweenT gondii positivity and L infantum positivity in our studyThis may suggest a lack of cross-reactivity with Toxoplasmaparasites New vector-borne parasites have been found thataffect cats such as Ehrlichia spp Rickettsia felis Anaplasmaphagocytophilum and Babesia spp (according to the vector-borne disease ESCCAP guidelines) [32] that might be ableto cross-react with Leishmania This has been demonstratedin dogs in that IFAT cross-reactivity has been reported forL infantum and Trypanosoma cruzi Leishmania braziliensisand Ehrlichia canis infection [33]

5 Conclusions

Our results demonstrate high levels of seroreactivity toL infantum in cats in an area of northern Italy that hastraditionally been considered to be free of leishmaniasis andnonendemic for this infection in dogs Possible IFAT cross-reactivity and a lack of a validated serological method forfeline specie could explain our unexpectedly high seropreva-lence Additional studies that include parasite isolation areneeded to clarify our findings on feline leishmaniasis in thisgeographic area

ISRN Parasitology 5

Standard curve

Log starting quantity

StandardUnknownFAM

40

35

30

25

1 2 3 4 5 6

Cq

E = 829 R2 = 0992 slope = minus3814 y-int = 4393840

Figure 1 Standard curve in logarithmic scale

Amplification

Cycles

RFU

1200

1000

800

600

400

200

0

0 10 20 30 40

QuantificationStep number 4Analysis mode fluorophoreCq determination single thresholdBaseline methodFAM autocalculatedThreshold settingFAM 6405 autocalculated

Figure 2 Amplification curve amplification of the standards (from106 LeishmL to 100 LeishmL) Below the threshold the nonampli-fied samples (negative)

Disclosure

This research received no specific grant from any fundingagency in the public commercial or not-for-profit sectors

Conflict of Interests

All the authors (E Spada D Proverbio AMigliazzo A DellaPepa R Perego and G Bagnagatti De Giorgi) declare thatthey have no conflict of interests

References

[1] G Baneth ldquoLeishmaniasesrdquo in InfectiousDiseases of theDog andCat C E Greene Ed pp 685ndash698 Saunders Elsevier St LouisMo USA 5th edition 2006

[2] R Gothe I Nolte and W Kraft ldquoLeishmaniosis of dogsin Germany epidemiological case analysis and alternative toconventional causal therapyrdquo Tierarztliche Praxis Ausgabe Gvol 25 no 1 pp 68ndash73 1997

[3] C BogdanG SchonianA-L Banuls et al ldquoVisceral leishmani-asis in a German child who had never entered a known endemicarea case report and review of the literaturerdquo Clinical InfectiousDiseases vol 32 no 2 pp 302ndash306 2001

[4] M M Dıaz-Espineira and R J Slappendel ldquoA case ofautochthonous canine leishmaniasis in the Netherlandsrdquo Vet-erinary Quarterly vol 19 no 2 pp 69ndash71 1997

[5] P M Schantz F J Steurer Z H Duprey et al ldquoAutochthonousvisceral leishmaniasis in dogs in North Americardquo Journal of theAmerican Veterinary Medical Association vol 226 no 8 pp1316ndash1322 2005

[6] K Koehler M Stechele U Hetzel et al ldquoCutaneous leishman-iosis in a horse in southern Germany caused by Leishmaniainfantumrdquo Veterinary Parasitology vol 109 no 1-2 pp 9ndash172002

[7] L Lobsiger N Muller T Schweizer et al ldquoAn autochthonouscase of cutaneous bovine leishmaniasis in Switzerlandrdquo Veteri-nary Parasitology vol 169 no 3-4 pp 408ndash414 2010

[8] C Ozon P Marty F Pratlong et al ldquoDisseminated felineleishmaniosis due to Leishmania infantum in Southern FrancerdquoVeterinary Parasitology vol 75 no 2-3 pp 273ndash277 1998

[9] M G Pennisi ldquoA high prevalence of feline leishmaniasis insouthern Italyrdquo in Canine Leishmaniasis Moving Towards aSolution R Killick-Kendrick Ed pp 39ndash48 Intervet Interna-tional Boxmeer The Netherlands 2002

[10] A Poli F Abramo P Barsotti et al ldquoFeline leishmaniosis due toLeishmania infantum in Italyrdquo Veterinary Parasitology vol 106no 3 pp 181ndash191 2002

[11] M G Pennisi T Lupo D Malara M Masucci A Migliazzoand G Lombardo ldquoSerological and molecular prevalence ofLeishmania infantum infection in cats from southern ItalyrdquoJournal of Feline Medicine amp Surgery vol 14 pp 656ndash657 2012

[12] C Ozon P Marty and A Lelievre ldquoLe chat reservoir deLeishmania infantum dans le sud del a Francerdquo in Proceedingsof the 24thWorld Small Animal Veterinary Association CongressLyon France September 1999

[13] S Vita D Santori I Aguzzi E Petrotta and A LucianildquoFeline leishmaniasis and ehrlichiosis serological investigationin Abruzzo regionrdquo Veterinary Research Communications vol29 supplement 2 pp 319ndash321 2005

[14] J Martın-Sanchez C Acedo M Munoz-Perez B Pesson OMarchal and F Morillas-Marquez ldquoInfection by Leishmaniainfantum in cats epidemiological study in Spainrdquo VeterinaryParasitology vol 145 pp 267ndash273 2007

[15] L Solano-Gallego A Rodrıguez-Cortes L Iniesta et al ldquoCross-sectional serosurvey of feline leishmaniasis in ecoregionsaround the Northwestern Mediterraneanrdquo American Journal ofTropical Medicine and Hygiene vol 76 no 4 pp 676ndash680 2007

[16] T Ayllon M A Tesouro I Amusategui A Villaescusa FRodriguez-Franco and A Sainz ldquoSerologic and molecularevaluation of Leishmania infantum in cats from central SpainrdquoAnnals of the New York Academy of Sciences vol 1149 pp 361ndash364 2008

6 ISRN Parasitology

[17] A Diakou E Papadopoulos and K Lazarides ldquoSpecific anti-Leishmania spp antibodies in stray cats in Greecerdquo Journal ofFeline Medicine and Surgery vol 11 no 8 pp 728ndash730 2009

[18] L Cardoso A P Lopes K Sherry H Schallig and L Solano-Gallego ldquoLow seroprevalence of Leishmania infantum infectionin cats from northern Portugal based on DAT and ELISArdquoVeterinary Parasitology vol 174 no 1-2 pp 37ndash42 2010

[19] A Duarte I Castro I M Pereira da Fonseca et al ldquoSurveyof infectious and parasitic diseases in stray cats at the LisbonMetropolitan Area Portugalrdquo Journal of Feline Medicine andSurgery vol 12 no 6 pp 441ndash446 2010

[20] C Maia J Gomes J Cristovao et al ldquoFeline Leishmaniainfection in a canine leishmaniasis endemic region PortugalrdquoVeterinary Parasitology vol 174 no 3-4 pp 336ndash340 2010

[21] T Ayllon P P V P Diniz E B Breitschwerdt A VillaescusaF Rodrıguez-Franco and A Sainz ldquoVector-borne diseases inclient-owned and stray cats from Madrid Spainrdquo Vector-Borneand Zoonotic Diseases vol 12 no 2 pp 143ndash150 2012

[22] E Spada D Proverbio A Della Pepa et al ldquoSeroprevalenceof feline immunodeficiency virus feline leukaemia virus andToxoplasma gondii in stray cat colonies in northern Italy andcorrelation with clinical and laboratory datardquo Journal of FelineMedicine amp Surgery vol 14 pp 369ndash377 2012

[23] World Organization for Animal Health (OIE) ldquoManual ofDiagnostic Tests and Vaccines for Terrestrial Animalsrdquo CAP218 section B paragraph 2 2008 httpwwwoieintfile-adminHomeengHealth standardstahm20108 LEISHMA-NIOSISpdf

[24] M-D Tabar L Altet O Francino A Sanchez L Ferrer andX Roura ldquoVector-borne infections in cats molecular study inBarcelona area (Spain)rdquo Veterinary Parasitology vol 151 no 2-4 pp 332ndash336 2008

[25] M Maroli M G Pennisi T Di Muccio C Khoury L Gradoniand M Gramiccia ldquoInfection of sandflies by a cat naturallyinfected with Leishmania infantumrdquo Veterinary Parasitologyvol 145 no 3-4 pp 357ndash360 2007

[26] S Reale L Maxia F Vitale N S Glorioso S Caracappaand G Vesco ldquoDetection of Leishmania infantum in dogs byPCR with lymph node aspirates and bloodrdquo Journal of ClinicalMicrobiology vol 37 no 9 pp 2931ndash2935 1999

[27] E Spada D Proverbio C Giudice M DiGiancamillo M Lodiand R Perego ldquoPituitary-dependent hyperadrenocorticism andgeneralised toxoplasmosis in a cat with neurological signsrdquoJournal of Feline Medicine and Surgery vol 12 no 8 pp 654ndash658 2010

[28] M Maroli L Rossi R Baldelli et al ldquoThe northward spread ofleishmaniasis in Italy evidence from retrospective and ongoingstudies on the canine reservoir and phlebotomine vectorsrdquoTropical Medicine and International Health vol 13 no 2 pp256ndash264 2008

[29] E Spada and D Proverbio ldquoCanine leishmaniasis in non-endemic area incidence of positivity to IFAT test in dogsmovedto endemic areardquo in Proocedings of the LVI National CongressSISVET pp 289ndash290 Giardini Naxos Italy September 2002

[30] E Spada D Proverbio D Groppetti R Perego V Grieco andE Ferro ldquoFirst report of the use of meglumine antimoniate fortreatment of canine leishmaniasis in a pregnant dogrdquo Journalof the American Animal Hospital Association vol 47 no 1 pp67ndash71 2011

[31] R Perego D Proverbio E Spada and E Ferro ldquoCanineleishmaniasis a sero-epidemiological survey by indirect fluo-rescence antibody test (IFAT) in 313 dogs at sanitary public

kennel ofMilanrdquo in Proceedings of the LIX Congress SISVET pp273ndash274 Viareggio Italy September 2005

[32] European Scientific Counsel Companion Animal Parasites(ESCCAP) ldquoControl of Vector-Borne Diseases in Dogsand Catsrdquo Guideline 05 2nd edition October 2012 httpwwwesccaporg

[33] E D C FerreiraM de LanaM Carneiro et al ldquoComparison ofserological assays for the diagnosis of canine visceral leishma-niasis in animals presenting different clinical manifestationsrdquoVeterinary Parasitology vol 146 no 3-4 pp 235ndash241 2007

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