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A Serological and Molecular Study of Leishmania infantumInfection in Cats from the Island of Ibiza (Spain)
Kate Sherry,1 Guadalupe Miro,2 Michele Trotta,3 Carmen Miranda,2 Ana Montoya,2
Carmen Espinosa,2 Fernando Ribas,4 Tommaso Furlanello,3 and Laia Solano-Gallego1
Abstract
The aim of this study was to evaluate the prevalence of Leishmania infantum infection within a feline populationby serologic and molecular methods and to identify associated risk factors. One hundred five cats livingoutdoors were studied. Sera were tested for IgG antibodies against L. infantum, Toxoplasma gondii, and felineimmunodeficiency virus (FIV) and for the detection of feline leukemia virus (FeLV) p27 antigen by enzyme-linked immunosorbent assay (ELISA). L. infantum real-time polymerase chain reaction (PCR) was performedon DNA extracted from blood. L. infantum and T. gondii seroprevalence rates were 13.2% and 55.2%,respectively. The prevalence of L. infantum by PCR was 8.7%. The total rate of L. infantum infection derivedfrom seroreactivity and/or positive PCR was 15.4%. Serology and PCR results were positively associated, andmoderate agreement (kappa¼ 0.489) was found between Leishmania ELISA and PCR. No statistical associationwas found between positive Leishmania PCR results and gender, clinical status, or T. gondii seropositivity. Sixof the 105 cats (5.7%) displayed clinical signs compatible with feline cutaneous leishmaniosis, and 4 out ofthese 6 cats (66.7%) were found to have Leishmania infection by means of serology and/or PCR. Leishmaniaseropositivity was associated with clinical signs of feline cutaneous leishmaniosis ( p¼ 0.029). The prevalenceof FeLV p27 antigen was 16.2% (17/105) and of FIV antibody was 20.9% (22/105), with coinfection found in9.5% (10/105) of the cats. Leishmania ELISA seroreactivity and positive PCR results were statistically associ-ated with FeLV infection and with coinfection of both retroviruses but not with a positive FIV status. The highseroprevalence and molecular rates of Leishmania infection observed indicate that cats are frequently infectedwith L. infantum, and the association with FeLV suggests a potential role for this retrovirus in feline Leishmaniainfection in endemic areas.
Key Words: Antibodies—Cat—Leishmania infantum—Real-time PCR—Retroviruses, Toxoplasma gondii.
Introduction
Leishmaniosis caused by Leishmania infantum is awidespread sandfly-borne zoonotic disease with a great
impact on public health. Much of the veterinary literaturedescribes canine leishmaniosis with regard to epidemiologyand pathogenesis, and this has demonstrated a high preva-lence of infection in the Mediterranean Basin (Baneth et al.2008). Feline leishmaniosis was first described in 1912 inAlgeria (Sergent et al. 1912) and occurs sporadically especiallyin regions and countries where canine leishmaniosis isendemic (Hervas et al. 1999).
The Leishmania species shown to cause feline leishmaniosisare L. infantum in southern France (Laurelle-Magalon andToga 1996, Ozon et al. 1998), Italy (Poli et al. 2002, Pennisi et al.2004), Portugal (Costa Durao et al. 1994, Marcos et al. 2009),Spain (Hervas et al. 1999), Brazil (Savani et al. 2004), and Iran(Hatam et al. 2009); Leishmania mexicana in Texas (Craig et al.1986, Barnes et al. 1993); and Leishmania amazonensis (de Souzaet al. 2005) and Leishmania braziliensis (Schubach et al. 2004) inBrazil. Despite the high prevalence of L. infantum in dogs inthe Mediterranean basin with common manifestations ofclinical disease (Solano-Gallego et al. 2001), far fewer cases ofclinical feline Leishmania infection have been described in the
1Department of Pathology and Infectious Diseases, Royal Veterinary College, University of London, United Kingdom.2Departmento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Spain.3Laboratorio Privato Veterinario San Marco, Padua, Italy.4Clinica Veterinaria San Jorge, Ibiza, Spain.
VECTOR-BORNE AND ZOONOTIC DISEASESVolume 11, Number 3, 2011ª Mary Ann Liebert, Inc.DOI: 10.1089/vbz.2009.0251
239
same region. The most frequently described lesions in felineleishmaniosis are ulcerocrusting and nodular dermatitis,alopecia, and scaling (Ozon et al. 1998, Hervas et al. 1999, Poliet al. 2002, Pennisi et al. 2004, Rufenacht et al. 2005) with thevisceral form of the disease involving the spleen, liver, lymphnodes, bone marrow, eye, and kidney being less commonlydescribed (Ozon et al. 1998, Hervas et al. 1999, 2001, Leivaet al. 2005). Although clinical cases of leishmaniosis have beenreported in cats with coinfection of feline leukemia virus(FeLV) and feline immunodeficiency virus (FIV) (Pennisi1999, Hervas et al. 2001, Poli et al. 2002, Pennisi et al. 2004,Grevot et al. 2005), the true association between feline leish-maniosis and retroviral infection remains unclear.
The recent description of subclinical Leishmania infection incats in the Mediterranean basin countries (Martın-Sanchezet al. 2007, Maia et al. 2008) and the demonstration of infec-tivity of sand fly vectors from a cat (Maroli et al. 2007) raisethe possibility of a reservoir host role for this species. How-ever, the scientific literature available on feline Leishmaniainfection is limited (Martın-Sanchez et al. 2007, Solano-Gallego et al. 2007a, Maia et al. 2008, Hatam et al. 2009). Nostudies have been performed in cats living on the Island ofIbiza (Spain), where canine and human leishmaniosis are wellestablished (Riera et al. 2004). The objective of this study wasto evaluate the prevalence of L. infantum infection in an Ibizianfeline population by serologic and molecular methods and toidentify associated risk factors such as the animal’s clinicalstatus and concurrent infections including FeLV, FIV, andToxoplasma gondii.
Materials and Methods
Blood was collected from 105 cats living outdoors in twoshelters on the Island of Ibiza (Spain) between June and July2008. Blood was collected into ethylenediaminetetraaceticacid and plain tubes, and full routine hematologic and clinicalchemistry analyses were performed. All blood and sera werestored at �208 before usage. Breed, age, gender, and fullclinical history were recorded where known but were notavailable for all of the cats. A full physical examination wasperformed by veterinarians before blood sampling. Thepopulation was composed of 49 spayed females, 4 entirefemales, 37 neutered males, and 11 entire males with nogender recorded for 3 of the sampled cats. All cats were do-mestic short-haired. Sixty of the cats displayed a variety ofclinical signs. Sera from 76 cats presented for various medi-cal reasons at the Royal Veterinary College (University ofLondon, United Kingdom) were used for L. infantum sero-logical evaluation by enzyme-linked immunosorbent assay(ELISA). Since L. infantum infection is not endemic in theUnited Kingdom (Shaw et al. 2009), these samples were usedas negative controls to establish cut-off values for ELISA.
An ELISA protocol previously described for cat serawas used with some modifications (Solano-Gallego et al.2007a). Briefly, microtiter plates were coated with 0.1 mL ofL. infantum (MHOM/FR/78/LEM-75 zymodeme MON-1)antigen (1 mg/mL in 50 mL of 0.05 M carbonate-bicarbonate,pH 9.6) and incubated overnight at 48C. One hundred mi-croliters per well of cat sera, diluted 1:100 in phosphate-buffered saline–0.05% Tween 20 (PBST)–1% dried skimmedmilk (PBST-M), was incubated for 1 h at 378C. After threewashes with PBST and one wash with PBS, 100 mL per well of
anti-cat IgG (AbD; Serotec) diluted 1:7500 in PBST-M conju-gated to horseradish peroxidase was added and incubated for1 h at 378C. Then, the plates were rewashed. The substratesolution, ortho-phenylenediamine dichloride 0.4 mg/mL,tablets plus buffer of 0.4 mg/mL urea hydrogen peroxide, and0.05 M phosphate-citrate, pH 5.0 (SIGMAFAST�; Sigma-Aldrich), was added at 200mL per well and developed for10 min at 248C. The reaction was stopped with 50 mL of 2 MH2SO4. Absorbance values were read at 490 nm in an auto-matic micro-ELISA reader (Spectra Max M2 Plate Reader,Molecular Devices). The reaction was quantified as ELISAunits (EU) related to a positive cat sera used as a calibrator andarbitrarily set at 100 EU. The calibrator cat had confirmedleishmaniosis and serum from this cat has been used in aprevious study (Solano-Gallego et al. 2007a). All determina-tions included the calibrator serum as a positive control andserum of a cat from the United Kingdom as a negative control.The cut-off was established at 69 EU for IgG (mean� 4standard deviations of sera of 76 cats from a United Kingdomcat population).
L. infantum polymerase chain reaction (PCR) was per-formed on 104 of the Ibizian cats sampled as previously de-scribed (Solano-Gallego et al. 2007b). Briefly, DNA wasextracted using High Pure PCR Template Preparation Kit(Roche Applied Science) in accordance with the manufactur-er’s recommendations. Commercial L. infantum primers andhybridization probes LC set (TIB Molbiol) that amplify afragment of the kinetoplast minicircle were used. Amplifica-tions were conducted in sealed 20mL LightCycler glasscapillaries. Thermal cycling was performed according tomanufacturer’s instructions (TIB Molbiol). The glyceralde-hyde 3-phosphate dehydrogenase (GAPDH) housekeepinggene was used to ensure that negative results representedtruly negative samples rather than a problem with DNAloading, sample degradation, PCR inhibition, or absence offeline cells (Solano-Gallego et al. 2007b). Only samples with apositive result for the GAPDH gene were evaluated forL. infantum real-time PCR.
To evaluate the association with feline retrovirus infections,105 of the cats were tested for FeLV antigen and for FIV an-tibody. Detection of FeLV antigen (p27) and FIV antibody wasperformed with a commercial ELISA kit (PetChek* FIVAb/FeLV; IDEXX Laboratories). Sera were analyzed for an-tibodies to T. gondii with the microscopic agglutination test asdescribed by Desmonts and Remington (1980) using a directmicroagglutination commercial kit (Toxo-Screen DA; Bio-merieux). Sera were diluted from 1:20 to 1:320. Positive andnegative control samples were included in each plate. A titerof �1:40 was considered indicative of T. gondii exposure incats (Desmonts and Remington 1980).
Cats were categorized as positive or negative for eachparameter using assigned cut-off points for T. gondii andL. infantum, and positive or negative result for FeLV and FIVusing data generated by the above techniques. Cats werecategorized in terms of presence or absence of anemia(hemoglobin concentration<8g/dL), hyperproteinemia (totalprotein >8g/dL), hypoalbuminemia (albumin <2.3 g/dL),and signs compatible with feline cutaneous leishmaniosis orclinical signs not related with signs compatible with felinecutaneous leishmaniosis. Clinical signs compatible with felinecutaneous leishmaniosis included ulcerocrusted dermatitis,nodular dermatitis, alopecia, and scaling especially of face
240 SHERRY ET AL.
and ears (Hervas et al. 1999, Poli et al. 2002, Pennisi et al. 2004,Rufenacht et al. 2005). Clinical signs not specifically relatedwith signs of feline cutaneous leishmaniosis included anyclinical signs with the exception of the cutaneous signs de-scribed above. The most common clinical signs not specifi-cally compatible with feline cutaneous leishmaniosis werelethargy, feline respiratory complex, gingivostomatitis, anddiarrhea. Chi-square analysis and Fisher’s exact test (SPSS17.0) were used to test for associations between all of theparameters. Differences were considered significant if thep-value was <0.05. Any parameters statistically linked tothe ELISA seropositivity were used in a logistic regressionmodel (SPSS 17.0) to assess for risk factors associated with apositive ELISA result. A kappa test (GraphPad Software, Inc.)was used to establish agreement between positive LeishmaniaPCR and ELISA results.
Results
A seroprevalence of 13.2% was found using ELISA forLeishmania and the PCR positivity rate was 8.7%. The resultsare listed in Table 1. All PCR samples were positive for theGAPDH housekeeping gene. Levels of Leishmania-specificantibodies in positive cats ranged from 77% to 306% (with amean of 116%). The parasite load ranged from 250 kinetoplastcopies/mL of blood to 3.2�107 kinetoplast copies/mL ofblood with a mean and SD of 3�106� 1�107. Six cats werepositive by both serology and PCR with 10 cats showingdiscordant results between PCR and serology. Only threecats that were PCR positive had a negative serology result(Table 2). A low Pearson correlation was found betweenparasite load and degree of antibody levels (r¼ 0.16). Theserology and PCR results were positively associated bychi-square analysis (24.2, p< 0.001). In addition, logisticregression analysis identified a strong association betweenPCR and ELISA. A kappa value of 0.489 (with 95% CI) wasfound between ELISA and PCR, which demonstrated amoderate agreement (Landis and Koch 1977). No statisticalassociation was found between PCR-positive results andgender, clinical status (specific or nonspecific signs), orT. gondii status. Six of the 105 cats (5.7%) displayed clinicalsigns compatible with feline leishmaniosis (Fig. 1) and 4 out ofthese 6 cats (66.7%) were found to have Leishmania infectionby means of serology and/or PCR as shown in Table 2.Leishmania seropositivity was associated with clinical signsof feline cutaneous leishmaniosis by Fisher’s exact test( p¼ 0.029). Leishmania-positive status based on serologyand/or PCR was associated with clinical signs compatiblewith feline cutaneous leishmaniosis and clinical signs notrelated to feline cutaneous leishmaniosis by Fisher’s exact test( p¼ 0.028, p¼ 0.006, respectively).
Forty-four percent (7/16) of FeLV-positive cats and 25%(5/20) of FIV-positive cats were seropositive for Leishmania.Forty-four percent of cats that were coinfected with FeLV andFIV were Leishmania positive by ELISA (4/9). Leishmaniaseropositivity was associated with FeLV but not with FIVinfection by Fisher’s exact test (15.5, p¼ 0.001, 3.0, p¼ 0.132,respectively) and with coinfection of both FeLV and FIV (8.3,p¼ 0.017, Fisher’s exact test). In addition, logistic regressionidentified a positive association between Leishmania ELISAand FeLV status. Positive PCR results were also associatedwith FeLV infection (11.077, p¼ 0.006, Fisher’s exact test) and
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FELINE LEISHMANIA INFANTUM INFECTION 241
with coinfection of both retroviruses (13.752, p¼ 0.04, Fisher’sexact test) but was not statistically associated with a positiveFIV status.
Infection with each of the retroviruses was associated withgender by chi-square analysis (FIV: 6.2, p¼ 0.013; FeLV: 4.0,p¼ 0.05) with females more likely to be infected. The presenceof clinical signs not related with feline cutaneous leishma-niosis was associated with FIV infection on chi-square anal-ysis (4.6, p¼ 0.032) and with anemia (6.0, p¼ 0.021) by Fisher’sexact Test. There was no statistical association between sero-positivity for T. gondii and any of the other parameters.
Discussion
Subclinical feline infection with L. infantum and clinicalfeline leishmaniosis are far less frequently reported than thecanine counterparts. The Leishmania seroprevalence in dogsranges from 24% to 34% in the Balearic islands (Solano-Gallego et al. 2006b); therefore, it is much higher than theseroprevalence found in cats (13.2%) in the same area in thisstudy. In addition, higher rates of positive PCR are reported indogs (20%) (Tabar et al. 2008b) than in cats (8.7%) in similargeographical areas. The reported feline prevalence in Spain(established via IFAT, PCR, and/or cytology) ranges from0.9% to 70% (Portus et al. 2002, Martın-Sanchez et al. 2007,Solano-Gallego et al. 2007a, Ayllon et al. 2008). Ser-oprevalence ranges from 0.9% to 28% depending on thegeographical region in Spain with higher seroprevalence ratesencountered in Mediterranean basin regions (Portus et al.2002, Martın-Sanchez et al. 2007, Solano-Gallego et al. 2007a,Ayllon et al. 2008) as described in dogs (Solano-Gallego et al.2006b, Miro et al. 2007). Fewer studies have been carried outusing molecular techniques in blood with rates of positivePCR ranging from 0.4% to 30.4% (Martın-Sanchez et al. 2007,
Ayllon et al. 2008, Maia et al. 2008, Tabar et al. 2008a). The roleof the cat as a reservoir for L. infantum is still controversial, butit has been hypothesized (Martın-Sanchez et al. 2007, Solano-Gallego et al. 2007a) that the cat may be a secondary reservoirhost rather than an incidental host by virtue of the demon-stration of infectivity of sand fly vectors from a chronicallyinfected cat in Italy (Maroli et al. 2007).
This study found a statistically significant association be-tween Leishmania infection and clinical signs compatible withfeline cutaneous leishmaniosis described in previous publi-cations (Hervas et al. 1999, Poli et al. 2002, Pennisi et al. 2004,
Table 2. Gender, Retroviral Status, Toxoplasma gondii Titers, and Clinical Signs of Cats
Positive by Serology and/or Polymerase Chain Reaction for Leishmania infantum
Cat ID GenderELISA
(% positivity)PCR
(kinetoplast/mL)FeLV/FIV
statusToxoplasmagondii titers Clinical signs
Ig 15 MN þ (76.8) � �/þ — Generalized dermatitis, hyperkeratosis,labial ulcer, generalized ventral alopeciaa,nasal purulent discharge, ear mites
Ig 23 MN þ (108.8) þ (1.2�106) þ/þ >1:320 Feline respiratory complex, ear mitesIg 25 FN þ (132.9) þ (1.4�105) þ/þ — Ear mitesIg 30 FN þ (306.1) � �/þ >1:320 Chronic diarrheaIg 32 FN þ (147.3) � þ/þ — Mild gingivostomatitisIg 33 FN þ (93.9) � �/� >1:320 Marked gingivostomatitis, uveitisIg 41 MN þ (165.1) þ (1.1�105) �/� >1:320 Marked gingivostomatitisIg 45 b þ (110.4) � þ/� 1:320 No clinical signs notedIg 46 FN þ (106.6) þ (4.5�105) þ/� 1:320 Crusted-ulcerative cutaneous lesion in left ear
pinnaa, ear mitesIg 51 MN þ (80.5) � �/� >1:160 Hyperkeratosis, nasal ulcera
Ig 53 FN þ (123.0) þ (9.7�103) þ/þ >1:160 Chronic diarrhea, ear mitesIg 56 MN � (42.6) þ (250) �/� >1:160 Circling, ear mitesIg 66 FN � (67.9) þ (250) þ/þ <1:160 Crusted cutaneous lesions on ear tipa, mild
gingivostomatitis, ear mitesIg 77 ME þ (154.2) þ (3.2�107) �/� — No clinical signs notedIg 73 ME þ (78.1) � �/� >1:160 Diarrhea, ear mitesIg 95 FE � (16.3) þ (290) �/� — No clinical signs noted
aClinical signs suggestive of feline cutaneous leishmaniosis.bGender was not known for Ig 45.M, male; F, female, N, neutered, E, entire.
FIG. 1. Crusted-ulcerative cutaneous lesion in a left earpinna of cat that was positive to both Leishmania infantumpolymerase chain reaction and serology (ID Ig 46).
242 SHERRY ET AL.
Rufenacht et al. 2005). In addition, a strong statistical associ-ation was found between ELISA and PCR. Discordant resultscan be attributed to the inherent differences between sero-logical testing and molecular methods. For example, althoughPCR is a very sensitive technique, it relies upon the presenceof the organism within the tissue used for molecular analysis.In PCR from blood, as in this study, low numbers or absenceof the organism may decrease the sensitivity of this method. Itshould be taken into account that blood was found not to bethe tissue of choice for Leishmania PCR detection in the dogdue to the lower sensitivity when compared with other tissues(Rodrıguez-Cortes et al. 2007, Solano-Gallego et al. 2007b,Maia et al. 2009). In addition, in this study, the parasite load inblood was quite variable and poorly correlated with degree ofantibody levels. Other tissues that may have increased the rateof detection by PCR would be the skin, spleen, lymph nodes,or bone marrow as reported in canine Leishmania infections(Rodrıguez-Cortes et al. 2007, Solano-Gallego et al. 2007b,Maia et al. 2009). In particular, this would have been inter-esting in view of sampling tissue from the site of cutaneouslesions from those cats with clinical signs that were compat-ible with feline leishmaniosis but which were negative byELISA or PCR and to confirm Leishmania infection in suspi-cious lesions (Rufenacht et al. 2005).
An association was found between a positive Leishmaniastatus (by both serologic and molecular methods) and FeLV.These findings are in contrast to other studies where no suchassociation was found between retroviral infections andLeishmania infection in cats (Martın-Sanchez et al. 2007, Ayl-lon et al. 2008) with the exception of one study from Italywhere a significant higher L. infantum seropositivity rate inFIV-positive cats was reported (Pennisi et al. 1998). However,several clinical leishmaniosis cases have been reported in catswith concurrent retroviral infections (Pennisi 1999, Hervaset al. 2001, Poli et al. 2002, Pennisi et al. 2004, Grevot et al.2005). These findings suggest that a retroviral infection, inparticular FeLV, is a potential risk factor for feline L. infantuminfection. A positive association between Leishmania and ret-roviral infections may suggest the role of immunosuppressionin susceptibility of cats to Leishmania as has been recognized inhuman beings where Leishmania subclinical infection can de-velop to disease and even become disseminated and predis-pose to the visceral form (Alvar et al. 2008). It should also raisethe clinical suspicion for L. infantum in cats with FeLV inendemic areas especially if presenting with consistent clinicalsigns compatible with feline leishmaniosis.
Previous studies using experimental infection of cats withL. braziliensis species showed that peak antibody leveloccurred on resolution of skin lesions (Simoes-Mattos et al.2005). The immune response mediated by antibodies is notconsidered protective against Leishmania and the period dur-ing which cats showed active skin lesions that harbored par-asites could not be determined by serology. Lesion size wasalso not correlated to antibody titer (Simoes-Mattos et al.2005). These findings suggest the possibility of a misdiagnosisin a cat with skin lesions but negative serology. Therefore,serological titers are not frequently correlated with clinicalsigns as demonstrated in experimental L. infantum infection incats (Kirkpatrick et al. 1984). The pathogenic evolution ofL. braziliensis and L. infantum in the vertebrate host is verydifferent as well as the type of immune responses elicited bythe two parasites (Murray et al. 2005, Castro et al. 2007,
Dantas-Torres 2007, 2009). Further investigation is needed todetermine how similar are these experimental models withL. braziliensis and L. infantum to natural L. infantum infectionin cats. This raises another consideration, which is the im-portance of screening of feline blood donors for L. infantuminfection. Studies among canine (Tabar et al. 2008a) and hu-man (Riera et al. 2004) blood donors have demonstrated ahigh rate of infection by PCR in blood samples, often despitebeing seronegative.
The prevalence of FeLV antigenemia and FIV antibodieswere similar to other studies (Hartmann 2006, Sellon andHartmann 2006, Solano-Gallego et al. 2006a). Statisticalassociation was found between sick and FIV antibodies aspreviously described (Hartmann 2006, Sellon and Hartmann2006, Solano-Gallego et al. 2006a). In disagreement with pre-vious studies (Hartmann 2006, Sellon and Hartmann 2006),female cats were more likely to have FIV antibodies and FeLVantigenemia.
Recent studies have demonstrated T. gondii seroprevalencerates of 45% in Barcelona (Gauss et al. 2003), 30.8% in Madrid,and 33.7% in La Rioja (Miro et al. 2004) similar to the highseroprevalence found in the Ibiza island. No association wasfound between positive serology and/or PCR for Leishmaniaand T. gondii antibodies in this study. Although other studieshave described an immunologic relationship between FIVinfection and failure to control T. gondii infection (Levy et al.2004), no association between T. gondii seroreactivity and FIVinfection was found in this study. However, this cannotbe directly compared to presence or absence of T. gondiiantibodies.
In conclusion, the high seroprevalence and molecular ratesof Leishmania infection observed indicate that cats arefrequently infected with L. infantum, and the association withFeLV suggests a potential role for this retrovirus in thedevelopment of Leishmania infection in cats in endemic areas.
Acknowledgments
The authors would like to thank Dr. Alhelı RodrıguezCortes from Facultat de Veterinaria, Universitat Autonomade Barcelona, for the supplying the positive feline serumcontrols for setting up the ELISA and Aviva Petrie for as-sistance with the statistical analysis. Molecular testing waskindly supported by Laboratorio Privato Veterinario SanMarco (Padova, Italy).
Disclosure Statement
The authors do not have potential conflict of interest.
References
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Ayllon, T, Tesouro, MA, Amusategui, I, Villaescusa, A, et al.Serologic and molecular evaluation of Leishmania infantum incats from Central Spain. Ann N Y Acad Sci 2008; 1149:361–364.
Baneth, G, Koutinas, AF, Solano-Gallego, L, Bourdeau, P, et al.Canine leishmaniosis—new concepts and insights on an ex-panding zoonosis: part one. Trends Parasitol 2008; 24:324–330.
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Address correspondence to:Laia Solano-Gallego
Department of Pathology and Infectious DiseasesRoyal Veterinary College
University of LondonHawkshead Lane
North MymmsHatfield
Herts AL9 7TAUnited Kingdom
E-mail: [email protected]
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