Immunization of Experimental Dogs With Salivary Proteins From Lutzomyia longipalpis, Using DNA and Recombinant Canarypox Virus Induces Immune Responses Consistent With Protection Against Leishmania infantum

Metacyclic Leishmania promastigotes are transmitted by sand flies that inject parasites and saliva into the host's skin. Previous studies have demonstrated that DNA plasmids encoding Lutzomyia longipalpis salivary proteins LJM17 and LJL143, when used to immunize dogs, resulted in a systemic and local Th1 cell-mediated immunity that interfered in parasite survival in vitro. Here we evaluated the ability of these same salivary antigens to induce anti-Leishmania immunity and to confer protection by immunizing dogs using a novel vaccination strategy more suitable for use in the field. The strategy consisted of a single dose of plasmid followed by two doses of recombinant Canarypoxvirus (rCanarypoxvirus) expressing L. longipalpis salivary proteins (LJM17 or LJL143). Thirty days after the final immunization, dogs were intradermally challenged with 107Leishmania infantum promastigotes in the presence of L. longipalpis saliva. We followed the experimentally infected dogs for 10 months to characterize clinical, parasitological, and immunological parameters. Upon vaccination, all immunized dogs presented strong and specific humoral responses with increased serum concentrations of IFN-γ, TNF, IL-7, and IL-15. The serum of dogs immunized with LJM17 also exhibited high levels of IL-2, IL-6, and IL-18. L. infantum infection was established in all experimental groups as evidenced by the presence of anti-Leishmania IgG, and by parasite detection in the spleen and skin. Dogs immunized with LJM17-based vaccines presented higher circulating levels of IFN-γ, IL-2, IL-6, IL-7, IL-15, IL-18, TNF, CXCL10, and GM-CSF post-infection when compared with controls. Results demonstrated that relevant Leishmania-specific immune responses were induced following vaccination of dogs with L. longipalpis salivary antigen LJM17 administered in a single priming dose of plasmid DNA, followed by two booster doses of recombinant Canarypox vector. Importantly, a significant increase in pro-inflammatory cytokines and chemokines known to be relevant for protection against leishmaniasis was evidenced after challenging LJM17-vaccinated dogs as compared to controls. Although similar results were observed following immunization with LJL143, the pro-inflammatory response observed after immunization was attenuated following infection. Collectively, these data suggest that the LJM17-based vaccine induced an immune profile consistent with the expected protective immunity against canine leishmaniosis. These results clearly support the need for further evaluation of the LJM17 antigen, using a heterologous prime-boost vaccination strategy against canine visceral leishmaniosis (CVL)

Alterações na medula óssea e distúrbios hematológicos na leishmaniose visceral canina

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2017-06-07T17:37:34Z No. of bitstreams: 1 Valter dos Anjos Almeida Alterações na medula...2017.pdf: 4995398 bytes, checksum: 0a35ce89ed673cf501c263ddacb50492 (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2017-06-07T17:47:44Z (GMT) No. of bitstreams: 1 Valter dos Anjos Almeida Alterações na medula...2017.pdf: 4995398 bytes, checksum: 0a35ce89ed673cf501c263ddacb50492 (MD5)Made available in DSpace on 2017-06-07T17:47:44Z (GMT). No. of bitstreams: 1 Valter dos Anjos Almeida Alterações na medula...2017.pdf: 4995398 bytes, checksum: 0a35ce89ed673cf501c263ddacb50492 (MD5) Previous issue date: 2017CNPq, CPqGM, FIOCRUZ, CAPES e CYTEDFundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, BrasilINTRODUÇÃO: cães com leishmaniose visceral frequentemente apresentam uma ampla variedade de anormalidades hematológicas. Objetivo: neste estudo, objetivou-se associar os distúrbios hematológicos com a distribuição das linhagens celulares e o perfil de expressão de citocinas na medula óssea de cães naturalmente infectados por Leishmania infantum. MATERIAL E MÉTODOS: após obtenção do diagnóstico e exames clínico-laboratoriais, as amostras foram coletadas e analisadas por microscopia convencional. Feito isso, foram utilizadas técnicas de biologia molecular para detecção de L. infantum e verificação da expressão de citocinas que podem influenciar na diferenciação celular na medula óssea. RESULTADOS: diante das análises, a infecção por Leishmania foi detectada por ELISA em 47/58 (81,0%) e por cultura esplênica em 18/57 (31,6%). A maioria dos cães com cultura esplênica positiva apresentou detecção do DNA de L. infantum na medula óssea (p=0,0004) acompanhado por histiocitose (p=0,0046). Destes animais, os que também possuíam desorganização esplênica apresentaram diminuição de hemácias (p=0,0066), acompanhado pela menor quantidade de células da linhagem eritróide na medula óssea em relação aos com cultura negativa e baço organizado (p=0,0127). Essas alterações foram acompanhadas pela elevação da expressão gênica IFN-γ (p=0,0015) e TNF (p=0,0091) nos cães que possuíam L. infantum na medula óssea. CONCLUSÕES: desta maneira, cães com cultura esplênica positiva apresentam alterações de parâmetros hematológicos referentes a anemia e leucocitose com neutrofilia que são acompanhadas de hipoplasia eritróide e estão associadas a mudanças nas expressões de citocinas no microambiente medular, como o aumento de IFN- γ e TNF.INTRODUCTION: dogs with visceral leishmaniasis often have a wide variety of hematological abnormalities. Objective: this study aimed to associate hematological disorders with the distribution of cell lineages and the profile of cytokine expression in the bone marrow of dogs naturally infected by Leishmania infantum. MATERIAL AND METHODS: after obtaining the diagnosis and the clinical laboratory tests, samples were collected and analyzed by conventional microscopy. After that, molecular biology techniques were used for detection of L. infantum and verification of cytokines expression that could influence in cell differentiation in the bone marrow. RESULTS: on the analysis, the Leishmania infection was detected by ELISA in 47/58 (81.0%) and splenic culture in 18/57 (31.6%). On the analysis, the Leishmania infection was detected by ELISA in 47/58 (81.0%) and splenic culture in 18/57 (31.6%). The majority of dogs with positive spleen culture had detection of L. infantum DNA in the bone marrow (p = 0.0004) followed by histiocytosis (p = 0.0046). Of these animals, those with splenic disorganization, they also presented a decrease of red blood cells (p = 0.0066), accompanied by a decrease of erythroid lineage cells in bone marrow compared to those with negative culture and organized spleen (p = 0.0127). These changes were accompanied by elevated gene expression IFN-γ (p = 0.0015) and TNF (p = 0.0091) in dogs with L. infantum in the bone marrow. CONCLUSIONS: thus, dogs with positive spleen culture present anemia and leukocytosis with neutrophilia that are accompanied by erythroid hypoplasia and are associated with changes in the expression of cytokines in bone marrow microenvironment, such as the increase of IFN-γ and TNF

Detecção do DNA de Leishmania infantum em hamsters infestados com carrapatos coletados de cães naturalmente infectados

RESUMO. Almeida V. dos A., da Hora T.N., Leça Júnior N.F., Carvalho F.S., da Silva A.L., Wenceslau A.A., Albuquerque G.R. & Silva F.L. Detection of Leishmania infantum DNA in hamsters infested with ticks collected from naturally infected dogs. [Detecção do DNA de Leishmania infantum em hamsters infestados com carrapatos coletados de cães naturalmente infectados.] Revista Brasileira de Medicina Veterinária, 38(4):329-333, 2016. Departamento de Ciências Agrárias e Ambientais, Universidade Estadual de Santa Cruz, Campus Soane Nazaré de Andrade, Hospital Veterinário, Km 16, Rodovia Jorge Amado, Ilhéus, BA 45662-900, Brasil. E-mail: [email protected] O objetivo desse estudo foi investigar a participação de Rhipicephalus sanguineus na transmissão da Leishmania infantum. Para isso, foram utilizados 24 hamsters adultos da linhagem Golden, de ambos os sexos, os quais foram divididos em dois grupos: controle (n = 4) e grupo experimental (n = 20). Os animais do grupo experimental foram infestados com carrapatos obtidos de cães naturalmente infectados por L. infantum. Os hamsters do grupo controle não foram infestados e foram mantidos sob as mesmas condições dos outros animais. Após três meses de observação, os animais foram submetidos à eutanásia e necropsia para obtenção de amostras de sangue, 35 baço, fígado, linfonodos e pele para histopatologia, imuno-histoquímica e reação da polimerase em cadeia (PCR). Quatorze hamsters (70%) do grupo experimental tiveram resultados positivos na PCR para detecção do DNA de L. infantum em amostras de capa de leucócitos. Os resultados obtidos nesse estudo sugerem que carrapatos R. sanguineus podem transmitir algumas formas ou partes de L. infantum para hamsters parasitados

Frequency of species of the Genus Eimeria in naturally infected cattle in Southern Bahia, Northeast Brazil

The aim of this study was to determine the presence of species of the genus Eimeria species in naturally infected bovines in Southern Bahia, Northeast Brazil. The study population comprised 117 Zebu crossbred cattle that belonged to 10 dairy herds with extensive or semi-extensive production systems. The modified Gordon and Whitlock technique was used to determine positive samples and number of oocysts per gram of feces. Statistical analyses were performed using the chi-square test with Yates correction and a 95% confidence interval. Thirty-nine cattle (33.33%) were positive, and ten different species were identified in infected animals: E. bovis (24.79%); E. canadensis (8.55%); E. zuernii (6.83%); E. ellipsoidalis (5.99%); E. cylindrica (3.42%); E. auburnensis (3.42%); E. brasiliensis (2.56%); E. bukidnonensis (1.71%); E. alabamensis (0.85%), and E. subspherica (0.85%). Higher parasitism was observed in animals up to one year of age (p = 0.005), but no animal presented clinical signs of the disease. As the presence of clinical eimeriosis was not evidenced and all animals were Zebu crossbred cattle from extensive or semi-extensive production systems, further studies should be conducted to investigate the effects of these factors on disease development

First report of Trypanosoma cruzi infection in naturally infected dogs from southern Bahia, Brazil Primeiro relato de infecção natural por Trypanosoma cruzi em cães do sul da Bahia, Brasil

In order to verify the Trypanosoma cruzi infection in domestic domiciled dogs in a rural endemic area from the south region of the State of Bahia, Polymerase Chain Reaction (PCR) were performed using S35 and S36 primers in 272 dogs living in the district of Vila Operaria, in the municipality of Buerarema. All animals were clinically evaluated; 2.5 mL of blood were collected through venipuncture for the performance of molecular tests. None of these animals showed clinical signs of the illness and only two were identified with the DNA parasite. This result is the first report of natural infection by T. cruzi in domestic dogs in southern Bahia.<br>Com o objetivo de verificar a infec&#231;&#227;o por Trypanosoma cruzi em c&#227;es dom&#233;sticos domiciliados em &#225;rea rural e end&#234;mica do sul da Bahia, foi realizada a Rea&#231;&#227;o em Cadeia da Polimerase (PCR), utilizando-se os iniciadores S35 e S36 em 272 c&#227;es domiciliados no distrito da Vila Oper&#225;ria, cidade de Buerarema. Todos os animais foram avaliados clinicamente e, posteriormente, foram coletados 2,5 mL de sangue por pun&#231;&#227;o venosa para realiza&#231;&#227;o do diagn&#243;stico molecular. Nenhum dos animais apresentou manifesta&#231;&#227;o cl&#237;nica da doen&#231;a e, em apenas dois foram identificados DNA do parasito. Esse resultado &#233; o primeiro relato de infec&#231;&#227;o natural por T. cruzi em c&#227;es dom&#233;sticos no sul baiano

Antigenicity of Leishmania-Activated C-Kinase Antigen (LACK) in Human Peripheral Blood Mononuclear Cells, and Protective Effect of Prime-Boost Vaccination With pCI-neo-LACK Plus Attenuated LACK-Expressing Vaccinia Viruses in Hamsters

Leishmania-activated C-kinase antigen (LACK) is a highly conserved protein among Leishmania species and is considered a viable vaccine candidate for human leishmaniasis. In animal models, prime-boost vaccination with LACK-expressing plasmids plus attenuated vaccinia viruses (modified vaccinia Ankara [MVA] and mutant M65) expressing LACK, has been shown to protect against cutaneous leishmaniasis (CL). Further, LACK demonstrated to induce the production of protective cytokines in patients with active CL or cured visceral leishmaniasis, as well as in asymptomatic individuals from endemic areas. However, whether LACK is capable to trigger cytokine release by peripheral blood mononuclear cells from patients cured of CL due to Leishmania infantum (L. infantum) or induce protection in L. infantum-infected hamsters [visceral leishmaniasis (VL) model], has not yet been analyzed. The present work examines the ex vivo immunogenicity of LACK in cured VL and CL patients, and asymptomatic subjects from an L. infantum area. It also evaluates the vaccine potential of LACK against L. infantum infection in hamsters, in a protocol of priming with plasmid pCI-neo-LACK (DNA-LACK) followed by a booster with the poxvirus vectors MVA-LACK or M65-LACK. LACK-stimulated PBMC from both asymptomatic and cured subjects responded by producing IFN-γ, TNF-α, and granzyme B (Th1-type response). Further, 78% of PBMC samples that responded to soluble Leishmania antigen showed IFN-γ secretion following stimulation with LACK. In hamsters, the protocol of DNA-LACK prime/MVA-LACK or M65-LACK virus boost vaccination significantly reduced the amount of Leishmania DNA in the liver and bone marrow, with no differences recorded between the use of MVA or M65 virus vector options. In summary, the Th1-type and cytotoxic responses elicited by LACK in PBMC from human subjects infected with L. infantum, and the parasite protective effect of prime/boost vaccination in hamsters with DNA-LACK/MVA-LACK and DNA-LACK/M65-LACK, revealed the significance of LACK in activating human and hamster immune responses and support LACK to be a valuable candidate for inclusion in a vaccine against human VL.EC was supported by a contract from RD16CIII/0003/0002 Red de Enfermedades Tropicales, Subprograma RETICS del Plan Estatal de I + D + I 2013-2016, co-funded by FEDER “Una manera de hacer Europa” funds. ME was supported by AEI MINECO/FEDER grant SAF2013-45232-R.S

First report of Trypanosoma cruziinfection in naturally infected dogs from southern Bahia, Brazil

In order to verify the Trypanosoma cruzi infection in domestic domiciled dogs in a rural endemic area from the south region of the State of Bahia, Polymerase Chain Reaction (PCR) were performed using S35 and S36 primers in 272 dogs living in the district of Vila Operaria, in the municipality of Buerarema. All animals were clinically evaluated; 2.5 mL of blood were collected through venipuncture for the performance of molecular tests. None of these animals showed clinical signs of the illness and only two were identified with the DNA parasite. This result is the first report of natural infection by T. cruzi in domestic dogs in southern Bahia

Antigenicity of Leishmania-Activated C-Kinase Antigen (LACK) in Human Peripheral Blood Mononuclear Cells, and Protective Effect of Prime-Boost Vaccination With pCI-neo-LACK Plus Attenuated LACK-Expressing Vaccinia Viruses in Hamsters

Leishmania-activated C-kinase antigen (LACK) is a highly conserved protein among Leishmania species and is considered a viable vaccine candidate for human leishmaniasis. In animal models, prime-boost vaccination with LACK-expressing plasmids plus attenuated vaccinia viruses (modified vaccinia Ankara [MVA] and mutant M65) expressing LACK, has been shown to protect against cutaneous leishmaniasis (CL). Further, LACK demonstrated to induce the production of protective cytokines in patients with active CL or cured visceral leishmaniasis, as well as in asymptomatic individuals from endemic areas. However, whether LACK is capable to trigger cytokine release by peripheral blood mononuclear cells from patients cured of CL due to Leishmania infantum (L. infantum) or induce protection in L. infantum-infected hamsters [visceral leishmaniasis (VL) model], has not yet been analyzed. The present work examines the ex vivo immunogenicity of LACK in cured VL and CL patients, and asymptomatic subjects from an L. infantum area. It also evaluates the vaccine potential of LACK against L. infantum infection in hamsters, in a protocol of priming with plasmid pCI-neo-LACK (DNA-LACK) followed by a booster with the poxvirus vectors MVA-LACK or M65-LACK. LACK-stimulated PBMC from both asymptomatic and cured subjects responded by producing IFN-γ, TNF-α, and granzyme B (Th1-type response). Further, 78% of PBMC samples that responded to soluble Leishmania antigen showed IFN-γ secretion following stimulation with LACK. In hamsters, the protocol of DNA-LACK prime/MVA-LACK or M65-LACK virus boost vaccination significantly reduced the amount of Leishmania DNA in the liver and bone marrow, with no differences recorded between the use of MVA or M65 virus vector options. In summary, the Th1-type and cytotoxic responses elicited by LACK in PBMC from human subjects infected with L. infantum, and the parasite protective effect of prime/boost vaccination in hamsters with DNA-LACK/MVA-LACK and DNA-LACK/M65-LACK, revealed the significance of LACK in activating human and hamster immune responses and support LACK to be a valuable candidate for inclusion in a vaccine against human VL.Ministerio de Economía, Comercio y Empresa (España)Depto. de Medicina y Cirugía AnimalFac. de VeterinariaTRUEpu

Disruption of Splenic Lymphoid Tissue and Plasmacytosis in Canine Visceral Leishmaniasis: Changes in Homing and Survival of Plasma Cells

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2017-02-09T13:18:57Z No. of bitstreams: 1 O'Hare JS Disruption of splenic....pdf: 6751415 bytes, checksum: 538de97cdfa0b81cb18100e980cf6923 (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2017-02-09T13:47:07Z (GMT) No. of bitstreams: 1 O'Hare JS Disruption of splenic....pdf: 6751415 bytes, checksum: 538de97cdfa0b81cb18100e980cf6923 (MD5)Made available in DSpace on 2017-02-09T13:47:07Z (GMT). No. of bitstreams: 1 O'Hare JS Disruption of splenic....pdf: 6751415 bytes, checksum: 538de97cdfa0b81cb18100e980cf6923 (MD5) Previous issue date: 2016Fundação de Amparo à Pesquisa do Estado da Bahia (Fapesb). Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes) and Fundação Oswaldo Cruz (Fiocruz).Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilUniversidade Federal da Bahia. Faculdade de Farmácia. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilUniversidade Federal da Bahia. Faculdade de Farmácia. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / Universidade Federal da Bahia. Faculdade de Medicina. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilVisceral leishmaniasis (VL) is a disease caused by Leishmania infantum, which is transmitted by phlebotomine sandflies. Dogs are the main urban reservoir of this parasite and the disease presents similar characteristics in both humans and dogs. In this paper, we investigated the potential pathways involved in plasma cell replacement of normal cell populations in the spleen, with respect to disease severity in dogs from an endemic area for visceral leishmaniasis. To this end, canine spleen samples were grouped into three categories: TYPE1SC- (non-infected dogs or without active infection with organized white pulp), TYPE1SC+ (infected dogs with organized white pulp) or TYPE3SC+ (infected animals with disorganized white pulp). We analyzed the distribution of different plasma cell isotypes (IgA, IgG and IgM) in the spleen. The expression of cytokines and chemokines involved in plasma cell homing and survival were assessed by real time RT-PCR. Polyclonal B cell activation and hypergammaglobulinemia were also evaluated. The proportion of animals with moderate or intense plasmacytosis was higher in the TYPE3SC+ group than in the other groups (Fisher test, P<0.05). This was mainly due to a higher density of IgG+ plasma cells in the red pulp of this group. The albumin/globulin ratio was lower in the TYPE3SC+ animals than in the TYPE1SC- or TYPE1SC+ animals, which evidences VL-associated dysproteinemia. Interestingly, TYPE3SC+ animals showed increased expression of the BAFF and APRIL cytokines, as well as chemokine CXCL12. Aberrant expression of BAFF, APRIL and CXCL12, together with amplified extrafollicular B cell activation, lead to plasma cell homing and the extended survival of these cells in the splenic red pulp compartment. These changes in the distribution of immunocompetent cells in the spleen may contribute to the progression of VL, and impair the spleen's ability to protect against blood borne pathogens

Epidemiology of canine leishmaniasis in southern Bahia, Brazil Nilo

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2015-05-28T17:07:28Z No. of bitstreams: 1 Leça Junior NF Epidemiology....pdf: 313446 bytes, checksum: 2e1aa7a47df4454b56ddcf85b2ed3f6f (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2015-05-28T17:21:24Z (GMT) No. of bitstreams: 1 Leça Junior NF Epidemiology....pdf: 313446 bytes, checksum: 2e1aa7a47df4454b56ddcf85b2ed3f6f (MD5)Made available in DSpace on 2015-05-28T17:21:24Z (GMT). No. of bitstreams: 1 Leça Junior NF Epidemiology....pdf: 313446 bytes, checksum: 2e1aa7a47df4454b56ddcf85b2ed3f6f (MD5) Previous issue date: 2015Ciência Animal. Araguaína, TO, BrasilUniversidade Estadual de Santa Cruz. UESC. Pós-Graduação em Ciência Animal. Ilhéus, BA, BrasilVeterinária. Ilhéus, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilUniversidade Estadual de Santa Cruz. UESC. Pós—Graduação em Genética e Biologia Molecular. Ilhéus, BA, BrasilUniversidade Estadual de Santa Cruz. UESC. Hospital Veterinário. Departamento de Ciências Agrárias e Ambientais. Ilhéus, BA, BrasilUniversidade Estadual de Santa Cruz. UESC. Hospital Veterinário. Departamento de Ciências Agrárias e Ambientais. Ilhéus, BA, BrasilUniversidade Estadual de Santa Cruz. UESC. Hospital Veterinário. Departamento de Ciências Agrárias e Ambientais. Ilhéus, BA, BrasilUniversidade Estadual de Santa Cruz. UESC. Hospital Veterinário. Departamento de Ciências Agrárias e Ambientais. Ilhéus, BA, BrasiltLeishmaniosis is a zoonosis caused by protozoa of the genus Leishmania. American cutaneous leishman-iosis (ACL) is mainly caused by the species L. amazonensis and L. braziliensis, and American visceralleishmaniosis (AVL) is caused by L. infantum chagasi. In addition to their proven roles as reservoirs ofAVL, dogs are also suspected by researchers to be reservoirs of ACL due to reports of this infection indomestic environments and of infected dogs in endemic areas. The aim of this study was to detect Leish-mania sp. infection in dogs from Vila Operária, Buerarema, Bahia, using parasitological tests, indirectimmunofluorescent assay (IFA) and polymerase chain reaction (PCR). Furthermore, this study also aimedto identify risk factors associated with illness in dogs in this locality by conducting an epidemiologicalsurvey. For this purpose, 292 dogs were clinically evaluated for the presence of skin lesions, and thedogs that showed these changes were submitted to scarification injury to enable preparation of slidesfor microscopic study of amastigotes. Subsequently, the dogs underwent blood sampling for serological(IFA) and molecular (PCR) tests. Additionally, the owners of the dogs answered an epidemiological ques-tionnaire to facilitate the identification of risk factors for exposure of dogs to pathogens of ACL. Of the 292dogs studied, 13 (4.5%) had lesions suggestive of ACL, but with a negative parasitological examinationand 147 (50.3%) were seropositive according to the IFA. Of the 273 dogs studied using PCR test, 10 (3.66%)were positive for L. braziliensis, and all samples were negative for L. infantum chagasi. Wastelands in theperidomicile and the presence of light in the household were risk factors associated with ACL. The resultsshow that Vila Operária has asymptomatic dogs with ACL and that the detection sensitivity of the IFAwas higher than that of PCR for the infected dogs