Abstract
Background
: This study aimed to evaluate the occurrence of Leishmaniaspp. in dogs and cats from Botucatu, São Paulo state, and Campo Grande, Mato Grosso do Sul state, Brazil, by the association of three diagnostic tests: blood culture in liver infusion tryptose medium, immunofluorescent antibody test and polymerase chain reaction. Fifty blood samples of dogs and cats from the Center for Zoonosis Control in Campo Grande, an area endemic for canine visceral leishmaniasis, were collected randomly, as well as canine and feline blood samples from the Municipal Kennel and Animal Protection Association in Botucatu, currently considered a transmission-free, non-endemic area.
Results
: Of the 50 dog blood cultures from Botucatu, three (6%) were positive and of the 50 cats, two (4%) were positive. In Campo Grande, 29 dog blood cultures (58%) were positive and all (100%) cats negative by this test. Polymerase chain reaction detected Leishmania spp. in 100% of dog and cat samples from Botucatu but found all the cats from Campo Grande to be negative. On the other hand, 36 dogs from Campo Grande were positive (72%) by the same technique. Immunofluorescent antibody test in Botucatu found 100% of dogs and cats non-reactive, while in Campo Grande, it detected positivity in 32 dogs (64%) and 15 cats (30%).
Conclusions
: The results show the importance of not only continuous epidemiological surveillance in areas not endemic for leishmaniasis, but also research for accurate diagnosis of this zoonosis.
Dog; Cat; Diagnosis; Leishmaniasis; Zoonosis
Background
Leishmania infantum (syn. L. chagasi) is the causative agent of visceral leishmaniasis (VL) in the New World, with endemic areas ranging from the southern USA to northern Argentina [11 . Kuhls K, Alam MZ, Cupolillo E, Ferreira GEM, Mauricio IL, Oddone R, Feliciangeli MD, Wirth T, Miles MA, Schonian G: Comparative microsatellite typing of new world Leishmania infantum reveals low heterogeneity among populations and its recent old world origin. PLoS Negl Trop Dis 2011, 5(6):e1155.]. It is considered an important but neglected disease that affects many countries and, in recent years, has become an important public health problem, transmitted to humans through stings of phle-botomine sand flies Lutzomyia longipalpis. Brazil is a country with a high number of cases (~90%) [22 . Romero GA, Boelaert M: Control of visceral leishmaniasis in latin america - a systematic review. PLoS Negl Trop Dis2010,4(1):e584.].
Some behavioral features of cats, such as nocturnal predatory hunting, walking as far as 1.5 km from their homes, and cohabitating in wild and domestic areas, favor the dissemination of the parasite to this species [33 . da Silva AVM, de Souza Cândido CD, Pita-Pereira D, Brazil RP, Carreira JCA: The first record of American visceral leishmaniasis in domestic cats from Rio de Janeiro, Brazil. Acta Trop 2008, 105(1):92-94.].
The frequent failures to diagnose feline leishmaniasis (FL) are attributable not only to the widespread unaware-ness of the disease by health professionals including veterinarians, along with the diminished frequency of cats in veterinary clinics, but also to the scarcity of studies about the epidemiological and clinical-pathological aspects of the disease. This context has hampered understanding of the true role of cats as reservoirs of Leishmaniaspp. and their importance in public health [44 . Simões-Mattos L, Mattos MRF, Teixeira MJ, Oliveira-Lima JW, Bevilaqua CML, Prata-Júnior RC, Holanda CM, Rondon FCM, Bastos KMS, Coêlho ZCB, Coêlho ICB, Barral A, Pompeu MML: The susceptibility of domestic cats(Felis catus) to experimental infection withLeishmania braziliensis. Vet Parasitol2005, 127(3-4):199-208.
5 . Dantas-Torres F, Simões-Matos L, Brito FLC, Figueiredo LA, Faustino MAG: Leishmaniose Felina: revisão de literatura. Rev Clín Vet2006, 61:32-40.
6 . Quinnell RJ, Courtenay O: Transmission, reservoir hosts and control of zoonotic visceral leishmaniasis. Parasitology 2009, 136(14):1915-1934.-77 . Maia C, Campino L: Can domestic cats be considered reservoir hosts of zoonotic leishmaniasis? Trends Parasitol 2011, 27(8):341 -344.].
The greatest difficulty found is posed by the diagnosis of canine visceral leishmaniasis (CVL), since the - methods utilized to its control are based on antibody research, which has its limitations. Thus, the identification of infected dogs is the key point to interrupt the epide-miologic chain of the disease in urban areas.
Serological diagnosis of CVL previously recommended by the Program of Surveillance and Control of Leishmaniasis was comprised of ELISA as the screening method and im-munofluorescent antibody test (IFAT) as confirmatory [88 . Secretaria de Vigilância em Saúde: Departamento de Vigilância Epidemiológica: Manual de vigilância e controle da leishmaniose visceral. In Série A. Normas e manuais técnicos. Brasília: Ministério da Saúde. 2003:9-18.]. In order to improve the diagnostic technique of CVL, the Ministry of Health has established the replacement of the currently used protocol (screening with ELISA and confirmation with IFAT), with the deployment of rapid immunoassay with recombinant antigens (k26 and k39) as screening and ELISA as confirmatory [99 . Secretaria de Vigilância em Saúde: Departamento de Vigilância das Doenças Transmissíveis: Nota técnica conjunta N°1/2011 CGDT-CGLAB/ DEVST/SVS/MS.In Esclarecimentos sobre substituição do protocolo diagnóstico da leishmaniose visceral canina (LVC). Brasília: Ministério da Saúde; 2011.].
The isolation of promastigote forms of Leishmania spp. by means of culturing any of several tissues, such as blood in the case of blood cultures, though laborious, is also a possible technique [1010 . Greene CE: In Infectious diseases of the dog and cat.3rd edition. Edited by Greene CE. Canadá: Saunders/Elsevier; 2006:1387.].
Among molecular methods, the polymerase chain reaction (PCR) has been used as a tool in epidemiological research studies to identify species of Leishmaniaspp. by selective amplification of DNA sequences of the parasite. The DNA detection is possible in a variety of tissues, including bone marrow, skin biopsies, lymph node aspirates, blood, histological sections of paraffin-embedded tissues and also in the vector [1010 . Greene CE: In Infectious diseases of the dog and cat.3rd edition. Edited by Greene CE. Canadá: Saunders/Elsevier; 2006:1387.,1111 . Ikeda-Garcia FA, Marcondes M: Métodos de diagnóstico da leishmaniose visceral canina. Rev Clín Vet 2007, 71:34-42.].
For better diagnostic acuity of VL, it is necessary to employ a combination of techniques since there is no method that singly gathers all desirable features for the diagnosis, such as: easy execution, accessible cost, rapidity and especially high sensitivity and specificity. It is recommended that this disease be diagnosed based on clinical symptomatology, on the epidemiological features of the region and on laboratorial exams, thereby contributing to the correct treatment of truly positive animals. The present work aimed to verify the occurrence ofLeishmania spp. in dogs and cats from an area endemic for leishmaniasis (Campo Grande, Mato Grosso do Sul state) and another non-endemic area (Botucatu, São Paulo state). For both, we used the association of thee diagnostic methods: blood culturing, IFAT and the PCR from the blood cultures of these animals.
Methods
Animals
Two hundred animals were studied, one hundred from Botucatu (fifty dogs and fifty cats) and one hundred from Campo Grande (fifty dogs and fifty cats). The analysis performed was EpiInfo.
Blood cultures
The blood samples were collected randomly in Campo Grande, MS, at the Center for Zoonosis Control (CZC) and in Botucatu, SP, at the Municipal Kennel and Animal Protection Association (APA). A blood volume from 5 mL to 8 mL was collected from each animal, through jugular vein puncture, into tubes with EDTA, and kept refrigerated until their arrival at the laboratory, where they were immediately processed for blood culturing.
Processing sites and reading from blood cultures
The blood samples of animals from Botucatu, SP, were processed at the Laboratory of Animal Health of the São Paulo Agency of Agribusiness Technology (APTA/SAA), Bauru, SP, whereas those from Campo Grande, MS, were processed at the School of Medicine and Animal Husbandry of the Federal University of Mato Grosso do Sul (UFMS) in the same city. The readings were monitored at the Laboratory of Animal Health of APTA/SAA.
Blood culture in liver infusion tryptose (LIT)
The culture medium used for the blood samples was LIT. These blood samples were manipulated in a laminar flow cabinet, previously cleaned with 70% alcohol and kept under ultraviolet light for 20 minutes. For each collected blood sample, the plasmatic and leukocyte portion and the sediment of the erythrocytes were inoculated respectively in three sterile threaded tubes containing 5 mL of sterile LIT medium each. Then the cultures were incubated and maintained under a temperature of 28 to 30°C, until four months after inoculation, when they were submitted to PCR for Leishmania spp.
Reading of the blood cultures
After ten days of the samples inoculation, the cultures were observed every 15 days, for four months, using optical microscopy with 1000x magnification. Both positive and negative cultures were immediately processed for extraction of the parasitic DNA, after the readings were concluded.
Preparation of blood samples in LIT for the extraction of parasitic DNA
The positive and negative cultures were washed separately in sterile buffered saline (PBS), 0.01 M (pH 7.2) and centrifuged at 1000 rpm for ten minutes and the sediment stored in sterile DNAse- and RNAse-free microtubes at -20°C, until the moment of extraction of the parasitic DNA [1212 . Pinto PLS: Circulação e caracterização de Trypanosoma cruzi isolados de mamíferos silvestres capturados no Estado de São Paulo, Brasil. Rev Inst Med Trop Sao Paulo 2001, 43(1):44.].
DNA extraction
The DNA was extracted from 300 μΙ, of the stored sediment, subjected to an application of Illustra™ blood genomic Prep Mini Spin Kit (GE Healthcare®), and then stored in sterile DNAse- and RNAse-free microtubes and kept at -20°C
Polymerase chain reaction (PCR)
The primers LINR4 (5' GGGGTTGGTGTAAAATAG GG 3')and LIN19 (5' CAGAACGCCCCTACCCG 3') were used as described by Ikonomopoulos et al. [1313 . Ikonomopoulos J, Kokotas S, Gazouli M, Zavras A, Stoitsiou M, Gorgoulis VG: Molecular diagnosis of leishmaniosis in dogs. Comparative application of traditional diagnostic methods and the proposed assay on clinical samples. Vet Parasitol 2003, 113(2):99-113.]. All the reactions were performed in duplicate, with 2.5 μL of PCR buffer (50 mmol KCl, 10 mmol of Tris-HCl, 1.5 mM MgCl2), 0.2 mM of deoxynucleotide triphosphate, 1.0 U of Taq Polymerase, 10 pmol of each initiator, 2 μL of DNA and 17.8 μL of ultrapure water to compose a final volume of 25 μL.
The amplification steps were carried out in a thermal cycler (MasterCycler® Personal, Eppendorf, Germany) according to Ikonomopoulos et al. [1313 . Ikonomopoulos J, Kokotas S, Gazouli M, Zavras A, Stoitsiou M, Gorgoulis VG: Molecular diagnosis of leishmaniosis in dogs. Comparative application of traditional diagnostic methods and the proposed assay on clinical samples. Vet Parasitol 2003, 113(2):99-113.], as follows: initial denaturation in a cycle at 95°C for three minutes, followed by 33 cycles at 95°C for 30 seconds, 58°C for 30 seconds and 72°C for one minute and a final extension of 72°C for seven minutes.
The amplified products were identified by means of gel electrophoresis in 1.5% agarose prepared in Tris-borato-EDTA (TBE) buffer 1.0 X and stained with ethidium bromide. The size of the amplified products was compared with the 100 bp ladder and visualized through Gel Doc - It™ Imaging System, using VisionWorks® LS Software (UVP, USA).
Immunofluorescent antibody test (IFAT)
The IFAT for Leishmania spp. was performed according to Camargo [1414 . Camargo ME: Fluorescent antibody test for the sorodiagnosis of american tripanosomiasis: technical modification employing preserved culture forms of Trypanosoma cruzi in a slide test. Rev Inst Med Trop Sao Paulo 1966, 8(5):227-234.].
Ethics statement
This study was approved by the Ethics Committee on Animal Experimentation of the School of Veterinary Medicine and Animal Husbandry, São Paulo State - University (UNESP), Botucatu, SP, Brazil under protocol number 65/2007.
Statistical analysis
The diagnostic techniques in this study were evaluated through the estimation of accuracy, sensitivity, specificity, negative predictive value, positive predictive value and kappa coefficient for concordance among the diagnoses of the tests, using PCR as the gold standard. Chi-square test and Fischer's exact test were performed to estimate the association between clinical signs and the positivity for Leishmania spp. [1515 . Medronho AR, Bloch KV, Luiz RR, Werneck GL: Epidemiologia. 2° edição. Atheneu: São Paulo; 2009.].
Results and discussion
Blood culture, IFAT and PCR for Leishmania spp
Of the analyzed canine blood cultures from Botucatu, three (6%) were positive and forty-seven (94%) negative. But among cats, two samples (4%) were positive and 48 (96%) negative. Both the IFAT and PCR for Leishmania spp. found that 100% of both the dog and cat samples were negative. The blood cultures of animals from Campo Grande presented 29 (58%) dog samples positive and 21 (42%) negative. All (100%) of the feline samples were negative by blood culture as well by PCR for Leishmania spp. As for the dogs, 36 (72%) samples were positive and 14 (28%) were negative by PCR forLeishmania spp. By IFAT, 32 (64%) dogs were positive and 18 (36%) negative. Fifteen cats (30%) presented positivity by IFAT forLeishmania spp. whereas 35 (70%) were negative (Table 1). The serum titers varied from 80 to 640 in dogs and 40 to 320 in cats.
Blood cultures are considered positive when, through an optical microscope, it is possible to see the presence of flagellate parasites, which, kept in LIT, can be characterized through biochemical and/or molecular biology techniques [1616 . Avelar JB: Caracterização molecular de isolados deTrypanosoma cruzi obtidos de mulheres durante a fase crônica da doença de Chagas. In Dissertação de Mestrado.Universidade Federal de Goiás, Instituto de Patologia Tropical e Saúde Pública; 2008. Available at: http://www.openthesis. org/documents/molecular-de-isolados-Trypanosoma-cruziobtidos-287795.html.
http://www.openthesis. org/documents/mol...
]. Because of its high specificity, this tech-niquemakesthepositiveculturespresentahighvalue, which is important for the isolation and identification of the parasite [1111 . Ikeda-Garcia FA, Marcondes M: Métodos de diagnóstico da leishmaniose visceral canina. Rev Clín Vet 2007, 71:34-42.,1717 . Chiari E: Chagas disease diagnosis using polymerase chain reaction, hemoculture and serologic methods. Mem Inst Oswaldo Cruz1999, 94(Suppl 1):299-300.].
Out of 50 blood cultures of dogs from Campo Grande, 29 (58.0%) were positive, but the actual presence of Leishmania spp. parasites was confirmed by means of PCR in 25 (86.2%), revealing a blood culture sensitivity of 69.4% (Table 2). The flagellate forms seen in four samples were negative by PCR, as well as three positive dog cultures and two positive cat cultures from Botucatu. These positive samples were 100% negative by IFAT and PCR tests, suggesting that the parasites seen in the first culture were other trypanosomatids. However, there was no confirmation by PCR, because it was not the aim of this study.
The suspicion above is due to the phylogenetic proximity between these parasites that belong to the same family (Trypanosomatidae), because, despite several studies demonstrating the same cross-reaction by serological methods, the morphological presentation is highly similar in culture.
Of the 21 negative blood cultures of these dogs, ten (47.6%) presented negative results equivalent to those obtained by PCR, while the blood culture technique presented 71.4% specificity (Table 2). Similarly, eight (38.0%) serum samples from these animals presented negative IFAT. Given this context, we suggest the absence of the parasite. The two remaining samples displayed high IFAT titers, both with values of at least 640, indicating that the dogs may have been infected. The finding may be attributable to the low parasite load at the moment of collection.
Eleven dogs (22.0%) from Campo Grande were found negative by blood culturing, but positive by PCR for Leishmania spp., due to the low sensitivity of the former, which can present negative or non-conclusive results, especially when the parasitaemia is sufficiently low to hamper observation of the parasites through an optical microscope.
The Campo Grande feline samples presented 100% negativity in both blood cultures and PCR, whereas the IFAT titers varied from 40 to 320. In this case, a judicious analysis must be made, because it is believed that the disease in this species has been misdiagnosed, besides the possibility of cross-reactions with other try-panosomes [44 . Simões-Mattos L, Mattos MRF, Teixeira MJ, Oliveira-Lima JW, Bevilaqua CML, Prata-Júnior RC, Holanda CM, Rondon FCM, Bastos KMS, Coêlho ZCB, Coêlho ICB, Barral A, Pompeu MML: The susceptibility of domestic cats(Felis catus) to experimental infection withLeishmania braziliensis. Vet Parasitol2005, 127(3-4):199-208.,1818 . Luciano RM, Lucheis SB, Troncarelli MZ, Luciano DM, Langoni H: Avaliação da reatividade cruzada entre antígenos de Leishmaniaspp. e Trypanosoma cruzi na resposta sorológica de cães pela técnica de imunofluorescência indireta (RIFI). Braz J Vet Res Anim Sci 2009, 46(3):181-187.].
The seroprevalence for leishmaniasis in the 50 dogs evaluated at the CZC in Campo Grande was 64% (32/50), with antibody titers that vary from 80 to equal or superior to 640, considered high when compared to the prevalence indices from epidemiological surveys performed in other Brazilian endemic areas. It is quite important to highlight that, although the canine samples in the present work were collected randomly at the Center for Zoonosis Control in Campo Grande, the prevalence of euthanized dogs in endemic areas is higher than the actual prevalence in the canine population as a whole, because most animals sent to these centers that present clinical suspicion for VL are sick or have already been serologically identified in mass surveys.
Albuquerque et al. [1919 . Albuquerque AR Aragão FR Faustino MAG Gomes YM Lira RA Nakasawa M Alves LC: Aspectos clínicos de cães naturalmente infectados porLeishmania (Leishmania) chagasi na região metropolitana do Recife. Rev Clin Vet 2007, 71:78-80.] also verified seropositivity in 64% (16/25), but only using symptomatic animals from the city of Recife in the Brazilian state of Pernambuco (PE). In the same study, but in Paulista, also a city endemic for VL, Dantas-Torres and Brandão Filho [2020 . Torres FD, Brandão Filho SP: Soroprevalência da leishmaniose visceral canina no Município de Paulista, Pernambuco, Brasil.InAnclivepa Brasil. 3rd edition. 2005:343-344. http://www.uece.br/cienciaanimal/dmdocuments/ Comunicacao1.2006.1.pdf.
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] evaluated 322 dogs of which 130 (40.3%) were found serologically positive by IFAT.
In the current study, 29 (90.6%) of the 32 samples positive by IFAT were also positive by PCR for Leishmania spp. Out of the 18 negative samples, 11 (61.1%) were equivalent to PCR. The IFAT sensitivity and specificity were 80.6% and 78.6%, respectively (Table 2).
Although the Ministry of Health advocates euthanasia for ELISA-positive animals [99 . Secretaria de Vigilância em Saúde: Departamento de Vigilância das Doenças Transmissíveis: Nota técnica conjunta N°1/2011 CGDT-CGLAB/ DEVST/SVS/MS.In Esclarecimentos sobre substituição do protocolo diagnóstico da leishmaniose visceral canina (LVC). Brasília: Ministério da Saúde; 2011.], in our study two dogs presented low titers (80), which could represent cross-reaction with other pathogens phylogenetically similar to Leishmania spp., as observed in a work of Luciano et al. [1818 . Luciano RM, Lucheis SB, Troncarelli MZ, Luciano DM, Langoni H: Avaliação da reatividade cruzada entre antígenos de Leishmaniaspp. e Trypanosoma cruzi na resposta sorológica de cães pela técnica de imunofluorescência indireta (RIFI). Braz J Vet Res Anim Sci 2009, 46(3):181-187.]. The other 30 animals presented higher levels, which usually indicates infection. However, both of them reacted positively to genus-specific PCR of blood cultures, suggesting that low titers are also related to the infection.
Only three samples of dogs from Campo Grande were negative by PCR and positive by IFAT. This difference found among the results of serological, parasitological and molecular methods may be attributable to such factors as the permanence of circulating antibodies in the peripheral blood even after parasite elimination, low quantity of circulating Leishmania spp. at the collection moment and consequently not detected by PCR. Yet some serology cross-reactions may be due to the existence of antigenic determinants and proteins common to parasites such as Trypanosoma cruzi [1818 . Luciano RM, Lucheis SB, Troncarelli MZ, Luciano DM, Langoni H: Avaliação da reatividade cruzada entre antígenos de Leishmaniaspp. e Trypanosoma cruzi na resposta sorológica de cães pela técnica de imunofluorescência indireta (RIFI). Braz J Vet Res Anim Sci 2009, 46(3):181-187.,2121 . Troncarelli MZ, Camargo JB, Machado JG, Lucheis SB, Langoni H:Leishmania spp. and/or Trypanosoma cruziin dogs from endemic and nonendemic areas for canine visceral leishmaniasis. Vet Parasitol 2009, 164(2-4):118-123.].
The Leishmania spp. infection in dogs from Campo Grande was minor by IFAT when compared to PCR, which presented 72.0% positivity. Seven dogs were non-reactive by IFAT, but positive by PCR. The immunodeficiency of some dogs or the existence of animals whose -immune system can quell the infection and render the parasite inert in the organism may have contributed to these results. Lachaud et al. [2222 . Lachaud L, Machergui-Hammami S, Chabbert E, Dereure J, Dedet JP, Bastien P: Comparison of six PCR methods using peripheral flood for detection of canine visceral leishmaniasis. J Clin Microbiol2002,40(1):210-215.], comparing PCR to serology, obtained a 79.8% prevalence of canine infection through PCR versus 29.6% through serology and demonstrated that the antigens from kinetoplast were more sensitive, especially K13A-K13B and RV1-RV2, detecting 10-3 parasites per cubic millimeter of blood.
Leontides et al. [2323 Leontides LS, Saridomichelakis MN, Billinis C, Kontos V, Koutinas AF, Galatos AD, Mylonakis ME: A cross-sectional study of Leishmaniaspp. infection in clinically healthy dogs with polymerase chain reaction and sorology in Greece. Vet Parasitol 2002, 109(1-2):19-27.] studied 73 healthy dogs from an endemic area in Greece, using IFAT and PCR in serum and bone marrow. The authors found 46 (63%) positive samples by PCR, whereas only nine (12.3%) by IFAT, clearly demonstrating that most dogs from this endemic area had been infected, but were still seronegative.
An important aspect that is probably associated with the nonsuccess of VL control is the selection of dogs for euthanasia by only serologic techniques whose low sensitivity and specificity result in underestimated infection rates (false negatives), thus enabling the maintenance of infected animals in endemic areas and consequently interfering in the impact that the elimination of dogs produces in the control of VL [2424 . Silva AVM, Paula AA, Cabrera MAA, Carreira JCA: Leishmaniose em cães domésticos: aspectos epidemiológicos. Cad Saude Publ 2005, 21 (1):324-328.,2525 . Andrade HM, Reis AB, dos Santos SL, Volpini AC, Marques MJ, Romanha AJ: Use of PCR-RFLP to identify Leishmania species in naturally-infected dogs. Vet Parasitol 2006, 140(3-4):231 -238.].
It can be concluded that, by associating serological and molecular techniques, a greater number of infected animals can be detected and consequently eliminated, thus contributing to the control of the illness. On the other hand, many animals can be spared with a judicious analysis of their results by the employment of one more diagnostic test.
After the completion of the procedures for the evaluation of the detection threshold of Leishmania spp. DNA through PCR, it was verified through electrophoresis that bands with 720 bp were present in the sample of pure culture, as well as at the 103,102 and 101dilutions. There - was no detection of such a band at the 100dilution. Therefore, it is concluded that the analytic sensitivity of PCR, using the initiators LIN R4 and LIN19, was 10 parasites/mL.
In Campo Grande, out of 50 feline samples, 15 (30%) were positive by IFAT, with titers varying from 40 to 320. Similar positivity was found in a study by Martins et al. [2626 . Martins CO, Rossi CN, Marcondes M, Neto LS, Lima VMF: Detecção de anticorpos anti-Leishmania spp. em Felis catusna área endêmica de Araçatuba, São Paulo, Brasil. Vet Zootec2008, 15(2):56.], who found, through the ELISA serologic method, 27.6% positivity in 112 samples of feline serum from the CZC of Araçatuba, SP, another city endemic for leishmaniasis. Da Silva et al. [33 . da Silva AVM, de Souza Cândido CD, Pita-Pereira D, Brazil RP, Carreira JCA: The first record of American visceral leishmaniasis in domestic cats from Rio de Janeiro, Brazil. Acta Trop 2008, 105(1):92-94.], using IFAT to assess eight samples of cat serum, obtained two samples positive (25%) for VL, with titers of 40 and 320. In Spain,
Martín-Sanchez et al. [2727 . Martín-Sanchez J, Acedo C, Muñoz-Pérez M, Pesson B, Marchal O, Morillas-Márquez F: Infection by Leishmania infantum in cats: epidemiological study in Spain. Vet Parasitol 2007, 145(3-4):267-273.] found a seropositivity of 60% in an analysis of one 183 sera. And in a study conducted in Italy, Poli et al. [2828 . Poli A, Abramo F, Barsotti P, Leva S, Gramiccia M, Ludovisi A, Mancianti F: Feline leishmaniosis due to Leishmania infantum in Italy. Vet Parasitol 2002, 106(3):181 -191.] identified only one positive sample (0.9%) out of one hundred and ten analyzed by IFAT.
The serology of infected cats is usually less specific than it is in dogs, because the production of antibodies against Leishmania spp. is smaller, which can render them seronegative [2828 . Poli A, Abramo F, Barsotti P, Leva S, Gramiccia M, Ludovisi A, Mancianti F: Feline leishmaniosis due to Leishmania infantum in Italy. Vet Parasitol 2002, 106(3):181 -191.]. Serological surveys performed through different techniques have shown that the prevalence of antibodies for Leishmania spp. in cats examined around the world varies from zero to 68.0%. This sensitivity of feline seroprevalence can vary according to the methodology used (sampling, serological technique and adopted cutoff point) and the geographic region where the study is made.
The supposition that the Leishmania spp. infection, with or without clinical symptoms, is misdiagnosed in countries where the illness is endemic would account for the discrepancy between the high rates of infection obtained in epidemiological studies and the low number of clinical cases described. Perhaps this low quantity of reported cases is due to a scarcity of serological surveys in endemic areas, difficulties in distinguishing between FL and other common feline diseases and the fact that many cases are diagnosed only when the animals become symptomatic [33 . da Silva AVM, de Souza Cândido CD, Pita-Pereira D, Brazil RP, Carreira JCA: The first record of American visceral leishmaniasis in domestic cats from Rio de Janeiro, Brazil. Acta Trop 2008, 105(1):92-94.].
In our study, only dogs coming from Mato Grosso do Sul state were positive forLeishmania spp. by PCR, by which 36 (72%) of the 50 dogs evaluated were positive. Only four dogs (11.1%) with positive PCR were asymptomatic. Thirty-two animals (88.9%) were positive by PCR forLeishmania spp. and presented various clinical changes: 28 dogs (77.8%) had weight loss; 22 dogs (61.1%) had skin changes; 19 (52.8%) had lymphadenopathy; 16 (44.4%) had onychogryphosis; 12 (33.3%) had alopecia, and 11 (30.5%) had ocular lesions.
Fifty dog serum samples and 50 cat serum samples (Table 3) from Botucatu were 100% negative by IFAT, the same results obtained by Langoni et al. [2929 . Langoni H, Modolo JR, Souza LC, Araujo WN, Shimabukuro FH, Mendonça AO, Leite BLS, Padovani CR: Epidemiological vigilance for canine leishmaniasis in the county of Botucatu, SP, Brazil. ARS Vet2001,17(3):196-200.], using the same diagnostic technique to test 781 sera of dogs in this locality. This study contributed to the active surveillance of leishmaniasis in the city, according to measures suggested by the Ministry of Health, whose new focus is to incorporate the "silent" states and cities, in other words, those with no reports of the disease in humans or dogs, by monitoring that aims to avoid or minimize the problems related to this affliction in areas without transmission [3030 . Ministério da Saúde, Brasil, Secretaria de Vigilância em Saúde. Departamento de Vigilância Epidemiológica: Manual de vigilância e controle da leishmaniose visceral / Ministério da Saúde, Secretaria de Vigilância em Saúde, Departamento de Vigilância Epidemiológica.Brasília: Editora do Ministério da Saúde: il. color - (Série A. Normas e Manuais Técnicos); 2006:120p.].
Conclusions
In Campo Grande (MS), the IFAT technique for the diagnosis of Leishmaniaspp. in dogs demonstrated greater accuracy in relation to blood culture, whereas the PCR technique is considered the gold standard given its greater sensitivity and specificity. A higher number of these infected dogs were identified by means of PCR, thus improving and contributing to the identification of reservoir animals and, consequently, to the control of leishmaniasis.
The positivity of cats by IFAT in Campo Grande (MS) indicates their possible involvement in the leishmaniasis epidemiological cycle, which highlights the extreme importance of continuing the investigation of the disease in this species.
The positivity of blood culture observed in dogs and cats from Botucatu (SP) suggests the necessity of investigation for other parasites, such as Trypanosoma cruzi.
Acknowledgments
The authors would like to thank the State of São Paulo Research Foundation (FAPESP) for financial support, the Center for Zoonosis Control in Campo Grande, MS, Brazil and the Municipal Kennel and Animal Protection Association (APA) in Botucatu, SP, Brazil, for their help with the experimental procedures.
References
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Ethics committee approval
The present study was approved by the Ethics Committee on Animal Experimentation of the School of Veterinary Medicine and Animal Husbandry, São Paulo State University (UNESP - Universidade Estadual Paulista), Botucatu, SP, Brazil under protocol number 65/2007.
Publication Dates
-
Publication in this collection
2014
History
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Received
23 Oct 2013 -
Accepted
17 Feb 2014