"BAIXADEIROS" HORSES: PREVALENCE OF ANTI-Trypanosoma spp. AND ANTI-Leishmania spp. ANTIBODIES

CAVALOS "BAIXADEIROS": PREVALÊNCIA DE ANTICORPOS ANTI-Trypanosoma spp. E ANTI-Leishmania spp.

Fernanda Pinto Ferreira Eloiza Teles Caldart Danilo Rodrigues Barros Brito Daniel Praseres Chaves João Luis Garcia Italmar Teodorico Navarro About the authors

Abstract

A total of 138 horse blood samples were collected from 2012 to 2013 for the subsequent separation of serum collection. For detection of anti-Leishmania spp. and anti-Trypanosoma spp. antibodies, indirect immunofluorescence reactions and immunoenzymatic assays were performed. Samples with titers ≥40 were considered positive in the indirect immunofluorescence (IFAT), and the cut off for the enzyme immunoassay (ELISA) was calculated with three standard deviations. When tested for Trypanosoma spp., 50.37% (68/135) of the samples were ELISA reagents, 18.84% (8/136) were positive for IFAT and 2.89% (4/138) for both tests. A significant statistical association was observed for the municipality (p = 0.013) and collection period (p = 0.042) in the ELISA. When tested for Leishmania spp., 25.4% (35/138) of the samples were ELISA reagents, 13.00% (18/138) were positive for the IFAT and 4.34% (6/138) were positive for two tests, there were no variables associated with seropositivity. A statistical association (p = 0.0034) was observed between the presence of anti-Trypanosoma spp. and anti-Leishmania spp. antibodies when the ELISA was used. The results suggest that this horse breed is in contact with leishmaniasis and trypanosomiasis, which demonstrates the need for a more accurate investigation into the real role of horses in these diseases to assist in disease control measures.

Keywords:
leishmaniasis; trypanosomiasis; equines; serology

Resumo

Um total de 138 amostras de sangue de equinos foram coletadas nos anos de 2012 a 2013 para posterior obtenção de soro. Para detecção de anticorpos anti-Leishmania spp. e anti-Trypanossoma spp. realizou-se a reação de imunofluorescência indireta e o ensaio imunoenzimático. Consideraram-se positivas, na RIFI, amostras com títulos iguais ou superiores a 40; o ponto de corte do ELISA foi calculado com três desvios padrão. Quando testadas para Trypanosoma spp., 50,37% (68/135) das amostras foram reagentes ao ELISA, 18,84% (8/136) à RIFI e 2,89% (4/138) para os dois testes. Observou-se associação estatística significativa quanto ao município (p= 0.013) e período de coleta (p=0.042) no ELISA. Quando testadas para Leishmania spp., 25,4% (35/138) das amostras foram positivas ao ELISA, 13,00% (18/138) à RIFI e 4,34% (6/138) nos dois testes, não havendo variáveis associadas estatisticamente à soropositividade. Foi observada associação estatística (p=0,0034) entre presença de anticorpos anti-Trypanosoma spp. e anti-Leishmania spp., quando utilizada a técnica de ELISA. Os resultados obtidos sugerem que esta raça de cavalo está em contato com os agentes das leishmanioses e tripanossomíases, demonstrando a necessidade de uma investigação mais precisa sobre o real papel de cavalos nessas enfermidades, a fim de auxiliar nas medidas de controle da doença.

Palavras-chave:
leishmanioses; tripanossomíases; equinos; sorologia

Introduction

Baixada Maranhense is located in the northern mesoregion of the state of Maranhão, which is characterized by vast low plains that flood in the rainy season and create huge lakes between January and July. It is also called Pantanal Maranhense. This region stands out due to a breed of native horses called "baixadeiros", which are characterized by their small size, robustness, strength and resistance to work(11 Chaves DP, Rodrigues D, Brito B, Clara A, Francin7sca J, Vaz R, et al. Soroprevalência de mormo , anemia infecciosa equina e brucelose do cavalo baixadeiro * The seroprevalence of glanders , equine infectious anemia and brucellosis of equine race “ baixadeiro .” Rev Bras Ciência Veterinária. 2015;221:39-42.).

The genus Trypanosoma comprises parasitic species of fish, amphibians, reptiles, birds and mammals, and these parasites are transmitted mainly by hematophagous invertebrate vectors. The main species responsible for trypanosomiasis in horses are Trypanosoma equiperdum and T. evansi, which are endemic in the Pantanal of Mato Grosso due to the animal, climate and environmental conditions that favor the development of the vectors(22 Silva RA, Arosemena NA, Herrera HM, Sahib CA, Ferreira MS. Outbreak of trypanosomosis due to Trypanosoma evansi in horses of Pantanal Mato-grossense, Brazil. Vet Parasitol [Internet]. 1995 Nov [cited 2017 Sep 2];60(1-2):167-71. Available from: http://www.ncbi.nlm.nih.gov/pubmed/8644453
http://www.ncbi.nlm.nih.gov/pubmed/86444...
).

Cutaneous leishmaniasis (CL) in horses has been reported in several Brazilian states since the 1950s; however, there are no characteristic clinical patterns. Usually, papules and nodules occur with single and multiple ulcerated lesions as well as proliferative lesions or crusts(33 Ramos-Vara JA, Ortiz-Santiago B, Segalès J, Dunstan RW. Cutaneous Leishmaniasis in Two Horses. Vet Pathol [Internet]. 1996 Nov 26 [cited 2017 Sep 2];33(6):731-4. Available from: http://www.ncbi.nlm.nih.gov/pubmed/8952039
http://www.ncbi.nlm.nih.gov/pubmed/89520...
). With regard to visceral leishmaniasis (VL), seropositive animals were found in endemic areas(44 Feitosa FLF, Leal J, Mendes LCN, Peiró JR, Perri SH V, Lima VMF, et al. Estudo soroepidemiológico de leishmaniose em equinos na região de Araçatuba-SP , Brasil , área endêmica para leishmaniose visceral. Brazilian J Veteterinary Res Anim Sci. 2012;49(6):500-2.). The role of horses as reservoirs has not been definitively confirmed(55 Soares IR, Silva SO, Moreira FM, Prado LG, Fantini P, Maranhão R de PA, et al. First evidence of autochthonous cases of Leishmania (Leishmania) infantum in horse (Equus caballus) in the Americas and mixed infection of Leishmania infantum and Leishmania (Viannia) braziliensis. Vet Parasitol. 2013 Nov 8;197(3-4):665-9.); however, studies have shown that Equidae can be infected by species of Leishmania and are preferred food sources for sandflies(66 Oliveira-Pereira YN, Moraes JLP, Lorosa ES, Rebêlo JMM. Preferência alimentar sanguínea de flebotomíneos da Amazônia do Maranhão, Brasil. Cad Saúde Pública. 2008;24(9):2183-6.). Experimentally, they do not cause infection to the vector, which suggests that they are not important reservoirs in the transmission chain(77 Cerqueira EJL, Sherlock I, Gusmão A, De Almeida Barbosa A, Nakatani M, Cerqueira E J L, Sherlock I, Gusmão A, Barbosa Junior A A N. Inoculação experimental de Equus asinus com Leishmania chagasi Cunha & Chagas, 1937. Rev Soc Bras Med Trop. 2003;36(6):695-701.).

The objective of this study was to determine prevalence of anti-Leishmania spp. and anti-Trypanosoma spp. antibodies in "Baixadeiro" horse in the region of Baixada Maranhense, state of Maranhão, Brazil.

Material and Methods

The Baixada Maranhense (01°59'-04°00'S, 44°00'-45°33'W), is located in Maranhão northern mesoregion and has a total area of approximately 18 thousand km2 and an estimated population of 518,241 inhabitants. It is comprised of 21 municipalities and its climate is classified by Köppen as Aw (Tropical Climate with dry season in winter)(88 IBGE. Maranhão, Cidades [Internet]. Instituto Brasileiro de Geografia e Estatísticas. 2017 [cited 2017 Sep 2]. p. 1. Available from: http://www.cidades.ibge.gov.br/xtras/uf.php?coduf=21&search=maranhao&lang=
http://www.cidades.ibge.gov.br/xtras/uf....
). A total of 138 horse blood samples from the municipalities of Pinheiro, Arari and Anajatuba (Figure 1), which belong to the protected area of Baixada Maranhense, were collected between April 2012 and March 2013.

Figure 1
Geographic location of the cities of Pinheiro, Arari and Anajatuba, Baixada Maranhense, Maranhão, Brazil.

The determination of anti-Trypanosoma spp. and anti-Leishmania spp. antibody titers was performed using an indirect immunofluorescence reaction according to Marzochi et al.(99 Marzochi MCA, Coutinho SG, Sabroza PC, Souza WJS. Reação de imunofluorescência indireta e intradermorreação para leishmaniose tegumentar americana em moradores na área de Jacarepaguá (Rio de Janeiro). Estudo comparativo dos resultados observados em 1974 e 1978. Rev do Inst Med Trop. 1980;22:97-155.) with some adaptations as well as an antigen for the epimastigote forms of Trypanosoma cruzi and promastigotes of L. amazonensis. During the analysis, the equine anti-IgG conjugate (FITC - Sigma Chemicals®) was used, and positive and negative patterns were included in each slide. Samples with titers ≥40(55 Soares IR, Silva SO, Moreira FM, Prado LG, Fantini P, Maranhão R de PA, et al. First evidence of autochthonous cases of Leishmania (Leishmania) infantum in horse (Equus caballus) in the Americas and mixed infection of Leishmania infantum and Leishmania (Viannia) braziliensis. Vet Parasitol. 2013 Nov 8;197(3-4):665-9.) were considered positive and diluted in ratio two until a negative titer was obtained.

The indirect enzyme-linked immunosorbent assay (ELISAi) was performed as described by Szargiki et al.(1010 Szargiki R, Castro EA de, Luz E, Kowalthuk W, Machado ÂM, Thomaz-Soccol V, et al. Comparison of serological and parasitological methods for cutaneous leishmaniasis diagnosis in the state of Paraná, Brazil. Bjid. 2009;12(1):47-52.) with adaptations. The standard concentrations of T. cruzi and L. amazonensis antigen, serum and peroxidase-conjugated protein A conjugate (Sigma Aldrich, USA)(1111 Fernández-Bellon H, Solano-Gallego L, Bardagí M, Alberola J, Ramis A, Ferrer L. Immune response to Leishmania infantum in healthy horses in Spain. Vet Parasitol. 2006 Jan 30;135(2):181-5.) were 2.5 µg/mL, 1:25 and 1: 500, respectively. All samples were tested in duplicate. The optimum conditions concerning dilutions of antigen, serum and conjugate were established by the highest ratio between the mean absorbances of the known positive and negative samples used as controls. The cutoff of each plate was obtained by the mean absorbance of the negative sera added by three standard deviations. After the number of calculations per plate, the corrected cutoff was estimated using the ROC curve constructed by the MedCalc Statistical Software program (version 13.2.0)(1212 Schoonjans F, Zalata A, Depuydt CE, Comhaire FH. MedCalc: a new computer program for medical statistics. Comput Methods Programs Biomed [Internet]. 1995;48(3):257-62. Available from: http://www.sciencedirect.com/science/article/pii/0169260795017038
http://www.sciencedirect.com/science/art...
).

The statistical association between the results in the above tests and the characteristics of the horses, such as gender, age, municipality and date of collection were analyzed using the Epi Info program 7.1.5.2(1313 Dean AG, Dean JA, Coulombier D, Brendel KA, Smith DC, Burton AH, et al. Epi Info, Version 6: a word processing, data bases, and statistic program for epidemiology on microcomputers. Atlanta, Georgia: Center for Diseases Control and Prevention; 1994.). Chi-square or Fisher exact tests were conducted with a level of significance of 5%. For tables 2 x 2, the odds ratio (OR) calculation with a 95% confidence interval (CI) was used as a measure of association. For larger tables, standardized residue analysis (ARP)(1414 Cervi EU. A Análise de Dados Categóricos em Ciência Política: Uso de testes estatísticos em tabelas de contingência com fontes secundárias de dados. Curitiba: PPGCP/UFPR. 2014;98 p.) was used with the objective of verifying which categories contributed most to the statistics.

Results

Among the 138 horses studied, their age were between seven and 120 months old (mean of 49 months), 61.6% (85/138) were female, 4.3% (3/138) were from the municipality of Anajatuba, 6.5% (9/138) were from Arari and 89.1% (123/138) were from Pinheiro.

For Trypanosoma cruzi., 50.4% (68/135) were positive for ELISA, 18.84% (8/136) for IFAT and 2.96% (4/135) for both tests. The RIFI titers ranged between 40 and 160, 75.00% (6/8) 40; 12.5% (1/8) 80; 12.5% (1/8) 160 (Figure 2). The Fisher exact test showed a statistically significant association with municipality in which there was a higher seropositivity in the ELISA for T. cruzi (P = 0.013) Arari. The differences in the total number of samples analyzed between the tests are due to the insufficient quantity of samples.

Figure 2
Prevalence of anti-Trypanosoma spp. and anti-Leislnnania spp. antibodies by IFAT, ELISA and both tests in "baixadeiro" horses from Baixada Maranhense, MaranMo, Brazil.

When tested for L. amazonensis, 25.4% (35/138) of the samples were ELISA positive, 13.00% (18/138) were IFAT positive and 4.34% (6/138) were positive for both tests. Titers varied between 40 and 320 with 66.67% (12/18) 40; 16.67% (3/18) 80; 11.11% (2/18) 160 and 5.55% (1/18) 320.

A statistical association (p = 0.0034) was observed between the presence of anti-T. cruzi and anti- L. amazonensis antibodies in 18.38% (25/136) of the samples when the ELISA technique was used. Of these animals, 64.00% (16/25) presented serum with a higher optical density for T. cruzi, while 36.00% (9/25) had a higher optical density for L. amazonensis.

The samples were collected in four periods: April/2012, August/2012, September/2012 and March/2013, with 81 (60.0%), 6 (4.4%), 39 (28.9%) and 9 (6.7%) samples. A significant association (p = 0.042) was observed between the collection period and the seropositivity for T. cruzi in the ELISA (Figure 3).

Figure 3
Equine seropositivity for Trypanosoma spp., according to the collection month. Baixada Maranhense, Brazil, 2012-2013.

Discussion

This is the first seroepidemiological investigation of anti-Trypanosoma spp. and anti-Leishmania spp. antibodies in horses in the Baixada Maranhense region. There was no statistical association between seropositivity and sex and age.

Regarding Trypanosoma spp., the ELISA technique had an antibody prevalence of 50.4%, which was lower than the prevalence observed by Dávila et al.(1515 Dávila AM, Souza SS, Campos C, Silva RA. The seroprevalence of equine trypanosomosis in the pantanal. Mem Inst Oswaldo Cruz [Internet]. 1999 [cited 2017 Sep 2];94(2):199-202. Available from: http://www.ncbi.nlm.nih.gov/pubmed/10224528
http://www.ncbi.nlm.nih.gov/pubmed/10224...
) in a study in Pantanal, Mato Grosso do Sul, in which a prevalence of 79.2% was observed. When analyzed by RIFI, the samples had a prevalence of 18.84%, which was also lower than the prevalence found by Herrera et al.(1616 Herrera HM, Dávila AMR, Norek A, Abreu UG, Souza SS, D’Andrea PS, et al. Enzootiology of Trypanosoma evansi in Pantanal, Brazil. Vet Parasitol. 2004 Nov 10;125(3-4):263-75.), who observed 73.50% seropositivity in horses from Nhecolândia, Mato Grosso do Sul. Studies were carried out in Pantanal regions with similar characteristics as those of the cities of this study; yet, the high prevalence results may be due to the collection season being the rainy season, unlike our study in which a large part of the samples were collected during the dry season and may have led to a low number of vectors as well as low seropositivity.

Among the three cities studied, a higher prevalence of antibodies against Trypanosoma spp. were observed in the city of Arari (p = 0.013) in which the horse permanence was close to the floodplain and forest areas as well as farther from the urban area, this proximity to the wild environment may facilitate greater contact with Triatominae, which are vector transmitters of Trypanosoma spp. and naturally occupy wild environments as their habitat(1717 Barreto MP. Ecologia de Triatomíneos e Transmissão do Trypanosoma cruzi, com Especial Referência ao Brasil. Rev Soc Bras Med Trop. 1976;10(6):339-53.). In addition, the collections in this municipality were all carried out in the month of March, which was the rainiest season. Mendes and Lima(1818 Mendes PC, Lima S do C. Influência do clima na ocorrência de triatomíneos sinantrópicos no município de Uberlândia-MG. Cad Prudentino Geogr. 2011;2(33):5-20.) evaluated the influence of climate on the occurrence of triatomines over a period of three years in Uberlândia, state of Minas Gerais. They observed a higher catch rate in the rainy season. Some studies indicate that the dispersion and formation of colonies occurs in the rainy season, while mating and oviposition are more concentrated during the dry season(1818 Mendes PC, Lima S do C. Influência do clima na ocorrência de triatomíneos sinantrópicos no município de Uberlândia-MG. Cad Prudentino Geogr. 2011;2(33):5-20.)

The role of equine in the transmission chain of leishmaniasis has not yet been fully clarified, and it is known that there is seroconversion(44 Feitosa FLF, Leal J, Mendes LCN, Peiró JR, Perri SH V, Lima VMF, et al. Estudo soroepidemiológico de leishmaniose em equinos na região de Araçatuba-SP , Brasil , área endêmica para leishmaniose visceral. Brazilian J Veteterinary Res Anim Sci. 2012;49(6):500-2.) and disease development(33 Ramos-Vara JA, Ortiz-Santiago B, Segalès J, Dunstan RW. Cutaneous Leishmaniasis in Two Horses. Vet Pathol [Internet]. 1996 Nov 26 [cited 2017 Sep 2];33(6):731-4. Available from: http://www.ncbi.nlm.nih.gov/pubmed/8952039
http://www.ncbi.nlm.nih.gov/pubmed/89520...
). In addition, a study conducted in the state of Maranhão showed that the horse is the animal of choice as a food preference for sand flies(66 Oliveira-Pereira YN, Moraes JLP, Lorosa ES, Rebêlo JMM. Preferência alimentar sanguínea de flebotomíneos da Amazônia do Maranhão, Brasil. Cad Saúde Pública. 2008;24(9):2183-6.). Using ELISA for screening and IFAT as confirmation for Leishmania spp., we obtained 4.34% of reactive animals, which was a result lower than the result found by Soares et al.(55 Soares IR, Silva SO, Moreira FM, Prado LG, Fantini P, Maranhão R de PA, et al. First evidence of autochthonous cases of Leishmania (Leishmania) infantum in horse (Equus caballus) in the Americas and mixed infection of Leishmania infantum and Leishmania (Viannia) braziliensis. Vet Parasitol. 2013 Nov 8;197(3-4):665-9.) in a study with horses from Belo Horizonte, Minas Gerais, where they observed a seroprevalence of 30.48% using both techniques. When the ELISA was used, 25.40% of reagent samples were found by Duarte et al.(1919 Duarte R, Theophilo FAO, Marzochi MCA, Ferreira FC, Oliveira MRF, Mendes FA, et al. Sorologia para leishmaniose em eqüinos no Município do Rio de Janeiro. 2000. 11 p.) in a serological survey in the municipality of Rio de Janeiro, which is an endemic region for the two clinical forms of leishmaniasis. Researchers observed 11.6% of positive horses, which is a result lower than the outcome of this study, and the difference possibly occurred due to the use of heterologous antigens of Leishmania major-like parasites(2020 Barroso-Freitas APT, Passos SRL, Mouta-Confort E, Madeira MF, Schubach AO, Santos GPL, et al. Accuracy of an ELISA and indirect immunofluorescence for the laboratory diagnosis of American tegumentary leishmaniasis. Trans R Soc Trop Med Hyg. 2009 Apr;103(4):383-9.).

The statistical association (p = 0.003) observed between the presence of anti-Trypanosoma spp. and anti-Leishmania spp. antibodies in ELISA can indicate cross-reaction between the tests, or even a possible co-infection. The cross-reaction in serological tests for parasites of species belonging to the Trypanosomatidae family is quite common because they are phylogenetically close species(2121 Sundar S, Rai M. Laboratory Diagnosis of Visceral Leishmaniasis. Clin Diagn Lab Immunol. 2002 Sep;9(5):951-8.). Of these animals, 64.00% (16/25) presented sera with higher optical density for Trypanosoma spp., while 36.00% (9/25) presented higher optical density for Leishmania spp., and it is possible that the most likely agent is that with higher optical density, although more studies are needed to confirm that.

As for antibody titers, for the two diseases, most of the tested animals showed titer 40. Studies with other species of mammals show that exposure to CL agents may not trigger a production of antibodies with high titers. This trend likely occurred because humoral immunity in cutaneous leishmaniasis is not as expressive as in VL and trypanosomiasis, in which immune responses are more exacerbated and low titers suggest that it is an early exposure(2222 Solano-Gallego L, Fernández-Bellon H, Serra R, Gállego M, Ramis A, Fondevila D, et al. Cutaneous leishmaniosis in three horses in Spain. Equine Vet J. 2003 May;35(3):320-3.

23 Dantas-Torres F. The role of dogs as reservoirs of Leishmania parasites, with emphasis on Leishmania (Leishmania) infantum and Leishmania (Viannia) braziliensis. Vet Parasitol [Internet]. 2007 Nov 10 [cited 2017 Jul 3];149(3-4):139-46. Available from: http://www.ncbi.nlm.nih.gov/pubmed/17703890
http://www.ncbi.nlm.nih.gov/pubmed/17703...
-2424 Arraes SMAA, Marini MT, Martello D, Silveira TGV, Lonardoni MVC, Nanni MR. Investigação sorológica de casos subclínicos de leishmaniose tegumentar após um surto em uma localidade endêmica. Rev Soc Bras Med Trop. 2008 Apr;41(2):205-8.).

Conclusions

The results obtained in this study suggest that this horse breed is in contact with the protozoa that cause leishmaniasis and trypanosomiasis, which demonstrates the need for a more accurate investigation of the real role of horses in these diseases to enhance control measures for the disease. The protozoa of the family Trypanosomatidae cross-react with each other in indirect diagnostic methods.

References

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  • 2
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    » http://www.ncbi.nlm.nih.gov/pubmed/8644453
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    » http://www.ncbi.nlm.nih.gov/pubmed/8952039
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  • 10
    Szargiki R, Castro EA de, Luz E, Kowalthuk W, Machado ÂM, Thomaz-Soccol V, et al. Comparison of serological and parasitological methods for cutaneous leishmaniasis diagnosis in the state of Paraná, Brazil. Bjid. 2009;12(1):47-52.
  • 11
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    Schoonjans F, Zalata A, Depuydt CE, Comhaire FH. MedCalc: a new computer program for medical statistics. Comput Methods Programs Biomed [Internet]. 1995;48(3):257-62. Available from: http://www.sciencedirect.com/science/article/pii/0169260795017038
    » http://www.sciencedirect.com/science/article/pii/0169260795017038
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  • 15
    Dávila AM, Souza SS, Campos C, Silva RA. The seroprevalence of equine trypanosomosis in the pantanal. Mem Inst Oswaldo Cruz [Internet]. 1999 [cited 2017 Sep 2];94(2):199-202. Available from: http://www.ncbi.nlm.nih.gov/pubmed/10224528
    » http://www.ncbi.nlm.nih.gov/pubmed/10224528
  • 16
    Herrera HM, Dávila AMR, Norek A, Abreu UG, Souza SS, D’Andrea PS, et al. Enzootiology of Trypanosoma evansi in Pantanal, Brazil. Vet Parasitol. 2004 Nov 10;125(3-4):263-75.
  • 17
    Barreto MP. Ecologia de Triatomíneos e Transmissão do Trypanosoma cruzi, com Especial Referência ao Brasil. Rev Soc Bras Med Trop. 1976;10(6):339-53.
  • 18
    Mendes PC, Lima S do C. Influência do clima na ocorrência de triatomíneos sinantrópicos no município de Uberlândia-MG. Cad Prudentino Geogr. 2011;2(33):5-20.
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    Barroso-Freitas APT, Passos SRL, Mouta-Confort E, Madeira MF, Schubach AO, Santos GPL, et al. Accuracy of an ELISA and indirect immunofluorescence for the laboratory diagnosis of American tegumentary leishmaniasis. Trans R Soc Trop Med Hyg. 2009 Apr;103(4):383-9.
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Publication Dates

  • Publication in this collection
    08 Nov 2018
  • Date of issue
    2018

History

  • Received
    20 Feb 2018
  • Accepted
    20 Aug 2018
Universidade Federal de Goiás Universidade Federal de Goiás, Escola de Veterinária e Zootecnia, Campus II, Caixa Postal 131, CEP: 74001-970, Tel.: (55 62) 3521-1568, Fax: (55 62) 3521-1566 - Goiânia - GO - Brazil
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