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PARASITOLOGY • Cienc. Rural 55 (6) • 2025 • https://doi.org/10.1590/0103-8478cr20240123 linkcopy

Open-access Anti-Leishmania spp., anti-Toxoplasma gondii and anti-Neospora spp. IgG antibodies in horses from Alagoas, Northeastern Brazil

Anticorpos IgG anti-Leishmania spp., anti-Toxoplasma gondii e anti-Neospora spp. em cavalos de Alagoas, nordeste do Brasil

AuthorshipSCIMAGO INSTITUTIONS RANKINGS

    ABSTRACT:

    Determined the seroprevalence and epidemiological profile of Toxoplasma gondii, Neospora spp. and Leishmania spp. infection in horses in Alagoas, northeastern Brazil. Blood samples were collected from 376 equines (horses) in rural areas of six municipalities. The serum samples were subjected to the Indirect Immunofluorescence Assay (IFA) to detect the presence of IgG antibodies anti-T. gondii, Neospora spp. and Leishmania spp. The cut-off points for T. gondii, Neospora spp. and Leishmania spp. were 1:64, 1:50 and 1:40, respectively. All horse owners answered an epidemiological questionnaire. Among the analyzed samples, 44.94% (169/376) were seroreactive for at least one of the three parasites. The prevalence values found were 17.3% (65/376) for T. gondii, 16.2% (61/376) for Neospora spp. and 11.4% (43/376) for Leishmania spp. No risk factors were associated with infection by T. gondii, Neospora spp. and Leishmania spp. The data obtained in this research could be considered in a future review of the Brazilian Equine Health Program to include technical standards for monitoring zoonosis in horses.

    Key words:
    horses; protozoan; risk factor.

    RESUMO:

    O objetivo deste estudo foi determinar a soroprevalência e o perfil epidemiológico da infecção por Toxoplasma gondii, Neospora caninum e Leishmania spp. em cavalos em Alagoas, nordeste do Brasil. Foram coletadas amostras de sangue de 376 equinos (cavalos) em áreas rurais de seis municípios. As amostras de soro foram submetidas à reação de imunofluorescência indireta (RIFI) para investigar a presença de anticorpos IgG anti-T. gondii, Neospora spp. e Leishmania spp. Os pontos de corte para T. gondii, Neospora spp. e Leishmania spp. foram 1:64, 1:50 e 1:40, respectivamente. Todos os proprietários de cavalos foram solicitados a responder a um questionário epidemiológico. Entre as amostras analisadas, 44,94% (169/376) eram sororreativas para pelo menos um dos três parasitos. As taxas de prevalência encontradas foram de 17,3% (65/376) para T. gondii, 16,2% (61/376) para Neospora spp. e 11,4% (43/376) para Leishmania spp. Nenhum fator de risco foi associado à infecção por T. gondii, Neospora spp. e Leishmania spp. Os dados obtidos nesta pesquisa podem ser considerados em uma futura revisão do Programa Brasileiro de Saúde Equina para incluir padrões técnicos para o monitoramento de zoonoses em equinos.

    Palavras-chave:
    cavalos; protozoário; fator de risco

    INTRODUCTION

    Horses are present in the daily lives of human beings, participating in many activities such as work, sport and leisure. The health management of these animals is; therefore, fundamental for the prevention and control of diseases (MAPA, 2016; TRUPPEL et al., 2014). Equines are classified as sentinel animals for different diseases, including several zoonoses, thus providing information on environmental contamination by pathogens and enabling the assessment of epidemiological risks to human populations (ALMEIDA, 2001).

    Toxoplasmosis is a cosmopolitan zoonosis that affects mammals and birds, and is caused by the obligate intracellular coccidium Toxoplasma gondii (DUBEY, 1995). In horses, the disease is usually asymptomatic, with the presence of tissue cysts of the parasite (COIRO, 2012; TASSI, 2007). Similar to T. gondii, Neospora caninum is also an obligate intracellular protozoan. In humans, this parasite is not considered a zoonosis; although, the presence of antibodies has been described in humans with immunodeficiency virus and in hunters and animal handlers (OSHIRO et al., 2015; BENETTI et al., 2009). In equines, Neospora hughesi was initially described in association with myeloencephalitis (MARSH et al., 1996), with later differentiation to N. caninum.

    Leishmaniasis is an anthropozoonosis that can affect a wide range of hosts, including horses, humans and canines, and is transmitted in the New World by phlebotomines of the genus Lutzomyia. Leishmaniasis can be divided into two large groups: a) American tegumentary leishmaniasis, caused mainly by Leishmania brasiliensis; and b) visceral leishmaniasis, caused by L. infantum/chagasi (CENTERS FOR DISEASE CONTROL AND PREVENTION, 2020). Equines are used as a means of locomotion, transporting loads and helping in farming activities in rural areas, which facilitates their exposure to phlebotomines, as well as direct contact with domestic dogs and wild animals that are reservoirs of leishmaniasis (LOPES et al., 2021; ESCOBAR et al., 2019).

    Considering the human-animal interface in equine farming and the risk of transmitting zoonoses (KHURANA et al., 2015; SACK et al., 2020), the aim of this study was to determine the seroprevalence of anti-Leishmania spp., anti-Toxoplasma gondii and anti-Neospora spp. IgG antibodies in horses in Alagoas, northeastern Brazil.

    MATERIALS AND METHODS

    Study area and sampling

    The study area, which has a tropical climate (temperature between 20-35 ºC), is located in the Atlantic forest at an average altitude of 238m (A: -10.65682 -38.23777; L: -8.81313 -34.96344). The highest rainfall in this region occurs in autumn/winter (1,000-1,500 mm), favoring the presence of parasites (ALAGOAS, 2010; EMBRAPA, 2012; IBGE, 2011; BEZERRA, 2015).

    Sample collection and processing

    The sample size was calculated considering the following: a) the number of horses in Alagoas (n > 20,000); b) an estimated prevalence of 50%; c) a margin of error of ± 5%; and d) a 95% confidence level, resulting in 376 animals (THRUSFIELD, 2007). Only properties where it was allowed to collect blood samples from all equines in the herd over 6 months of age, and where the animal caretaker agreed to respond to the epidemiological questionnaire, were selected for the research. Thus, six properties that met this requirement were selected for this investigation (Figure 1). The animals belonged to the coastal regions and the inland regions. Horses were chosen by convenience, mainly to avoid stress. Blood samples were collected by jugular venipuncture into sterile tubes without anticoagulants, centrifuged and the serum stored at -20 ºC until serological analysis.

    thumbnail of Figure 1
    Figure 1
    Geographical location of the municipalities of the stud farms and horses used in this study in the state of Alagoas, Brazil.

    Serological analysis

    The Indirect Immunofluorescence Assay (IFA) was the technique chosen to test for IgG class antibodies in the serum samples, using Rabbit FITC-conjugated anti-horse IgG F7759 (whole molecule) (Sigma Aldrich, St. Louis, MO). The conjugated anti-horse IgG was used at a dilution of 1:100, according to the standardized protocol for this antibody in the laboratory.

    The protocol established by CAMARGO (1964) was used to test for anti-T. gondii antibodies, using multipost slides sensitized with tachyzoites of the ME49 strain. The cut-off points for T. gondii were diluted in 1X phosphate-bufered saline (PBS) solution (pH 7.4) at a ratio of 1:64.

    To carry out the anti-Neospora spp. IFA, multipost slides were sensitized with tachyzoites of the NCSpain7 strain, following the protocol proposed by PADILLA-DÍAZ (2021). The cut-off points for Neospora spp. were diluted in 1X phosphate-bufered saline (PBS) solution (pH 7.4) at a ratio of 1:50 (PADILLA-DÍAZ et al., 2021).

    Multipost slides containing promastigote forms of L. infantum were used to test for anti-Leishmania antibodies (SOARES, 2013). The cut-off points for L. infantum were diluted in 1X phosphate-bufered saline (PBS) solution (pH 7.4) at a ratio of 1:40 (SOARES, 2013).

    To ensure the reliability of the reaction, a known positive and negative sample were used as controls in all reactions. After vortex mixing, 8 μL of all diluted samples were placed in each well of the multipost slides and incubated at 37 °C for 1 hour, at approximately 90% humidity. The total peripheral fluorescence of the antigens was considered a positive sample. Samples with apical fluorescence or without fluorescence were considered negative.

    Epidemiological survey and statistical analysis

    For the statistical analysis using the EpiInfo™ 7.2.1.0 software, descriptive statistics were employed to determine absolute and relative frequencies. To analyze the risk factors associated with T. gondii, Neospora spp. and Leishmania spp. infection, univariate analysis of relevant variables was conducted utilizing Pearson’s chi-square test, supplemented by Fisher’s exact test where necessary. Subsequently, logistic regression analysis was employed, with the result of the IFA, categorized as reagent or non-reagent, serving as the dependent variable for protozoan infection. Independent or explanatory variables included in the model were those demonstrating statistical significance < 0.05 (THRUSFIELD, 2007)

    RESULTS AND DISCUSSION

    An analysis of 376 equine serum samples revealed a frequency value of 44.9% (169/376) for at least one of the three parasites investigated. Positivity values by pathogen were: 17.2% (65/376) antibodies for anti-T. gondii, 16.2% (61/376) for anti-N. caninum and 11.4% (43/376) for anti- Leishmania spp. Eight horses showed simultaneous seroreactivity (co-infection) to Neospora spp. and Leishmania sp.; and nine animals to Neospora spp. and T. gondii. There were no co-infections with T. gondii and Leishmania spp. or with the three parasites at the same time. The presence of dogs (reservoir of Leishmania and definitive host of Neospora spp.) and cats (definitive host of T. gondii) was observed at all the farms involved in this study. Feed, water and/or environmental contamination with oocysts from the faeces of these species would help explain the occurrence of seropositive animals.

    The magnitude of T. gondii, Neospora spp. and Leishmania spp. infections varies among different locations, animal exhibitions and study sites. Therefore, management conditions, as well as the epidemiological status and seroprevalence, are intrinsic to each region. Among the animals that tested positive for some form of the protozoa under investigation, 86.9% (147/169) were females; 80.4% (136/169) were purebred (predominantly Mangalarga Marchador); 85.8% (145/169) of them were young (1-6 years old), 13.6% (23/169) were adults (6-12 years old), and 2.3% (4/169) were elderly (≥ 13 years old).

    Prevalence and univariate analysis and logistic regression of risk factors associated with Toxoplasma gondii (Table 1), Neospora spp. (Table 2) and Leishmania spp. (Table 3) infection in horse in the state of Alagoas, Brazil are described below. In the univariate analysis, two variables demonstrated statistical significance (P < 0.05, Table 2). Nevertheless, after conducting a multivariate logistic regression analysis, none of these variables were validated as risk factors for Neospora spp. infection.

    Thumbnail
    Table 1
    Prevalence and univariate analysis and logistic regression of risk factors associated with Toxoplasma gondii, infection in horse in the state of Alagoas, Brazil.

    Thumbnail
    Table 2
    Prevalence and univariate analysis and logistic regression of risk factors associated with Neospora spp., infection in horse in the state of Alagoas, Brazil.

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    Table 3
    Prevalence and univariate analysis and logistic regression of risk factors associated with Leishmania spp., infection in horse in the state of Alagoas, Brazil.

    Previous studies in Alagoas have shown that horses are exposed to T. gondii infections, making them an interesting animal model to gain a better understanding of the environmental epidemiology of these two protozoa (VALENÇA, 2015). In the states of Pará and Paraíba, seroepidemiological studies to detect anti-T. gondii antibodies in horses revealed infection values of 10.3% and 11.6%, respectively (ALMEIDA et al., 2017; MOREIRA et al., 2019). In the states of Pará and Minas Gerais, the frequency of anti- Neospora spp. antibodies was reported to be 5.6% and 23.9%, respectively (ALMEIDA et al., 2017; LANGONI, 2007). Findings similar to those of our study were described by COSTA et al. (2023) in Paraíba, with a prevalence of 13.7% for anti-T. gondii antibodies and 5% for anti- Neospora spp. antibodies.

    It is important to highlight the detection of anti-T. gondii antibodies in horses, as Brazil is the eighth largest exporter of equine meat, supplying Japan and EU countries such as France, Holland, Belgium and Italy. The safety of equine meat is a cause for concern, since a several cases of congenital toxoplasmosis was reported in France and was attributed to the probable consumption of horse meat imported from Brazil. (EVERS et al., 2013; BÁRTOVÁ, 2015; POMARES et al., 2011; BRASIL, 2016).

    The seropositivity found in this study for Leishmania spp. in horses is similar to recent findings in the state of Pernambuco, bordering Alagoas, and in other states in the country such as Minas Gerais and São Paulo (BENASSI et al., 2018; SOARES et al., 2013). Until now, there are no studies confirming the transmission of Leishmania spp. from infected horses to the phlebotomine. Nevertheless, one aspect of the epidemiological chain of leishmaniasis transmission that needs to be clarified is the role of horses for the disease. A possibility that cannot be ruled out is that the use of repellent collars on rural dogs would force phlebotomines to feed on the blood of horses, ensuring oviposition and maintaining the vector population in the area. In such a scenario, the risk of zoonotic transmission would continue to exist (PRADELLA et al., 2020; ESCOBAR et al., 2020; COSTA, 2011).

    CONCLUSION

    The possibility of using horses as a model for epidemiological surveys for Toxoplasma gondii and Leishmania spp. needs to be studied and validated. The data from this study could be used to update the National Equine Health Plan and include regulations for monitoring zoonoses in horses.

    ACKNOWLEDGEMENTS

    The authors would like to thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); the Fundação de Amparo à Pesquisa do Estado de Alagoas (FAPEAL - Process no. E:60030.0000000120/2021) ; the Fundação de Amparo à Ciência e Tecnologia de Pernambuco (FACEPE) - IBPG Process no. 0180-5.05/22; and the Fundação Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) - Process no. (23038000830/2021-48) for their financial support of this research at the time of the study.

    REFERENCES

    • CR-2024-0123.R2

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    • AUTHORS’ CONTRIBUTIONS
      The authors contributed equally to the development and writing of the manuscript.

    Publication Dates

    • Publication in this collection
      03 Mar 2025
    • Date of issue
      2025

    History

    • Received
      04 Mar 2024
    • Accepted
      23 Sept 2024
    • Reviewed
      17 Dec 2024
    Creative Common - by 4.0
    This is an open-access article distributed under the terms of the Creative Commons Attribution License
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