Anti-Toxoplasma gondii and Anti-Neospora caninum Antibodies in Urban Traction Equids in Northeast Brazil: Seroprevalence and Risk Factors

The aim of this study was to describe the prevalence of anti-T. gondii and anti-N. caninum antibodies in equids that carry out traction work in Northeastern Brazil, and to establish the potential risk factors associated with seropositivity for these agents. Blood samples were collected from 322 traction equids (horses, donkeys and mules) in urban areas of 16 municipalities in the State of Paraíba, Brazil. The samples were sent for serological diagnosis using the Immunofluorescence Antibody Test (IFAT). Epidemiological questionnaires were given to the owners to assess the possible risk factors associated with infections. It was observed that 13.7% (44/322, CI: 10.9–16.5) of the equids tested positive for anti-T. gondii antibodies and 5% (16/322, CI: 2.6–7.4) tested positive for anti-N. caninum antibodies. Conducting traction work for over four years was considered a risk factor associated with T. gondii infection (odds ratio: 6.050; CI: 4.38–8.54, p = 0.025). There were no risk factors associated with N. caninum infection. It was concluded that traction equids have a significant prevalence of anti-T. gondii and anti-N. caninum antibodies in urban areas in the State of Paraíba, with an identified risk factor for seropositivity for anti-T. gondii as conducting traction work for more than four years.


Introduction
Traction equids have a relevant social and economic role for vulnerable families, especially in Northeast Brazil, as these animals carry out long journeys daily [1]. Among the several diseases that can affect equids, we highlight Toxoplasma gondii and Neospora caninum infections, which are caused by obligate intracellular protozoa parasites. As a result of these diseases, equids can be affected as intermediate hosts and suffer reproductive and neurological disorders, such as miscarriage, neonatal mortality and encephalomyelitis [2][3][4].
In Brazil, equids are highly exposed to infections by T. gondii and N. caninum, being important in the epidemiology of these parasites' infections [5]. Epidemiological studies to detect anti-T. gondii antibodies in rural horses showed frequencies ranging from 10.3% to 11.6% in the states of Pará and Paraíba, respectively [6,7]. For infections by N. caninum, the positivity frequencies in rural horses ranged from 5.6% to 23.9% in the States of Pará and Minas Gerais, respectively [7,8]. However, there is a scarcity of studies evaluating T. gondii and N. caninum infections in donkeys and mules, or evaluating the epidemiological profile of these infections in traction equids raised in urban environments. gondii and N. caninum infections in donkeys and mules, or evaluating the epidemiological profile of these infections in traction equids raised in urban environments.
The aim of this study was to evaluate the prevalence of anti-T. gondii and anti-N. caninum antibodies and to find potential risk factors associated with seropositivity for these agents in urban traction equids in Northeast Brazil.

Study Area and Sampling
Serum samples from traction equids were collected in 16 municipalities in the semiarid region of the state of Paraíba, Brazil ( Figure 1). To determine the minimum number of animals to be sampled, simple random sampling was used: = × 1 − n = sampling number; Z = normal distribution value for the 95% confidence level; P = expected prevalence of 10.3%-T. gondii; 5.6%-N. caninum [7]; d = 5% sampling error.
To perform adjustments for finite populations, the following formula was applied: = × + najus = adjusted sample size; N = total population size; n = initial sample size.
The adjustment of the population sample size considered the total population of equids in the state of Paraíba, which was taken to be 98,584 animals (IBGE; available at https://cidades.ibge.gov.br/brasil/pb/pesquisa/24/27745, accessed on 5 March 2023). Thus, the minimum number of animals required for participation in the study was 142 for anti-T. gondii prevalence and 81 for anti-N. caninum prevalence. However, 322 samples were collected. To determine the minimum number of animals to be sampled, simple random sampling was used: n = Z 2 × P(1 − P) d 2 n = sampling number; Z = normal distribution value for the 95% confidence level; P = expected prevalence of 10.3%-T. gondii; 5.6%-N. caninum [7]; d = 5% sampling error.
To perform adjustments for finite populations, the following formula was applied: najus = N × n N + n najus = adjusted sample size; N = total population size; n = initial sample size. The adjustment of the population sample size considered the total population of equids in the state of Paraíba, which was taken to be 98,584 animals (IBGE; available at https://cidades.ibge.gov.br/brasil/pb/pesquisa/24/27745, accessed on 5 March 2023). Thus, the minimum number of animals required for participation in the study was 142 for anti-T. gondii prevalence and 81 for anti-N. caninum prevalence. However, 322 samples were collected.

Sample Selection
Convenience sampling was used, meaning that the equids included in the research were performing traction work in urban areas during active searches in the municipalities. The minimum age of the horses used in the study was one year old, regardless of race or sex ( Figure 2).

Sample Selection
Convenience sampling was used, meaning that the equids included in the research were performing traction work in urban areas during active searches in the municipalities. The minimum age of the horses used in the study was one year old, regardless of race or sex ( Figure 2). After taking assigned consent from the owner of the animal, external jugular venipuncture was used to collect 5 mL of blood from each animal. The samples were individually identified, packaged and sent to the Laboratory of Immunology and Infectious Diseases (LIID) at the Veterinary Hospital at the Instituto Federal da Paraíba (IFPB), Sousa campus, where they were centrifuged at 2500 rpm for 10 min, and then the sera were stored in 2 mL microtubes and frozen at −20 °C [6].

Serological Analyses
The Immunofluorescence Antibody Test (IFAT) was used to detect anti-T. gondii antibodies based on the work of Langoni et al. [8]. T. gondii taquizoytes (RH strain) were used as antigens. Positive and negative control sera from horses were included in each slide. Sera were considered positive when T. gondii tachyzoites showed total peripheral fluorescence at a 1:64 cut-off [9].
To detect anti-N. caninum antibodies, the IFAT was based on the work of Conrad et al. [10]. N. caninum taquizoytes (NC-1 strain) were used as antigens. Positive and negative control sera from horses were included in each slide. Sera were considered positive when N. caninum tachyzoites showed total peripheral fluorescence at a 1:50 cut-off [11]. After taking assigned consent from the owner of the animal, external jugular venipuncture was used to collect 5 mL of blood from each animal. The samples were individually identified, packaged and sent to the Laboratory of Immunology and Infectious Diseases (LIID) at the Veterinary Hospital at the Instituto Federal da Paraíba (IFPB), Sousa campus, where they were centrifuged at 2500 rpm for 10 min, and then the sera were stored in 2 mL microtubes and frozen at −20 • C [6].

Serological Analyses
The Immunofluorescence Antibody Test (IFAT) was used to detect anti-T. gondii antibodies based on the work of Langoni et al. [8]. T. gondii taquizoytes (RH strain) were used as antigens. Positive and negative control sera from horses were included in each slide. Sera were considered positive when T. gondii tachyzoites showed total peripheral fluorescence at a 1:64 cut-off [9].
To detect anti-N. caninum antibodies, the IFAT was based on the work of Conrad et al. [10]. N. caninum taquizoytes (NC-1 strain) were used as antigens. Positive and negative control sera from horses were included in each slide. Sera were considered positive when N. caninum tachyzoites showed total peripheral fluorescence at a 1:50 cut-off [11].
The conjugate (anti-horse IgG, labelled with fluorescein isothiocyanate, Sigma ® , St. Louis, MO, USA) was used at a 1:1500 dilution in pH 7.2 phosphate-buffered solution (PBS) containing 0.01% Evans blue. Positive samples were titrated from sequential dilutions on the basis of two until negative.

Epidemiological Questionnaire
Pre-structured questionnaires were given to owners to assess the risk factors associated with T. gondii and N. caninum infections. The variables included for analysis were related to species (donkey, mule, horse), breed, sex, age and time of traction activity. Questions related to environmental management, contact with other species (dogs, cats, sheep, cattle, pigs, wild animals and other equids), nutritional management, type of food and storage, as well as health and reproductive information were asked.

Statistical Analysis
Descriptive statistical analysis was used to calculate the frequencies of the results obtained in the serological test. Risk factors associated with T. gondii and N. caninum infections were assessed using data from epidemiological questionnaires in two stages: univariate and multivariate analysis. In the univariate analysis, each independent variable was crosscorrelated with the dependent variable (seropositivity), and those with a p-value ≤ 0.2, according to the chi-square test or Fisher's exact test [12], were selected for multivariate analysis using multiple logistic regression [13]. In order to verify some collinearity between the data, a correlation test was applied whereby if the correlation coefficient was greater than 0.9, one of the variables was eliminated using the criteria of biological plausibility. To verify the fit of the model, the chi-square parameters and the omnibus test were used. The significance level adopted in the multiple analyses was 5%. The results were analysed using GraphPad Prism version 8.0.0 for Windows, GraphPad Software, San Diego, CA, USA.

Results
It was observed that, among the 322 traction equids evaluated, 13.7% (44/322, CI: 10.9-16.5) tested positive for anti-T. gondii antibodies, and for anti-N. caninum antibodies the prevalence was 5% (16/322, CI: 2.6-7.4). The seropositivity by species for both anti-T. gondii and anti-N. caninum antibodies is shown in Table 1. There were no animals simultaneously positive for anti-T. gondii and anti-N. caninum antibodies. The anti-T. gondii antibody titrations ranged from 1:64 to 1:1024 and from 1:50 to 1:100 for the anti-N. caninum antibody titrations ( Table 2). Table 2. Distribution of anti-Toxoplasma gondii and anti-N. caninum antibody titrations by Immunofluorescence Antibody Test (IFAT) in traction equids in the semi-arid region of Northeastern Brazil. In the univariate analysis for the anti-T. gondii antibodies' positivity, only the variables of age and time of work were selected for multiple logistic regression (p ≤ 0.20) (Table 3). In the multivariate analysis, a time of work variable higher than four years was considered a risk factor associated with anti-T. gondii positivity (odds ratio: 6.05). In the univariate analysis for the anti-N. caninum antibodies positivity, only the variable sex was selected for multiple logistic regression (p ≤ 0.20) (Table 4). However, there were no risk factors associated with anti-N. caninum antibodies.

Discussion
The prevalence rates of 13.7% for anti-T. gondii and 5% for anti-N. caninum antibodies were similarly observed in the northern region of Brazil, with a prevalence of 10.3% (134/1298) for anti-T. gondii and 5.6% (73/1298) for anti-N. caninum [7]. Lower seroprevalence in horses was observed in the State of São Paulo, Southeast Brazil, with prevalence values of 0.9% (1/116) for anti-T. gondii and 2.6% (3/116) for anti-N. caninum [14]. On the other hand, in studies conducted in the Northeast and Midwest regions of Brazil, the prevalences were 28.5% (129/453) for anti-T. gondii in horses and 2% (7/333) for anti-N. caninum in donkeys [15,16]. The intensity of T. gondii and N. caninum infections varies between different locations, exposures and study focuses. Surveyed population, management conditions and environmental characteristics may contribute to the variation in seropositivity between studies [17,18].
This study provides relevant information about the seroprevalence of anti-T. gondii (12%) and anti-N. caninum (5.1%) in mules, since, among the equids, it is the least studied [7,19,20]. However, mules, despite having a predisposition to infertility [21], are able to travel long distances in urban and rural areas [22]. As animals that use force to perform traction work, exposure to these agents may decrease their production and productivity.
The results of these anti-T. gondii and anti-N. caninum antibody studies demonstrated that most seropositive animals had low titers, mainly 1:64 (70.4%) for anti-T. gondii and 1:50 (93.7%) for anti-N. caninum. Low antibody titers were also observed by James et al. [23], in which 73% (239/328) of the animals titrated below 1:100 for anti-T. gondii, and by Bartová et al. [24], who observed that all positive animals for anti-T. gondii (3%; 19/643) and for anti-N. caninum (2.3%; 15/6430) antibodies titrated up to 1:100. Low titers for anti-T. gondii in horses should not be ignored [25], as they commonly develop lower antibody titers [26,27]. In infections by other pathogens, they found that high titers of antibodies were observed after recent contact with the antigen, indicating acute infection, in addition to low titers in chronic infections, denoting old contact with the agent [28,29].
A time of work of more than four years was considered a risk factor associated with T. gondii infection in traction equids (Odds ratio: 2.89). Li et al. [30] observed that the older horses become, the more they are exposed to jobs that involve travelling long distances, and therefore are predisposed to have contact with food and water contaminated by T. gondii oocystis. In our study, the results may have occurred because traction equids are commonly exposed to a wide variety of urban environments, making long journeys, carrying a high load weight, maintaining contact with other species of animals, and are associated with precarious care provided by owners, all of which increase the possibility of being infected by T. gondii and/or N. caninum.
There were no risk factors associated with positive results for anti-N. caninum antibodies in the studied animals. However, it was observed that the majority of positive equids maintained contact with dogs (87.5%). It is possible that oocysts can be excreted by dogs in food, water and pasture, becoming sources for N. caninum infections [31,32].
The expressive prevalence of anti-T. gondii antibodies in equids denotes that adequate prevention and control measures must be taken. Although it does not occur frequently, the literature mentions that there may be human infections from the ingestion of T. gondiiinfected equine meat [25,33], and that older animals are most commonly used for food [24]. Even though the consumption of equid meat is not common in Brazil, there is an emphasis on the export of this product [17]. The prevalence found in the present study for infections by N. caninum emphasises the need for prevention, since it can cause reproductive neosporosis [34,35].

Conclusions
It was concluded that traction equids had a significant prevalence of anti-T. gondii and anti-N. caninum antibodies in urban areas in the semi-arid region of Northeast Brazil. Traction work for more than four years was a risk factor associated with seropositivity for anti-T. gondii. Surveillance and epidemiological control measures, such as registration and periodic serological testing, should be taken in order to establish what actions are required to reduce infections by these protozoa.