Occurrence and risk factors associated with infection by Toxoplasma gondii in goats in the State of Alagoas , Brazil

Introduction: Goats are considered very susceptible to infection by Toxoplasma gondii and when this occurs during pregnancy, it may cause fetal death with subsequent fetal resorption, abortion, mummification, stillborn or the birth of weak goats. The objective of this work was to determine the occurrence of and identify risk factors for T. gondii infection in goats in different mesoregions in the State of Alagoas. Methods: The research was conducted on 24 goat breeding farms in 10 municipalities. A total of 454 blood samples were examined for anti-T. gondii antibodies by indirect immunofluorescence antibody test. To evaluate the risk factors of toxoplasmosis in goats, questionnaires were applied analyzing the farm’s production system and nutritional, reproductive and sanitary management. Results: Disease occurrence was 39% with 95.8% of farms presenting seropositive animals. Significant associations were observed for mesoregion (OR = 0.23; 95%CI = 0.09 0.57), age (OR = 0.36; 95%CI = 0.20 0.64), semi-intensive herd management (OR = 8.70; 95%CI = 1.87 40.43), access of cats to water provided for goats (OR = 3.38; 95%CI = 1.89-6.02) and cats feeding on placental remnants (OR = 2.73; 95%CI = 1.38 5.40). Conclusions: Toxoplasma gondii infection is disseminated in the State of Alagoas and the adoption of a program that monitors infection foci is required to reduce the risk factors identified in the present study.

Toxoplasma gondii is the most studied coccid in the world, with more than 15,000 original research articles and over 500 published revisions, since it is one of the most common agents in parasitic infections of homeothermic animals, including humans 1 .
Among domestic animals, the ingestion of sporulated oocysts derived from cat feces is the most important infection route.Cats are considered the principal reservoir of the cycle of toxoplasmosis, which is considered rare or nonexistent in environments without cats 2 .
Goats are considered very susceptible to infections by T. gondii and when infected during pregnancy, they present parasitemia in the first week postinfection and, consequently, in the second and third week, the placenta and fetus are infected, respectively 3 .Infection causes fetal death, with subsequent resorption, miscarriage, mummification, stillbirth or the birth of weak goats 4 .
Numerous studies of anti-T.gondii antibody prevalence in goats have been developed in several countries, including those conducted in the United Sates 5 , Venezuela 6 , Brazil 7 , Spain 8 , Ghana 9 , Uganda 10 and Thailand 11 .
Data regarding infection and the prevalence of antibodies anti-T.gondii in research conducted in Brazil highlighted the need to include the protozoan among the causes of reproductive losses in goat herds in the country 12 .However, few countries in the world regularly monitor toxoplasmosis in different hosts, including humans and particularly in animals 1 .
As demonstrated by several studies, the principal risk factors for infection by T. gondii in goats are: sex and age, in which the principal affected group is adult females; the pluviometric index, high humidity and mild temperatures in the regions where the herds are raised 9,11 ; management systems 13 ; common feeders and drinkers for young and adult goats; the presence of cats; and goats with reproductive disorders on the property in question 4,14,15 .

RESULTS
Anderlini GA et al -Risk factors associated with Toxoplasma gondii infection in goats The purpose of this work was to determine the occurrence of infection by T. gondii in goats in the State of Alagoas and identify the risk factors associated with infection.
The State of Alagoas is located in the macroregion of northeastern Brazil between the parallels 8º48'12" and 10º30'12" south and the meridians 35º09'36" and 38º13'54" west, covering a territorial area of 27.767,7 km² (approx.10720 mi²), corresponding to 0.3% of the national territory.Situated in the intertropical strip, the state receives a large quantity of solar energy throughout the year, varying from 2200 to 2600h of sunlight, which determines the hot climate, with annual temperatures around 22ºC to 28ºC 16 .The State of Alagoas is divided into three mesoregions: East, Agreste and Sertão and into 13 geographic microregions 17 .
In order to calculate the sample size, an expected prevalence of 50% was considered, to maximize the sample size, and a minimum confidence interval of 95% and statistical error of 5% was established.Thus, the working sample size (n) was determined as 358 goats 18 ; however, a total of 454 goats (40 males and 414 females) belonging to 24 properties and from 10 municipalities were eventually studied.
The goats were subdivided into three age groups: ≤ 12 monthsold, 13-24 months-old and > 24 months-old.Regarding the region of origin, the study included herds from properties located in all three Alagoas mesoregions: East, Agreste and Sertão.
The reaction of indirect immunofluorescence (RIFI) technique was used for serological assays, in accordance the previously described method 19 .The serological reactions were considered positive when total fluorescence occurred at a dilution of 1:64 20 .
To study the possible risk factors of infection, questionnaires were applied consisting of objective questions designed to elicit information regarding the breeder, general characteristics regarding the property and herd, the management system, the sanitary situation of the herd and reproductive management.From these data, identification of the risk factors associated with infection by T. gondii was conducted by univariate analysis of the variables of interest using Pearson's Chi square test or the Fisher exact test when necessary 21 and when a positive association was identified in the univariate analysis, multivariate analysis was performed using the logistic regression model, considering the serological status of the animal (positive or negative) for T. gondii as a dependent variable.The SAS (Statistical Analysis System) program was used to perform the statistical calculations, with significance determined as p > 0.05.
Of the 24 properties analyzed in this study, the presence of goats positive for T. gondii was a determined on 95.8%.In addition, of the 10 municipalities analyzed, nine (90%) showed the presence of positive animals.Considering sex, predominance of females was verified, which showed 40.3% (167/414) positivity against 25% (10/40) for males.
Risk factors; i.e., whether the variables sex, age, region, property size, number of animals and rearing system were associated or not with infection by T. gondii, were determined by univariate and multivariate analyses, as presented are in Tables 1 and 2. A significant association was not observed for the variable sex, despite the fact that prevalence in females was considerably higher than in males.
According to age distribution, high occurrence was observed among adult goats (27.6%, aged between 13 and 24 months-old and 50.4% for age > 24 months-old); however, no significant association was determined for this factor.In addition, the indices of occurrence verified in the mesoregions East (66.2%) and Agreste (41.2%) showed no association with infection by T. gondii in the multivariate analysis.
Considering property size, analysis verified that goats bred and raised in areas between 20 and 30ha had significantly higher chance of being infected (OR = 3.46) compared to those living on properties larger than 200ha.Considering herd size, higher occurrence was observed in herds with more than 100 (80%) heads of goat compared with those numbering less than 50 (48%) and between 50 and 100 (15.4%).In relation to rearing system, the goats maintained using semiintensive management showed a higher risk of infection by T. gondii (OR = 8.7) compared to extensive management.
The results of the univariate and multivariate analyses regarding the variables water source, feed, common drinkers for young and adult goats and common feeders for young and adult goats are presented in Tables 2 and 3.Although univariate analysis verified significant associations for the variables water source, common drinkers and common feeders for young and adult goats, these were not confirmed in the multivariate analysis.
In relation to the reproductive management and hygienic-sanitary variables, the presence of goats with reproductive disorders, the age at which the disorders appeared and the kind of reproductive management adopted were significant in the univariate analysis (Table 4).However, only the variable reproductive management showed a significant association in the multivariate analysis.The adoption of a natural mating system increased the chance of infection (OR = 6.49).Of the 24 properties, 17 (70.8%)showed reproductive disorders, with miscarriage being the most common problem observed (88.2%), followed by retention of the placenta (5.9%) and repetition of the rut (5.9%).
Analysis of the risk factors involving the definitive host of the agent verified that access to water provided for the goats by cats (OR = 3.38) and cats feeding on placental remnants (OR = 2.73) were significant variables and associated with the risk of infection by T. gondii, as demonstrated in Table 2.
Given the geographical dimensions of Brazil, significant climatic diversity can be a factor of influence in prevalence variations, as are the test used and the respective cutoff point adopted.Moreover, according to some authors 14 , goats maintained in suburban areas under precarious conditions of hygiene and in contact with people and their domestic animals are factors that can explain the increase in seropositivity of the herd.
Though not statistically significant in the multivariate analysis, the higher prevalence observed in females (40.4%) corroborates some studies 7,10 , but is in disagreement with research in which the variable sex was a significant factor (OR = 2.91 11 and OR = 1.73 25 ) as a determinant of infection by T. gondii , .
The hypothesis that females are more susceptible to infection, to severe forms of the disease and show higher rates of mortality by T. gondii has mainly been reinforced in herds in forest areas that are characteristically hot and humid, which coincides with the results of specific investigations 9 .Under controlled conditions in the laboratory, a dichotomy in the susceptibility between males and females is evident, revealing that physiological differences between males and females, mainly hormonal, play an important role in determining susceptibility to parasitic infections 30 .
The results of the multivariate analysis demonstrate a positive association for adult goats in relation to young.Similar results were described by others, who also verified that goats over one yearold presented a higher percentage of seropositivity in relation to those aged less than 12 months-old 11,22 .Analysis confirmed that seropositivity was directly related to age 14 and the age factor was significant for the occurrence of infection by T. gondii in goats aged over 24 9 and 36 months-old 27 .Given the time required to achieve subsistence goat breeding in the State of Alagoas, it is probable that The authors declare that there is no conflict of interest.

REFERENCES
adult animals in the goat herds evaluated have been more frequently exposed to numerous predisposing factors or sources of infections in the environment and, therefore, present a high infection index.
Although no significant findings were determined in the multivariate analysis, among the mesoregions studied, East and Agreste showed significant associations with infection by T. gondii in the univariate analysis.The Agreste region of Alagoas is a climatic transition zone between the East and the Sertão, it has a semiarid climate that is considerably humid and the volume of rainfall and the period of humidity is around 2.5-fold higher than the Sertão 31 .This could be a relevant factor for higher rates of infection determined in this study, in agreement with previous studies in the State of Bahia in Brazil 24 and in countries like Spain 8 and Thailand 11 , which showed that regions of high humidity and milder temperatures favor higher prevalences.However, in Africa, it would be a mistake to attribute differences in prevalence to higher pluviometric indices, such that attention should be focused on the kind of soil and its vegetable cover, which affect local humidity and provide microclimates favorable to the maintenance of the oocysts 10 .
In East Alagoas, the climate is humid, coastal and tropical and the mean annual temperature of around 25°C, typical of the Atlantic Forest, is propitious for the maintenance of oocysts of T. gondii, in agreement with results reported for the State of Pernambuco 25 , Brazil, which has a climate similar to Alagoas.Most notable are comparisons between the Zona da Mata in Pernambuco and the East region of Alagoas, where higher indices of seropositivity were registered for both.
Concerning the size of the properties studied, those between 30 and 200 hectares showed a significant association with infection (OR = 3.46) in the multivariate analysis.In these kinds of properties, the goats generally pasture at greater distances, facilitating their contact with areas that can be frequented by domestic or wild feline and contamination by sporulated oocysts.
The management of a property oriented by animal production constitutes a fundamental role in prophylaxis or as potential factor regarding the dissemination of disease in the herd.In this study, the semi-intensive management system proved to be a risk factor that was significantly associated with infection by T. gondii (OR = 8.7).This rearing system permits contact between goats and oocysts excreted in the feces of the wild or domestic felines, thus increasing the probability of infection.Similarly, in the State of Rio Grande do Norte, it was observed that the system of extensive management (OR = 1.18) was associated with infection 13 , also previously demonstrated in the State of Ceará 15 , particularly in regions with poor socioeconomic conditions, where goat raising consists subsistence breeding and where it is common for the goats to be confined overnight.
The presence of goats with reproductive disorders and aged between one and three years-old on the properties showed a significant association in the univariate analysis, even though this was not confirmed in the multivariate analysis.Females of this age are more susceptible and reproductive disorders in this phase become more evident in the herd 9,27 .In most herds, fetal losses occur in females of similar age, particularly among those over three years-old 4 .
The property variable regarding the adoption of a natural mating system increased the risk of infection (OR = 6.49) by T. gondii.Even in the light of this result, is not possible to definitively conclude that venereal transmission occurred in this study, although one study involving goats experimentally infected with T. gondii verified that males can excrete the parasite in semen for up to 52 days 33 .
Although the mere presence of cats on the property did not constitute a risk factor in this study, certain characteristics associated with the habitual behavior of cats were significant, including access to water consumed by the herd (OR = 3.38) and the fact that cats feed on the placental remnants (OR = 2.73).The presence of cats has been considered a risk factor for toxoplasmosis in goats 15 .In a study on goats in Texas, USA, nondetection of anti-T.gondii antibodies was attributed to the absence of cats on the properties of that region 33 .In other similar studies, the higher prevalences verified in goat herds was associated with the presence of a high density population of cats, pets, rodents and humans on the properties, providing high availability of definitive intermediate hosts for the parasite, thus increasing the chances of infection 10,15,24 .
The occurrence of antibodies against T. gondii in goats in the State of Alagoas was reported for the first time.Based on the risk factors identified, a state-wide monitoring program that focus on preventing infection is required and should include determination of the serological status of all herds and the development of educational projects emphasizing the risk factors identified in this study, in order to promote better sanitary conditions for goat herds in Alagoas.

TABLE 2 -Multivariate analysis for risk factors associated or not with infection by Toxoplasma gondii in goats in the State of Alagoas, 2008.
OR: odds ratio, CI: confidence interval.

TABLE 3 -Risk factors according to feed management, associated or not with infection by Toxoplasma gondii in goats in the State of Alagoas, 2008.
n: number of goats positive by RIFI, RIFI: reaction of indirect immunofluorescence, OR: odds ratio, CI: confidence interval.base: 454 goats, *number of goats tested, **statistically significant.

Acquisition of breeder males for reposition in the last five years
n: number of goats positive by RIFI, RIFI: reaction of indirect immunofluorescence, OR: odds ratio, CI: confidence interval.+base: 328 goats, *number of goats tested, **statistically significant.