Toxoplasma gondii in goats from Curitiba , Paraná , Brazil : risks factors and epidemiology Toxoplasma gondii em caprinos de Curitiba , Paraná , Brasil : fatores de risco e epidemiologia

Toxoplasmosis is a zoonosis caused by Toxoplasma gondii, a protozoan with wide geographical distribution and minimal parasitic specificity that affects many species of wild and domestic animals. In livestock, especially in small ruminants like goats, toxoplasmosis can cause abortion and the birth of weak animals, leading to economic losses to farmers, and is a major source of human infection. This is a seroepidemiological study of toxoplasmosis in goats in the state of Paraná, Brazil. Sera from 405 goats from the metropolitan mesoregion of Curitiba, eastern state, were tested by the enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence antibody test (IFAT). Information on properties and goat characteristics was also collected using questionnaires. The prevalence of toxoplasmosis was 39.41 and 35.96% by ELISA and IFAT, respectively. T. gondii antibody prevalence increased with age. The risk factors for T. gondii infection in goats were: age over one year; exposure to cats, type of management and purpose of breeding. Other epidemiological factors and relevant control measures are discussed in the current study.


Introduction
Toxoplasma gondii is a coccid parasite for which cats are the definitive host and warm blood animals act as intermediate hosts (FRENKEL et al., 1970).It is present almost worldwide and is of medical and veterinary importance because it can cause miscarriages and birth defects in intermediate hosts (TENTER et al., 2000).Feldman and Miller (1956) were the first to report toxoplasmosis in goats, examining livestock in New York State.From then on, the disease has been identified as a major cause of reproductive problems in sheep and goats in many countries (SKJERVE et al., 1998;BORDE et al., 2006).T. gondii infection can cause neonatal mortality and abortion at different gestational ages, fetal mummification, stillbirth or perinatal mortality (BLEWETT, 1983).
Studies conducted in Uruguay showed that toxoplasmosis is an important problem in sheep, causing annual losses of US$ 1.4 to 4.7 million (FREYRE et al., 1999).Munday and Mason (1979) were the first to describe toxoplasmosis as an important cause of reproductive losses in goats.Despite less documented in goats, it seems to cause much more damage in these animals and adult goats are also clinically affected (DUBEY, 1987).
Several epidemiological studies have been performed in Brazil where toxoplasmosis prevalence varies from 10 to 92.4%, according to geographical region and diagnostic technique used (Table 1).
There is concern about the transmission of T. gondii through in natura goat milk and its products as well as goat meat and byproducts when these are consumed by humans.The consumption of poorly pasteurized goat milk is a great public health concern.Toxoplasmosis has become one of the most widespread zoonoses as goats with acute infection can eliminate tachyzoites through their milk (SKINNER et al., 1990;VITOR et al., 1991).Silva Filho et al. (2008), in the municipality of Guarapuava, southern state of Paraná, described an outbreak of abortion in a goat herd occurred between June and July 2005, there were in the property 304 goats and 89.1% of them showed anti-T.gondii antibodies by IFAT.From 136 pregnant 61 miscarried, and of these, 59 had titers positive for T. gondii.The epidemic outbreak in this property was an atypical event.However, little data is available on the importance of this animal species for the maintenance of T. gondii life cycle in the state of Paraná where goat meat and milk production is a major industry.There is a lack of data on the current prevalence of anti-T.gondii antibodies in goats and whether the rate of anti-T.gondii antibodies is similar in all herds.ELISA and IFAT techniques were used in this serological study and the main risk factors for goat toxoplasmosis were assessed.

Materials and Methods
Sera from 405 goats of 12 livestock in the metropolitan mesoregion of Curitiba, state of Paraná, southern Brazil (25° 25' 40" S and 49° 16' 00" W), were analyzed.The minimum number of sera to be tested was calculated assuming a 50% prevalence, 95% confidence interval and 5% precision (THRUSFIELD, 2004).Blood samples were collected by jugular venipuncture and sera were separated, identified and stored at -20 °C until their use.Information on the epidemiology of toxoplasmosis including property location, facilities, presence of other animals, sanitary conditions, type of handling and livestock exploration and sex and age of goats were collected.Two techniques were used to detect anti-T.gondii IgG antibodies in the sera, indirect immunofluorescence antibody test (IFAT) and immunoenzymatic assay (ELISA).
For both techniques, antigens were from tachyzoites obtained by in vitro culture.The standard RH strain was grown in Vero cell and RPMI Medium 1640 (GIBCO™) was the culture medium containing 2% sodium bicarbonate with 10% fetal calf serum.Culture bottles were then left in a carbon dioxide heater at 37 °C and 5% CO 2 until cell growth was visible and the monolayer had been established.Next, cells were infected with tachyzoites and growth or contamination of culture bottles was examined under an inverted microscope at 250 and 400× magnification.After more than 75% of the monolayer had been destroyed, the medium was removed to a 50 mL Falcon tube, the bottle was scraped with a rubber rod and washed with sterile PBS, pH 7.2, and this PBS was placed in the same Falcon tube.The contents were passed through a 15 × 6 needle to rupture the Vero cells.Fragments were placed between a slide and cover slip and examined under a light microscope to check for cell rupture and tachyzoite release.The Falcon tube containing the liquid fraction of the bottle was centrifuged at 2500 x g for 10 minutes.The supernatant was discarded and the pellet was diluted in PBS, pH 7.2, and used for the production of intact (IFAT) or soluble antigen (ELISA).
IFAT was performed according to Camargo (1964).Fixed slides with standard amounts of antigen were used.The parasites were counted in a Thoma chamber, several concentrations of tachyzoites per µL (1600,800,400,200,100,50,25) were tested, and 20 µL of tachyzoite solution per spot were added.The best concentration was 200 tachyzoites per µL or 4000 parasites per spot, i.e., 100 to 150 parasites per field at 400 x magnification, which allows reading and visualization of parasite outline fluorescence.Sera were initially diluted at 1:16 and 1:64.Sera that were reactive at 1:64 were also tested at dilutions of 1:256, 1:1024, 1:4096 and 1:16384.Following evaluation in triplicate of different conjugate dilutions against different serum dilutions, a dilution of 1:200 was selected as it allowed good differentiation between positive and negative sera.The conjugate used was anti-caprine IgG FITC (whole molecule, SIGMA).The reaction was considered positive when titers were equal to or greater than 1:64.
For ELISA antigens were obtained from tachyzoites disrupted by five cycles of freezing (-196 °C) and thawing (+37 °C) followed by ultrasound (for 1 minutes three times at 60 Hz, Bandelin Sonoplus HD 2070), centrifuged (9000 x g 30 minutes) and filtered (0,22 µM).The protein was dosed by Bradford method (BRADFORD, 1976).The soluble antigen obtained was diluted in sodium carbonate-bicarbonate buffer, pH 9.6, at concentrations of 250, 500, 1000 and 2000 ng.mL -1 , which were tested for standardization.To each plate wells (Nunclon, cat nº 167008) 100 µL of the soluble antigen were added and incubated in a humidity chamber at 4 °C for 12 to 14 hours.The plate was washed three times with 0.01 M PBS, pH 7.4, containing 0.05% Tween 20 or 80 (PBST).Blockage of the wells was performed with 200 µL of 0.05 M sodium carbonate-bicarbonate buffer solution, pH 9.6, which was removed by three washes with PBST.First, two positive and two negative control sera were tested at different concentrations of the antigen protein (1000 and 2000 ng per well) and different conjugate dilutions (1:5,000, 1:10,000, 1:20,000 and 1:40,000) and sera (1:100, 1:200 and 1:400).Standardization was achieved by assessing the best combination of serum dilution, conjugate dilution and antigen concentration with the least amount of expensive reagents (conjugate) or those difficult to obtain (antigen and serum).
The purpose of the test was considered to establish the cutoff for ELISA.A high sensitivity was chosen because the purpose was to assess the seroprevalence of anti-T.gondii antibodies in goats.Thus, the cutoff was calculated from the mean of eight negative control sera plus twice the standard deviation.
For statistical analysis, a database was created based on epidemiological data and serological results.Comparisons were made between positive and negative rates, and goat sex and age.Data were analyzed by z-test probabilities (mutually exclusive categories) and the chi-square test (χ 2 ), followed by z-test of standardized residuals (HABERMAN, 1973).The risk factors for goat toxoplasmosis were also defined according to ELISA, by calculating the relative risk for each factor (THRUSFIELD, 2004).The statistical significance was set at p ≤ 0.05.
This study was approved by the Animal Experimentation Ethics Committee of the Universidade Federal do Paraná Biological Sciences Department under protocol nº 427.

Results
Seropositive goats were found in all herds.The prevalence rates varied among the farms studied (Figure 1) for both IFAT and ELISA.Of 405 sera studied, 36.05% were positive and 63.95% negative for IFAT and 39.5% were positive and 60.5% negative for ELISA, varying according to goat sex and age (Table 2).In regard to sex, 24.6% of bucks and 38.4% of does were seropositive by IFAT (χ 2 = 4.69; p < 0.05) while 23.1% of bucks and 42.9% of does were positive for toxoplasmosis by ELISA (χ 2 = 9.26; p < 0.05); the differences were statistically significant in both methods (Table 2).
As for age, by IFAT, 16% of goats younger than 1 year old, 34.1% of those 1 to 3 years old and 48.5% of those older than 3 years old were positive for toxoplasmosis (χ 2 = 28.4;p < 0.05) while by ELISA 20.2, 34.1, and 54.3 of goats, respectively, were positive (χ 2 = 32.3;p < 0.05) (Figure 2); again, the differences were statistically significant in both methods.The amount of serum reagents increased with goat age (z-test, p < 0.05).

Discussion
The prevalence of anti-T.gondii IgG antibodies in goats in the metropolitan mesoregion of Curitiba was 39.5% by ELISA and 36.05% by IFAT.However, the prevalences ranged from 14.3 to 80.0% in individual farms.
The mean prevalence found in the current study was greater than that reported in Londrina, State of Paraná (30.71%) (SELLA et al., 1994), in Rio Grande do Sul (30%) (MACIEL; ARAÚJO, 2004) and São Paulo (23.4%) (MODOLO et al., 2008).However, the prevalence found was lower than that reported in Minas Gerais, 68 and 43% reported by Chiari et al. (1987) and Carneiro et al., (2009), respectively.These differences could be due to different serological techniques and cutoffs used in the studies, different type of animal handling and geographical region, and climate changes over the last few years.
The analysis of the results showed that the prevalence of toxoplasmosis increase with goat age, and that age is a risk factor for infection.This is because older goats have been exposed to oocysts present in the environment for a longer period of time, which increases the likelihood of exposure to the parasite (SELLA et al., 1994;JITTAPALAPONG et al., 2005;CAVALCANTE et al., 2008).As for sex, the proportion of seropositive does was greater than that of bucks.This can be explained by the fact that the mean age of bucks was lower than that of does because, while bucks are slaughtered before reaching one year of age, does are kept for reproduction and milk production.
A positive association was found between high toxoplasmosis prevalence and presence of cats living close to livestock.Felines play an important role in the transmission of infection due to the elimination of oocysts in their feces, thus contaminating the environment.The access of cats to the area where goat food is stored was also a major risk factor, as well as the type of feed cats were provided; in farms where cats were fed with cat food, the positive rates were lower than those in farms where cats hunted for food.
A higher prevalence of toxoplasmosis was seen in farms located in periurban compared to rural areas.This is due to the fact that population density and dynamics of definitive and intermediate hosts vary between urban and rural environments.Bisson et al. (2000) also reported similar differences, as did Kamani et al. (2010) andThomaz-Soccol et al. (2009) for this or other animal species.Lower infection rates were seen in goats under intensive handling system.Figueiredo et al. (2001) and Araújo Neto et al. (2008) have both reported that extensive/semi-extensive handling systems are risk factors associated with anti-T.gondii antibodies.Goats raised under these farming systems are more likely to be exposed to oocysts shed by wild and domestic felines on pastures.
Greater serum prevalence was also seen among dairy compared to meat goats.Goats used for milk production have a higher mean  age than those raised for slaughter, thereby increasing their likelihood of contamination.Exclusively dairy herd farms were not studied in the current study, but high seropositivity was determined in mixed-exploration herds (milk and meat).
The current study showed that does are a group at risk of infection.As abortions and fetal damage only occur during the acute phase of infection, does with chronic infection are no longer a reason for concern for the breeder since they can be considered immune to the disease.However, it is necessary to monitor food and water provided to serum negative goats, particularly during breeding and gestational periods, when problems may occur.
Since herbivorous are mainly infected through oocyst ingestion, the elimination of cats from goat farms is recommended, or cats should be provided cat food, because serum prevalence was lower in farms where cats were fed with cat food.However, this is not a solution to the problem as other cats living in the area or wild felines can also eliminate oocysts and most breeders keep cats to control rodents and pests.Thus, it is very difficult to convince them of the need to eliminate the presence of felines.It should also be emphasized that chronically infected cats can hardly excrete oocysts, and therefore they do not pose a risk in this case.The focus should be on younger cats.These recommendations should be given to breeders because knowledge of the risks can help implement control measures to reduce infection during gestational periods.Thus, the best way to control infection is to protect goat feed from exposure to cats and the herd should be provided with water from a reliable source.
The current study identified the risks of toxoplasmosis infection in goats, and one should bear in mind that this animal species is part of the human food chain and people can become infected by drinking milk or eating raw or undercooked meat.This species should not be overlooked as a source of infection, especially for pregnant women.Vaz et al. (2010) showed that the group at risk of infection is around 46.07% and the seroconversion rate was 0.16%.To minimize the risks of prenatal infection and consequently fetal sequelae, pregnant women should be educated about the risks of drinking milk or eating raw or undercooked meat of this species and other animals.
The analyses of serological and epidemiological data in the current study allow to conclude that the mean prevalence of toxoplasmosis infection in goat livestock in the metropolitan mesoregion of Curitiba varied according to property studied, from 11.4 to 80%, and that the prevalence of T. gondii antibodies in goats increased with age.Besides age, the other main risk factors for goat toxoplasmosis are presence of cats living close to livestock, access of cats to goat feed, semi-intensive handling and mixedexploration herds.Knowing serum prevalence of toxoplasmosis in goat livestock can help develop and implement infection control measures.

Figure 1 .
Figure 1.Prevalence of anti-Toxoplasma gondii antibodies in goats of different ages from 12 farms in the metropolitan mesoregion of Curitiba, State of Paraná, southern Brazil.

Figure 2 .
Figure 2. Seroprevalence of anti-Toxoplasma gondii antibodies determined by microplate-ELISA according to age.

Table 1 .
Prevalence of toxoplasmosis in goats in Brazil according to state and year.

Table 2 .
Serology results according to goat sex and age.

Table 3 .
Risk factors associated with anti-Toxoplasma gondii antibodies, by ELISA.