EPIDEMIOLOGICAL STUDIES ON TOXOPLASMOSIS IN SMALL RUMINANTS AND EQUINE IN DAKAHLIA GOVERNORATE, EGYPT.

YOUNIS, E.E.^*; ABOU-ZEID, N.Z.^**; ZAKARIA, M.^* and MAHMOUD, M.R.^*

^* Department of Internal medicine, infectious and fish diseases (Infectious diseases) Faculty of Veterinary Medicine, Mansoura University, Mansoura. Egypt.

^** Department of Animal medicine (Infectious diseases), Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt.

Email: yasserelnaker@yahoo.com                                                                           Assiut University Email: www.aun.edu.eg

INTRODUCTION

Toxoplasma gondii (T. gondii) was an intracellular cyst-forming apicomplexans protozoan organisms occurring in domestic animals and man throughout the world. It has an indirect life cycle, with feline as the definitive hosts (El-On and Peiser, 2003).

The main source of infection of herbivores is infected omnivores feces, by ingestion of T. gondii sporulated oocysts in contaminated food and water. Therefore toxoplasmosis in these animals were significantly associated with the presence of omnivores in the farms. T. gondii cysts may persist in the tissues of the host for years (Dubey et al., 1989 and Dubey, 2010).

Most acquired toxoplasmosis in ovine, caprine and equine are subclinical, however fever, ataxia, retinal degeneration and encephalomyelitis may develop. Severity of toxoplasmosis in sheep and goats are associated with the stage of pregnancy. Infection during the early stage of gestation can result in fetal death, resorption and abortion, while infection in the later stage of gestation may have no clinical effect and lambs are usually born normal but infected and immune (Dubey and Beattie, 1988 and Buxton et al., 2007).

Due to the microscopic size and intracellular localization of the proliferative forms of this protozoan parasite and the relative difficulty in its laboratory cultivation, the development of immunodiagnostic tools such as serology and immunohistochemistry are essential in the demonstration of infection (Uggla and Buxton, 1990). The aim of the present study was to determine the Incidence and possible risk factors associated with toxoplasmosis in small ruminants (sheep & goats) and equine (horses and donkeys) from Dakahlia governorates, Egypt.

MATERIALS and METHODS

1. Animals

a)    Slaughtered animals: 25 slaughtered sheep at Mansoura abattoir and 25 donkeys slaughtered at Mansoura Zoo were used in serological diagnosis of toxoplasmosis; histopathology and tissues bioassay in cats.

b)    Surveyed animals: 267 sheep, 81 goats, 54 horses and 54 donkeys of different sex from different localities in Dakahlia province aged between 3 months to 8 years were used for determination of seroprevalence of toxoplasmosis in sheep and goats and identification of risk factors associated with toxoplasmosis in the period from October 2013 – October 2014.

c)     Cats:  100 cats 2-3 months age were used for bioassay of tissue samples taken from slaughtered sheep at abattoir and slaughtered donkeys at zoo (2 cats were used for each tissue sample). They were previously proved free from; Nematodae; Cestodes; Isospora, Eimeria and T. gondii oocysts through fecal examination. Cats were fasted overnight, each 2 cats fed one tissue sample (diaphragm muscles) cut into small pieces using disposable scalpel and then faecal samples of cats were collected daily for 14 days post infection and examined microscopically for oocysts as described by Dubey, (2001) by fecal floatation technique.

1. Samples:

a)    Blood Samples:  10 ml of blood were collected from examined sheep, goats, donkeys and horses. After clotting at room temperature, blood was centrifuged at 3000 rpm for 10 minutes and the collected sera were stored at -20^oC until assayed for antibodies against toxoplasmosis.

b)    Tissue Samples: About 50 grams of freshly tissue specimens from diaphragm of slaughtered sheep and donkeys were used for bioassay in cats (2 cats were used for each tissue sample) according toDubey, (2001) and 50 gm preserved in formalin 10% used for histopathological examination according to Bancroft and Stevens, (1996).

c)     Faecal Samples of cats were collected daily for 14 days post infection and examined microscopically for oocysts as described by Dubey, (2001).

1. Serological examination of serum samples from sheep, goats, horses and donkeys for toxoplasmosis

(1)      Latex agglutination test (LAT).

Latex antigen preparation: The antigen prepared through sensitization of latex particles with locally isolated T. gondii strain sonicated tachyzoites as procedures of Lunde and Jacobs (1967). The test procedures were adopted as methods of Holliman      et al. (1989).

(2)      Indirect haemagglutination test (IHAT). Using Toxo-HAI Fumouze kit (Fumouze, France). Procedure was done according to the manufacturer's instructions, according to (Camargo and Leser, (1976).

(3)               Enzyme linked immunosorbent assay (ELISA). According toVoller et al. (1976) .

Antigen preparation: It was kindly obtained from Zoonotic Diseases Department, Veterinary Research Division, National Research Centre, El-dokki, Giza, Egypt). Whole soluble tachyzoites antigens were prepared as described byWaltman et al. (1984).

The technique was performed as previously described by Voller et al. (1976). In brief, ELISA plates were coated over night at 4^oC with 10 µg/ml of T. gondii antigen diluted in carbonate-bicarbonate buffer (0.1 M, pH 9.6, 100 µl/ well). The plates were washed three times with 0.05% PBS-Tween 20 (PBS-T) and blocked by addition of 100 µl/well bovine serum albumin in PBS for one hour at 37^oC. After repeated washing, 100 µl / well of duplicate dilution of sera (positive, negative or tested serum sample) [Sera diluted 1:100 in PBS-T] was added per well and incubated for one hour at 37^oC. The sera were removed by repeated washing and 100 µl / well of conjugate, which was anti-sheep/ anti-horse IgG horseradish peroxidase enzyme conjugated then incubated for one hour at 37^oC. After three washings of the plates with PBS-T, 100 µl/well of p-nitrophenyl phosphate in substrate buffer were added and re-incubated for one hour at 37^oC. Stopping the reaction was done parallel for the tested sera. Results were expressed as OD and the positive threshold value was determined to be equal to the mean of the negative control sera value + two folds the standard deviation.

Statistical analysis:

The obtained data were computed and analyzed by using repeated measures ANOVA and the results were tested for significance using t-test (SAS, 1996). To identify risk factors associated with infection by T. gondii, a bivariate analysis was carried out using Chi-square and Fisher’s exact tests with significance level of 5%, using the statistical program EPI-INFO, version 3.5.1.

RESULTS

1 - Seroprevalence rate of toxoplasmosis:

Seroprevalence rate of toxoplasmosis were 41.7%, 66.1% and 62% in sheep, 49.4%, 64.2% and 50.6% in goat, 50%, 72.2% and 72.2% in horse and 44.3%, 67.1% and 68.4% in donkeys, by LAT; IHAT and ELISA respectively (Table 1). The peak levels of IHAT titers in both sheep and goats was >1:1280, whereas in horses and donkeys was 1:160.

2 - Bioassay in cats

The results of tissues bioassay in cats revealed that 8 out of 25 slaughtered sheep (32.0%) and 9 out of 25 slaughtered donkeys (36.0%) were positive(Table 2).Oocysts of T. gondii were observed after a prepatent period (P.P.P.) 3 – 4 days. The cats continued shedding toxoplasma oocysts for 9 -13 days from the appearance of oocysts (Picture 1).

Table 1: Seroprevalence of toxoplasmosis in sheep, goats, horses and donkeys using LAT, IHAT and ELISA tests.

3. Histopathological examination

Histopathological examination of bioassay positive diaphragm muscles revealed T. gondii tissue cysts in only 3 (37.5%) sheep samples and 4 (44.4%), donkeys samples (Picture 2).

4. Sensitivity and specificity of different serological tests in relation to bioassay in cats

The comparison between results of serological tests and results of bioassay in cats, the sensitivity of LAT, IHAT and ELISA were 87.5%, 100% and 100% in sheep respectively and 55.7%, 88.9% and 100% in donkeys respectively, while the specificity of LAT, IHAT and ELISA were 76.5%, 100% and 100% in sheep respectively and 93.8%, 93.8% and 100% in donkeys respectively (Table 2).

5. Risk factors associated with the infection with T. gondii in sheep, goats, horses and donkeys in sheep, goats, horses and donkeys:

a. Host: The seropositivty of toxoplasmosis by ELISA was higher in equine (horses 72.2% and donkeys, 68.4%) than in small ruminants (sheep, 62.0% and goats, 50.6%).There was a highly significant differences in T. gondii infection among animal species, seropositivity in horses was 4.88 times (P. value <0.000), in donkeys was 2.54 times (p= 0.013) and in sheep was 1.8 (p=0.023) higher than was in goats.

b. Gender: T. gondii infection was significantly higher (P- value < 0.01) in female sheep (67.3%), goats (62.5%), horses (80.0%) and donkeys (90.3%) than in male sheep (48.8%), goats (24.0%), horses (57.9%) and donkeys (73.9%). 

c. Age: Toxoplasmosis in small ruminants (sheep and goats) and equine (horses and donkeys) was significantly higher (P- value < 0.05) at age >5 years in sheep (80.7%) [OR = 17.5], goats (66.7%) [OR = 6], horses (89.5%) [OR = 13] and donkeys (83.7%) [OR = 18] than that at age < 2 year sheep (41.6%), goats (31.3%), horses (50.0%) and donkeys (30.8%).

d. Abortion: There was non-significant increase (p. value >0.05) in incidence of toxoplasmosis in aborted ewes 15/17 (88.2%) [OR= 1.22] and goats 5/6 (83.3%) [OR=3.33] than those with normal birth 92/153 (60.1%) and 21/35 (60.0%) in ewes and goats respectively, While  in equine, toxoplasmosis in both aborted mares and she-donkeys (75%) was less than in normally birthing  mares (79.17%) [OR=0.79] and she-donkeys (86.9%) [OR=0.43].

e. Rearing system: Toxoplasmosis was highly significantly increased in sheep and goats which raised in intensive management system (76.4% &56.8%) [P- value, < 0.01] than those raised in extensive (46.5% & 33.3%) and semi-intensive system (62.1% & 46.4%).Moreover sheep & goats reared under intensive system were 2.6 times more likely to have toxoplasmosis than those reared under extensive system [Table 3].

Table 2: Showing sensitivity and specificity of LAT, IHAT and ELISA in slaughtered sheep and donkeys using tissues bioassay in cats as a gold standard test.

Table 3: Showing some risk factors related to toxoplasmosis in small ruminants (sheep and goats) and equine (horses and donkeys).

Picture (1): Toxoplasma gondii non sporulated oocysts. (A) and T. gondii sporulated oocysts (B). X 1200

Picture (2): photomicrograph from diaphragm muscle of sheep showing T. gondii tissue cyst (arrow) {H & E. X 1200}.

DISCUSSION

Toxoplasmosis has a very wide geographic distribution and is considered to be one of the most common parasitic infections of man and warm blooded animals. Clinical symptoms of toxoplasmosis are not specific. Therefore, evaluation of serological tests becomes important in order to use sensitive and specific tests in serological surveys (Uggla et al., 1983 and Moreno     et al., 1991).

With regard to the seroprevalence of toxoplasmosis, 122 (41.7%), 193 (66.1%) and 181 (62.0%) out of 292 sheep, 40 (49.4%), 52 (64.2%) and 41 (50.6%) out of 81 goats, 27 (50.0%), 39 (72.2%) and 39 (72.2%) out of 54 horses and 35 (44.3%), 53 (67.1%) and 54 (68.4%) out of 79 donkeys were sero-positive against toxoplasmosis by LAT; IHAT and ELISA respectively. The difference in antibody response observed by use of the three serological tests may be in part due to the type of antigen used and in other part due to the class of antibodies measured. These results were concordant with those reported by Shaapan (2005) who found that the seroprevalence of toxoplasmosis in slaughtered sheep were 34 % by DT, 37 % by IFAT, 41.7%  by ELISA and 43.7 % by MAT.

High seroprevalence of toxoplasmosis among sheep seems to be catches with those reported previously where 51.5% in Brazil by IFAT(Romanelli et al., 2007); 50.4% and 61.4% in Egypt by LAT and ELISA respectively(Hassanain et al., 2011); 71% in Libya byLAT(Al-mabruk et al., 2013) and67% in the Caribbean Dominica Islandby ELISA(Hamilton et al., 2014). In goats our results were nearly similar to those obtained by several authors where 46.0% in Brazil by IFAT (Carneiro et al., 2009); 50% in Turkey by IHAT(Sevinc et al., 2000a) and 52.8% in Romania by ELISA (Iovu et al., 2012). Moreover in equine similar results were described where 65.3% of donkeys from Monofia province, Egypt by ELISA (El-Ghaysh, 1998); 71.2% of horses in Iran by MAT (Hajialilo et al., 2010).

On the other hand, lower and higher seroprevalence of toxoplasmosis in sheep were observed elsewhere; for examples, 95.7% and 90.9% in Kars province, Turkey by ELISA and SFDT respectively (Mor and Arslan, 2007); 34.5% in Somaliaby LAT(Kadle, 2014); 29.9% in Mexico by MAT (Alvarado-Esquivel et al., 2013)and 3.3 % in Iran by IFAT (Derakhshan and Mousavi 2014). In goats lower seroprevalence of toxoplasmosis were reported by Figueiredo et al. (2001)in Brazil 18.4% by IHAT; Kandil and Abou-Zeina (2000)in Egypt 39% by ELISA andDerakhshan and Mousavi (2014) in Iran 1.7 % by IFAT, whereas higher results were reprted byBeyhan et al.  (2013)in Turkey 95.24% by (SFDT), Also variable seroprevalence of toxoplasmosis in equines, were observed elsewhere; for examples, 6.9% of horses in North America (MAT)(Dubey et al., 1999), 7.2% of horses in Turkey (SFDT) (Karatepe et al., 2010); 25% of horses and 23.6% of donkeys in China by MAT(Yang    et al., 2013); and 5% and 8% of donkeys in Italyby LAT and IFAT (Machacova et al., 2014). The difference in seroprevalence rates in the present work and the previous studies may be also attributed to abundance of infected cats and their contact with examined animals in farms and slaughtered houses.

With regard to tissue bioassays in cats the results revealed T. gondii from 32.0% & 36.0% of slaughtered sheep and donkeys. These results were in agreement with Esteban-Redondo et al. (1999) and Da Silva and Langoni, (2001). Also Shaapan and Ghazy, (2007) found that 52.6% of horses were        T. gondii infected by bioassays in mice and cats.

Results of histopathological examination of diaphragm of sheep and donkeys, which positive by tissues bioassay in cats, revealed T. gondii tissue cysts in only 3out of 8 (37.5%) and 4 out of 9 (44.4%), respectively. These results were in agreement with those recorded by Belbacha et al. (2004) and Hassanain et al. (2011).

The comparison between results of serological tests and results bioassay in cats, the sensitivity of both ELISA and IHAT in sheep and donkeys were 100%, whereas sensitivity of LAT was 87.5% and 55.7% in sheep and donkeys. Also specificity of ELISA in sheep and donkey were 100%, while specificity of IHAT and LAT were 100% & 76.5% in sheep and were 93.8% & 93.8% in donkey. These results coincided with those detected by Figueiredo et al. (2001) who reported that there was a high and significant positive correlation between IHAT versus IFAT, IHAT versus ELISA, and ELISA versus IFAT; Mcload and Remington, (1996) and Puije et al. (2000),who reported that ELISA was highly sensitive 92% and specific 91% when compared to IFAT which was used as reference.

With regard to species susceptibility, the seropositivty of toxoplasmosis by ELISA was higher in equine (horses 72.2% and donkeys, 68.4%) than in small ruminants (sheep, 62.0% and goats, 50.6%). The higher percentage of infection in equine may be attributed to the old infections of examined animals, where the peak levels of IHAT titers in horses and donkeys was 1:160 and was >1:1280 in both sheep and goats. These results were in agreement with those reported byPuije et al. (2000)in Ghana 32.2% & 26.8% of sheep & goats by ELISA and Ahmed et al. (2013) in Sudan 38% and 27.6% of horses and donkeys respectively by LAT. This result differs from those reported byGondim et al. (1999)in Brazil 28.93% and 18.75% in goats and sheep by LAT; Prelezov et al. (2008) in Bulgaria 48.2% & 59.8% of sheep and of goats by IHAT and Bocanegra et al. (2012) in Spain 10.8% & 25.6% of horses and donkeys by MAT. This variation in species susceptibility may be attributed to difference on the environmental and ecological condition, which effect on the biology of the parasite or the system of breeding and hygienic measures inside farms.

With regard to gender, T. gondii infection was significantly higher (P- value < 0.01) in female sheep (67.3%), goats (62.5%), horses (80.0%) and donkeys (90.3%) than in male sheep (48.8%), goats (24.0%), horses (57.9%) and donkeys (73.9%). These were in agreement with those reported by Hajialilo et al. (2010); Al-mabruk et al. (2013); andDubey et al. (2014).These results were not coincided with Esmat, (1997) and Boughattass et al. (2011). This variation may be attributed to the young age of male sheep and goats used in this study.

With regard to age susceptibility, toxoplasmosis was significantly higher (P- value < 0.05) in sheep, goats, horses and donkeys at age >5 years than that at age <1 year. This variation may be due to the exposure of old animals to T. gondii oocysts for long periods. These results were in agreement with those observed by Figueiredo et al. (2001); Boughattass et al. (2011) and Al-mabruk et al. (2013).

Moreover toxoplasmosis was significantly higher in sheep and goats which raised in intensive management system (76.4% &56.8%) than those raised in extensive (46.5% & 33.3%) and semi-intensive system (62.1% & 46.4%). This variation may be related presence of uncontrolled number of stray cats around farms. These results coincided with those recorded by Neto et al. (2008); Tzanidakis et al. (2012) and Al-mabruk et al. (2013).

Toxoplasmosis was highly significantly increased in aborted sheep 15/17 (88.2%) and 5/6 (83.3%) goats than those with normal birth 92/153 (60.1%) and 21/35 (60.0%) respectively. These differences may be due to multiplication of T.gondii in placenta and release of antigen into maternal circulation. These results were in agreement with those reported byAktas et al. (2000); Sevinc et al. (2000 b) and Hussein et al. (2011).

It could be concluded that IHAT in sheep, goats, horses and donkeys could be used as a screening test in seroepidemiological studies, and their results must be confirmed by ELISA. Species, gender, age, rearing system play an important role in incidence of toxoplasmosis. Seroprevalence of T. gondii infection indicated wide spread of toxoplasmosis in sheep, goats, horses and donkeys in Dakahlia province. Thus further studies will be needed to clarify the role of sheep, goats, horses and donkeys in transmission of infection to man and pet animals.

ACKNOWLEDGEMENTS

The authors are deeply indebted to Dr. Raafat M. Shaapan, Prof. of zoonotic Diseases, Veterinary Research Division, National Research Center, for providing T. gondii strain used, antigen preparation for LAT and ELISA. Also, the authors are thankful to Mohamed Afifi (Biostatistics Assistant Lecturer) faculty of Veterinary Medicine, Zagazig University, for his valuable assistance in performing the statistical analysis.

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