Evaluation of sero-prevalence of toxoplasma gondii infection in sheep using different immunodiagnostic methods

Toxoplasmosis is a significant zoonotic infection caused by the obligate intracellular protozoan parasite, Toxoplasma gondii which is cosmopolitan in distribution (Tenter et al., 2000). The main clinical sign of the disease in goats, sheep and humans is abortion. The definitive hosts of T. gondii are cats while warm blooded animals are the intermediate hosts (Abu-Dalbou et al., 2010).The sexual reproduction of T. gondii occurs only in cat which is the transmission vector and feaces are the main source for transmission of infection. In animals, the infection of T.gondiioccurs by consuming food or water contaminated with cat faeces or by eating meat infected with cysts. Whereas, humans get infection by ingestion of tissue cysts in undercooked or uncooked meat or it can even be transmitted from mother to fetus (Shah et al., 2013). Congenitally infected lambs have shown clinical signs of incoordination, weakness and a high mortality rate. The infection caused by T. gondii has significant veterinary implication because it causes miscarriage or congenital malformations in the definitive host as well as in intermediate hosts. Prevalence in sheep and goats is generally very high due to the continuous contamination of pastures by T. gondiioocysts (Dubey, 2009). A large numbers of mammal and bird species are susceptible to infection and act as an intermediate host (Thompson et al., 2009). After ingestion of oocysts by a secondary host (humans, birds, rodents and domestic animals) the oocysts release sporozoites which change into tachyzoites (Skariah et al., 2010). Among the ruminants, Toxoplasma tissue cysts are mostly found in sheep and goats and rarely in farmed deer, cattle and water buffalo (Bubalisbubalis) (Tenter et al., 2000). It has been found that about one-third of the human population worldwide is infected with T. gondii (Abu-Dalbou et al., 2010). But the frequency differs depending on geographical areas. Seroprevalence of toxoplasmosis of 20%-30% was found in USA, 25% in Japan, 60% in Netherland and Italy, 50% in Finland and 50%-60% in Poland. Some countries have incidence of more than 80%. The seroprevalence rate in Dera Ghazi Khan, Pakistan was detected to be 29.5%. Prevalence of T. gondii infection can vary from 0 to 100% in sheep flocks in different countries. The estimated worldwide seroprevalence of toxoplasmosis in livestock has been reported as 30% in sheep, 15% in goats and 9% in cattle. The present study was aimed to investigate the seroprevalence of Toxoplasma gondii infection in sheep of seven different regions of District Dir (Lower), Pakistan.

Study Design
 
District Dir Lower was divided into seven different areas such as LalQilla, Munda, Samarbagh, Balambat, Timergara, Khall, and Adenzai. One hundred and seventy five (175) blood samples were collected from sheep of all the seven areas (25 from each area). The data of all goats and sheep were taken in a suitable questionnaire format that identified the age gender and area. The research work was carried out in the Fazal Rahim Clinical Laboratory, Timer gara. All the samples was analyzed by Lateral Flow Chromatographic Immunoassay (LFCI), Latex Agglutination Test (LAT) and screened for specific IgM antibody against T. gondii using ELISA.
 
Blood samples collection
 
5 ml blood samples were taken from the jugular vein of sheep and allowed the blood to clot.The serum was separated by centrifugation and carefully withdrawn into a new pre-labeled tube and stored in a freezer at -20°C.
 
Lateral flow chromatographic immunoassay technique
 
The test device was placed on a clean surface and labeled with the specimen’s tag table number. Then one drop (30.5 - 45.5 micro liters) of serum was put into the sample wells and one drop of diluent was also put into the sample wells. The result was displayed in 10-15 minutes. If the M line was produced, the test showed the presence of IgM anti-T. gondii in the sample and if G line was produced the test showed the presence of IgG anti-T. gondii in the sample. If both the M and the G lines were produced, the test shows the presence of both IgG and IgM anti-T. gondii in the sample.
 
Qualitative technique of latex agglutination test
 
The reagents and the samples were stored at 18-25 degree Celsius before use and the Toxo latex performance was tested with the available positive and negative controls. Then 50.0 micro liter serum and 40.0 micro liter Toxo latex chemical was placed on one portion of the slide and mixed for 4.0-5.0 seconds to obtained uniform suspension. Then the slide was shaken at 100.0 rpm for 5 minutes and slide was observed under the microscope for the presence or absence of agglutination. Samples showing agglutination were considered as reactive.
 
Enzyme-linked immunosorbent assay (ELISA) technique
 
The samples tested by Lateral Flow Chromatographic Immunoassay and Latex Agglutination Test (LAT) were confirmed by Enzyme-Linked Immunosorbent Assay (ELISA) technique under the given protocol. All the samples were screened for specific IgM antibody against T. gondiiby Enzyme-Linked Immuno Sorbent Assay (ELISA). For this purpose PrioCHECK® Toxoplasma Antibody SR ELISA (Catalog number: 7610240) were used with coated micro well plate having purified Toxoplasma antigen.
 
Statistical analysis
 
Bio-statistical analysis was performed by Chi-square (c2) test (Graph Pad Prism 6.01 Version).

Area, age and gender wise prevalence (%) of toxoplasma gondii in sheep by LFCI
 
In all the seven different areas in DisttDir (Lower), prevalence of sheep indicated highest infection at Khall (40%) followed by Samarbagh (40%), Lalqilla (36%) Adenzai (36%) Balambat (32%), Timergara (32%) and lowest in Munda (28%) (Table 1). All Areas showed non-significant difference (c2 =1.309, d.f = 6, P =0.9712). It was noticed that total prevalence in older goats was significantly (P< 0.05) higher (41%) than younger ones (27.5%) (Table1). Age wise prevalence of sheep showed non-significant difference (P = 0.0796). Generally sex wise prevalence indicated that females (36%) were more sensitive than males (32%) (Table1). Statistical difference was not noticed (P = 0.7260).


 
Area, age and gender wise prevalence (%) of toxoplasma gondii in sheep by LAT
 
In all the seven different areas in DisttDir (Lower), prevalence of sheep indicated highest infection at Khall (48%) followed by Lalqilla (44%), Samarbagh (40%), Adenzai (40%), Balambat (36%), Timergara (36%) and lowest in Munda (32%), (Table 1). All Areas showed non-significant difference (c2 =1.818, d.f = 6, P =0.9356). It was noticed that total prevalence in aged sheep was significantly (P< 0.05) higher (46.31%) than younger ones (31.25%) (Table1). Age wise prevalence of sheep showed significant difference (P = 0.0453). Generally sex wise prevalence indicated females (40%) to be more sensitive than males (38%) (Table 1). Statistical difference was not noticed (P = 0.8650).
 
Area, age and gender wise prevalence (%) of toxoplasma gondii in sheep by ELISA
 
All around in seven different areas in District Dir (Lower), prevalence of sheep indicated highest infection at Khall (48%), followed by Samarbagh (44%), Lalqilla (44%), Adenzai (44%), Balambat (40%), Timergara (36%) and lowest in Munda (32%), (Table 1). All Areas showed non-significant difference (c2 =1.888, d.f = 6, P =0.9297). It was noticed that the total prevalence in older sheep was significantly (P< 0.05) higher (48.42%) than younger ones (32.5%) (Table1). Age wise prevalence of sheep showed significant difference (P = 0.044). Generally sex wise prevalence indicated females (42.4%) to be more sensitive than males (38%) (Table 1). Statistical difference was not noticed (P = 0.6146).
 
Comparison of LFCI, LAT and ELISA for the diagnosis of toxoplasma gondii in sheep of district Dir (Lower)
 
Over all prevalence of Toxoplasmosis was shown to be34.85% by LFCI, 39.42% by LAT and 41.14% by ELISA (Table 1). Correctness of different tests can be placed as LFCI < LAT < ELISA. The highest accuracy was shown by ELISA and lowest by LFCI. Statistical analysis was completed by Chi-square (c2). Overall comparison of LFCI, LAT and ELISA showed non-significant difference (c2 =1.561, d.f = 2, P =0.4582) in District Dir (Lower).

Out of total 175 samples of sheep collected from different areas of District Dir (Lower), 61 (34.85%) were detected positive by LFCI, 69 (39.42%) by LAT and 72 (41.14 %) by ELISA, and showed similarity with the results of other study. Sensitivity of LFCI, LAT and ELISA was100%, but specificity of the three tests was different because ELISA had 100% sensitivity followed by LAT (97.16%) and LFCI (90.35%). T.gondii infection in sheep is universal in distribution (Tenter et al., 2000). In current study, prevalence of toxoplasmosis in sheep was 41.14 % which is less than that reported from District Mardan (44%) and is higher than Mohmand agency (36.0%) (Shah et al., 2013) reported previously. The sero positivity rate of toxoplasmosis in sheep in District Dir (Lower) was higher than prevalence reported from Canada (57%) (Waltner-Toews et al., 1991) and Brazil (46%), however lower than that reported in Turkey (31%) and Northeastern China (4%) (Silva et al., 2013, Oncel and Vural 2006, Yang et al., 2013). Similarly present study indicated that the prevalence rate of T. gondii infection varied in different age groups of sheep (can be ranked as 0-1 year of age 32.5% < above 1 year of age 48.42). The highest prevalence rate of T. gondii infection was found in sheep above 1 year of age group (48.42%). Prevalence rate of T. gondii infection in female sheep was higher (42.4%) than male sheep (38%), indicating that female sheep were more susceptible to toxoplasmosis as compared to male sheep.

The present study revealed that toxoplasmosis was prevalent in both the sexes (male and female) and all age groups of sheep in District Dir (Lower), Pakistan. This study demonstrated that the frequency of toxoplasmosis was more in female sheep. The sheep of age more than 1 year were more likely to be seropositive with toxoplasmosis than the younger sheep. This study might be helpful in control of toxoplasmosis in respective area.

The authors are grateful to the Deanship of Scientific Research, King Saud University, Riyadh, Saudi Arabia, for funding the work through the research Group project No. RGP- 210.