Abstract: Comparative diagnosis of Toxoplasma gondii infection in sheep were carried out with PCR and some serological assays using 200 blood and serum samples collected from sheep of different ages and sexes slaughtered in the main abattoir in Cairo and Giza, Egypt. PCR showed the higher prevalence of toxoplasmosis (48.5%) followed by the Modified Agglutination Test (MAT) (45.5%) and the Latex Agglutination Test (LAT) (41.0%), while the lowest prevalence was detected with the Indirect Hem-Agglutination Test (IHAT) (38.5%). When the data of the serological tests were compared with that of the PCR, as a reference test for toxoplasmosis, MAT had the highest sensitivity (95.9%) followed by LAT (90.7%) and the lowest sensitivity by IHAT (80.4%). On the other hand IHAT had the highest specificity (91.3%) followed by MAT (88.3%) and the lowest specificity was by LAT (84.5%). The present study adopt PCR and serological survey of T. gondii antibodies in sheep by using more sensitive and specific antigens prepared from isolated T. gondii local strain and the obtained results suggests that MAT alone or with LAT can be used as a highly sensitive screening test followed by PCR as a specific confirmatory test for diagnosis of toxoplasmosis in sheep. Consequently, the high prevalence of sheep toxoplasmosis detected by this study scopes the public health significance of sheep’s meat as source of human infection.
INTRODUCTION
Toxoplasmosis is one of the most important infectious and zoonotic diseases worldwide, caused by an obligatory intracellular protozoan parasite; Toxoplasma gondii which can infect humans and almost all warm-blooded animals (Vaz et al., 2010). The parasite distributed on regions and weather condition of the environment where oocysts survive and is responsible for major economic losses in humans and most classes of livestock including sheep through abortions, still birth and neonatal losses. It causes mental retardation and loss of vision in children and abortion in pregnant women (Raeghi et al., 2011).
Cats are main reservoir of T. gondii because they are the only hosts that can excrete the resistant stage (oocyst) of the parasite in the feces. Humans become infected by eating undercooked meat from infected animals and food and water contaminated with oocysts (Sreekumar et al., 2005). Besides vertical infection during pregnancy, humans and animals can get infected either by oral uptake of sporulated Toxoplasma oocysts or by ingestion of tissue cysts upon consumption of raw or undercooked meat of infected slaughtered animals (Zhou et al., 2012).
Among livestock, toxoplasmosis causes great losses in sheep. Toxoplasma gondii may cause embryonic death and resorption, fetal death and mummification, abortion, stillbirth and neonatal death in these animals (Dubey, 2009). Most sheep acquire the infection post-natally by ingestion of oocysts from contaminated environment or ingestion of infected tissues of animals and the disease which cause serious economic losses among sheep industry all over the world especially at lambing time (Shaapan, 2015). Also, sheep are important to the economy of many countries because they are a source of food for humans; the infected sheep meat is a source of T. gondii infection for humans and animals (Ragozo et al., 2008).
Many studies investigate the T. gondii incidence in sheep from different regions in Egypt which were 45% using Sabin Feldman tests (Michael and El-Refaii, 1977), 18.30 and 14.8% by using SFT and IHAT, respectively (El-Menyawy, 1987), 47 and 50% for ELISA and IFAT, respectively (El-Ghaysh and Mansour, 1994), 49.5 and 52% in slaughtered sheep in Tanta abattoir using IHAT and IFAT, respectively (Ibrahim et al., 1997), 55.9 and 54.1% with IHAT and IFAT (Aal and Barakat, 2000) and 37% by ELISA by mean of patent Ag-coated plates from specific kit (Kandil and Abou-Zeina, 2000) and recently, comparative serological examination revealed that, MAT showed the higher prevalence of toxoplasmosis (43.7%) followed by ELISA (41.7%), using antigen prepared from isolated T. gondii local strain (Shaapan et al., 2008).
Molecular biology methods have been increasingly used in diagnosis of ovine toxoplasmosis. Polymerase Chain Reaction (PCR) amplification has become the preferred method. Most PCR assays used for Toxoplasma identification use primers targeting the B1 gene. It is a 35-fold-repetitive gene that is highly specific and conserved among strains of Toxoplasma (Burg et al., 1989), the P30 surface antigen (single copy) the ribosomal RNA (110 copies), both the small subunit rRNA gene (Tenter et al., 1994) and the 529 bp DNA fragment (Homan et al., 2000). Moreover, PCR had been shown to be useful and reliable confirmative technique for diagnosis of T. gondii infection in ovine and caprine placenta and aborted fetuses (Hurtado et al., 2001; Masala et al., 2003).
The present work was aiming to studying the prevalence of T. gondii infection among sheep slaughtered in Cairo and Giza municipality abattoirs using IHAT, LAT and MAT serological tests and followed by the confirmatory PCR screening assay and also the sensitivity, specificity, advantages and disadvantages of each test were be determined.
MATERIALS AND METHODS
Blood and serum samples: Blood samples were collected from 200 apparently healthy sheep slaughtered at the main abattoir of Cairo (El-Bassatin), Giza (El-Moneeb) and sera were obtained, labeled in serial number and stored at -20°C until used for serological and PCR testing.
Toxoplasma gondii strain: Virulent RH strain of T. gondii used for PCR and local T. gondii local strain tachyzoites used for antigens preparation for serological assays were obtained from colony kept in Department of Zoonoses, National Research Center. Both strains maintained by serial passage in mice according to the procedures described by Shaapan and Ghazy (2007).
Polymerase Chain Reaction (PCR)
DNA extraction: Genomic DNA was extracted from T. gondii RH strain (positive control) using a commercially available kit (Dneasy blood and Tissue kit, Qiagen Co., Cat. No. 69504). DNA was purified through the columns according to the manufacturer’s protocol and eluted in 50 μL of the supplied AE buffer and then stored at -20°C. Concerning DNA extraction from blood samples, the samples were collected in test tubes containing heparin as an anticoagulant then digested using the same kit using the blood protocol recommended by the manufacturer prior to assay by PCR.
PCR amplification: Primers used for PCR were targeting the repetitive 35-fold B1 gene (Burg et al., 1989). The outer primers were only used in this study. The reactions were set up to a final volume of 25 μL containing 2 μL of DNA sample (standard controls) or 5 μL of blood specimen DNA, 1 μL of each primer (100 pmol) and 12.5 μL Pyro-Start™ Fast PCR Master Mix (Fermentas Co., Cat. No. Ko211). The PCR was performed in a PTC-100 Peltier Thermal Cycler (MJ Research, Incline Village, USA). The first cycle included 1min of denaturation at 95°C. This first step was followed by 39 cycles of 2 sec of denaturation at 94°C, 5 sec of annealing at 48°C and 25 sec of primer extension at 72°C and a final extension step of 72°C for 10 min. A 10 μL aliquot of the amplified product was analyzed on 1% agarose gel and stained with Ethidium bromide. Every PCR run included positive and negative controls.
Serological assays
Indirect Hem-Agglutination Test (IHAT): The indirect hem-agglutination test was adopted using soluble tachyzoites antigen coated in tanned red blood cells and the procedures were carried out according the serology manual prepared by Palmer et al. (1976).
Latex Agglutination Test (LAT): The antigen prepared through sensitization of latex particles with T. gondii sonicated tachyzoites as procedures of Lunde and Jacobs (1967). The latex agglutination test was adopted as methods of Holliman et al. (1989), using negative and positive reference serum samples and reactions were performed by adding a drop of latex solution into a drop of serum on a microscope glass slide and mixed by side movement of slide. The agglutination appears within 2-3 min in positive cases.
Modified Agglutination Test (MAT): The formalized killed whole tachyzoites of T. gondii was prepared according to the method described by Desmonts and Remington (1980). The procedures were carried out according to the method described by Dubey and Desmonts (1987) at a dilution of 1:25.
Data analysis: The data of the various serological tests were analyzed. The sensitivity and specificity of each test were determined by comparing its results with that of PCR as a reference test according to the method described by Waltman et al. (1984) and Dubey et al. (1995).
RESULTS
Prevalence of T. gondii infection in sheep using PCR and serologic assays: Examination of the 200 serum samples of sheep by PCR, MAT, LAT and IHAT revealed that 97 (48.5%), 91 (45.5%), 82(41.0%) and 77 (38.5%) were positive reactors with T. gondii respectively which at the same time was considered the percentage of infection (Table 1 and Fig. 1).
Comparison between the PCR and serological tests: The sensitivity and specificity of LAT, MAT and IHAT serological tests were calculated by comparing their results with those of PCR test and reveled that MAT had the highest sensitivity (95.9%) followed by LAT (90.7%) and the lowest sensitivity by IHAT (80.4%). On the other hand IHAT had the highest specificity (91.3%) followed by MAT (88.3%) and the lowest specificity was by LAT (84.5%) (Table 2).
| Fig. 1: | Detection of T. gondii in sheep blood samples by PCR, M: Mol. wt. marker (100 bp ladder), C: Positive control, S1-S4: Positive PCR T. gondii samples at 193 bp |
| Table 1: | Prevalence of T. gondii antibodies among sheep sera using PCR and different serological tests* |
*No. of samples examined by each test were 200, PCR: Polymerase chain reaction, MAT: Modified agglutination text, LAT: Latex agglutination text, IHAT: Indirect hem-agglutination | |
| Table 2: | Comparison between the PCR and serological tests used for detection of T. gondii infection |
PCR: Polymerase chain reaction, MAT: Modified agglutination text, LAT: Latex agglutination text, IHAT: Indirect hem-agglutination | |
DISCUSSION
A few studies analyzed risk factors associated with T. gondii seropositivity in sheep, the presence of cats on the farm, using surface water for drinking water, altitude and farm size were factors associated with infection rates; prevalence was higher at low altitudes and on large farms (Vesco et al., 2007). Diagnosis of toxoplasmosis by demonstration of T. gondii in tissue is too much difficult and also the disease is manifested by mild clinical symptoms and develops an in-apparent infection. Therefore Malik et al. (1990) pointed out that the detection of antibody response by screening of slaughtered sheep sera serologically appears to be the conclusive tool for proper diagnosis of toxoplasmosis.
The present reported prevalence rate of T gondii infection in our study using PCR, MAT, LAT and IHAT were 48.5, 45.5, 41.0 and 38.5%, respectively. Higher incidence of toxoplasmosis (64 and 62.5%) were recorded by Dubey et al. (1986) and Malik et al. (1990) in adult sheep in USA, respectively; while lower incidence of toxoplasmosis (28.5 and 3.6%) were detected by Savio and Nieto (1995) and Dubey and Foreyt (2000) in adult sheep, respectively.
The difference between the obtained results of PCR and serological tests during the present study and those reported by other investigators might be attributed to the host-parasite relationship which depends upon the virulence of T. gondii strain; the immune status of the different infected sheep, the age and management of sheep in different localities and the time of exposure to infection and biology of the parasite. This is agreed with that concluded by Shaapan et al. (2008) who added that the prevalence of infection may vary strongly in some country from one locality to another due to difference in certain ecological factors and breeding system in these areas.
Generally the higher infection rate among sheep with T. gondii found in this study may be attributed to the feeding habit of sheep which usually graze short grasses and lick soil around them thus, are liable to contract the infection with T. gondii oocysts. Moreover, stray cats may easily enter to the environment of sheep. This agreed with that obtained by El-Menyawy (1987), Malik et al. (1990) and Lunden et al. (1993).
It is highly significant to determine the specificity and sensitivity of serological diagnosis of T. gondii infection in sheep. Highest sensitivity (95.9%) by MAT, highest specificity by IHAT (91.3%) and lowest specificity (84.5%) by LAT were obtained in this study when the data of the serological tests were compared with that of the PCR, as a reference test for toxoplasmosis. Also Dubey, (2010) concluded that detection of Toxoplasma DNA by PCR was considered the most specific and standard test by which all other tests should be judged. LAT revealed lowest specificity, is frequently produce false positive LAT titers and so technical considerations involving accuracy of reading weakly positive reactions could account for some of the differences (Holliman et al., 1989). On other hand, IHAT demonstrates great specificity, is quantitative, low cost and may be easily adopted but requires further refinement with regard to procedures and standardization of the antigen used (Dubey et al., 1995). Regarding MAT, Dubey (1997) and Shaapan et al. (2008) found that MAT has the highest sensitivity among all serological tests for detection of Toxoplasma gondii infection in naturally infected sows and sheep, respectively which is easy to perform and does not require sophisticated equipment.
CONCLUSION
The present report revealed high prevalence of this zoonotic parasite infection with T. gondii in Egyptian sheep which suggests that the risk of humans contracting infection through consumption of sheep meat. In addition this study suggested that MAT alone or with LAT can be used as a highly sensitive screening test followed by PCR as a specific confirmatory test for diagnosis of toxoplasmosis in sheep.