Isolation And Genotyping Of Local Strains Of Toxoplasma Gondii From Free Range Chickens And Goats
Loading...
Date
2014-07
Authors
Ibrahim, Norrulhuda
Journal Title
Journal ISSN
Volume Title
Publisher
Universiti Sains Malaysia
Abstract
Toxoplasma gondii is a protozoan parasite which is distributed worldwide. It is the causative agent of toxoplasmosis in humans, domesticated and wild animals. Until today, not much is known of the T. gondii genetic characteristics from domesticated animals in Malaysia. In this study, PCR-RFLP method was used for identification of local strains of T. gondii isolated from free range chickens and goats using eight RFLP markers i.e; 3’SAG2, 5’SAG2, SAG3, BTUB, cB21-4, GRA1, GRA6 and SRS1. All the markers were optimized for annealing temperatures (Ta) and magnesium chloride (MgCl2) concentrations. A modified method was used to separate contaminant-free viable T. gondii cysts from brains and hearts of free range chickens and goats. The isolates from both animals were inoculated and propagated in mice. Then, the identification of T. gondii strain was performed by isolating the T. gondii from infected mice organs. The optimized PCR conditions were found to be different than previously reported. The optimal condition for each markers were; 3'SAG2 (63.0°C and 1.5 mM MgCl2), 5'SAG2 (59.9°C and 2.0 mM MgCl2), SAG3 (58.7°C and 2.0 mM MgCl2), BTUB (58.7°C and 1.5 mM MgCl2), cB21-4 (53.7°C and 1.5 mM MgCl2), GRA1 (61.1°C and 1.5 mM MgCl2), GRA6 (57.8°C and 1.5 mM MgCl2) and SRS1 (64.2°C and 2.5 mM MgCl2). The half-brains from the mice which were fed 235 mg/kg prednisole showed presence of an average of 10 cysts under microscopic examination, compared to an average of two cysts in the mice used as a control. This indicates that prednisolone has caused the immune system of the mouse to be suppressed, which subsequently increased its susceptibility to the T. gondii infection. The best condition for the isolation of tissue cysts using dextran modified method was 21% dextran solution and two-step filtration (100-160 μm and < 10 μm). The isolated cysts in the filtrate were found to be contaminant-free, viable and able to infect healthy mice. Blood, brains and hearts from 213 free range chickens were obtained from three different states in Peninsular Malaysia and 31 (14.6%) serum were found to be seropositive when assayed with MAT (MAT ≥ 1:6). All the positive samples were inoculated into mice and 12 samples had MAT ≥1:24 (cutt off titer) after 21 days post- infection (p.i.). These 12 samples were subjected to PCR-RFLP analysis and two different T. gondii strains were identified namely type I (41.7%) and non-type III (58.3%). A total of 225 blood samples from goats were obtained from two states and 30 (13.3%) were found to be seropositive when assayed with MAT (MAT ≥ 1:6). The brains and hearts of positive samples were inoculated into mice and T. gondii was isolated from only 10 samples out of 30 samples that had MAT titer of ≥1:24. The isolates were than subjected to PCR-RFLP analysis and were identified as T. gondii type I (10%) and non-type III (90%).
In summary, there were 14.6% and 13.3% seropositivity in free range chickens and goats respectively. Out of the above positive samples, only 38.7% and 33.3% of free range chickens and goats samples, respectively, showed positivity for RFLP analysis. It can be concluded that among the animal population studied, non-type III T. gondii was the more dominant genotype compared to type I and there was absence of type II genotype. Therefore, there is substantial environmental contamination of T. gondii oocysts in the study areas of this project.
Description
Keywords
Toxoplasma gondii