The Use Of Molecular And Serological Methods For Toxoplasmosis Investigation In Sheep
Toxoplasma gondii were diagnosed in 80 blood samples of sheep at Al-Diwaniya city in Al-Qadisiyah governorate by using latex test and molecular diagnosis with Polymerase Chain Reaction depending of presence B1 gene of T. gondii, the results showed that antibodies were detected in 13 samples out of 80 (16. 25%). of sheep in Al-diwaniyah city, Since significant(P≤ 0. 05) in highest titration indicated at 1/80 (38. 17%), while the lowest titer indicated at 1/40 (10. 09), the results of Polymerase chain reaction for detection of B1 gene in blood of sheep showed that (7 of 80)(8. 75%) of positive samples from the sheep and (73 of 80) negative samples. It had been concluded that sheep were found infected with T. gondii with possible transmission this disease to human and extends to public health through the consumption of infected meat that facilitates transmission of the disease to humans, the economic importance of livestock in general and sheep in particular and in cooperation with the human environment because of the spread of toxoplasmosis in Al-Diwaniyah city.
Introduction
Toxoplasmosis is a common animal-human disease that is widespread worldwide, the clinical expression of the disease varies from mild to severe. Toxoplasma can cause serious complications of the fetus when primary infection occurs in pregnant females, as well as individuals who have an immune disorder exposed to severe forms of disease. Toxoplasma gondii is an intracellular parasite that has the ability to infect various animals and humans, Toxoplasma gondii is one of the most common parasites in the world, a common cause of infertility, the birth of abnormal embryos in animals and humans. The incidence of small ruminants is not limited to fetal loss but extends to public health through the consumption of infected flesh that facilitates transmission of the disease to humans. The diagnosis of this disease does not depend on the symptoms and depends on the examination of the body's various fluids (blood, tissue, amniotic fluid, etc. ) by serological and molecular methods. Therefore, it is very important to study the common diseases between humans and these animals. Hence the idea of the current study to diagnose the parasite in sheep using serological and molecular methods to give a clear picture of the economic damage caused by the parasite when infected with sheep for the possibility of the occurrence of abortions have a negative impact On the economic side as well as give an idea of the role of sheep infected with the transfer of parasites to humans and the occurrence of disease.
Material and methods
Blood samples from sheep herds were randomly collected in some areas of the governorate and from animals in Al-Diwaniyah city, after the samples were collected; they were brought to the Parasitology Laboratory in biology department/ college of sciences/ University of Al-qadisiyah. Blood samples were divided into two parts, for obtaining serum and EDTA added for PCR and kept at -20c until used, diagnostic procedures divided two way, Latex agglutination test (Toxocell-latax, Spanish, Biokit) and Polymerase Chain Reaction (PCR), this test was conducted and processed by BIONEER depending on primers used by (AL-Khalidy, 2013), Primers Sequence Forward (5-GAACCACCAAAAATCGGAGA-3), and the reverse is (5-GATCCTTTTGCACGCACGGTTGTT-3) with product size (399bp), through three steps: extraction DNA from the samples, ensure the extraction the serum of blood samples and amplification of DNA using a (Primers) specialized the parasite T. gondii and showed the outcome of doubling in the gel Alagaros. DNA was extracted by using AccuPrep ® Genomic DNA Extraction, kit (Bioneer, Korea) according to protocol described by the manufacturer instructions. Data were analyzed by program (10. 5 SPSS version software) and test X2- Square to determine a moral difference under the level of probability (P ≤ 0. 05).
Results and Dissection
The Results showed that antibodies were detected in 13 samples out of 80 (16. 25%). of sheep in Al-Diwaniya city, Since significant(P≤ 0. 05) in highest titration indicated at 1/80 (38. 17%), while the lowest titer indicated at 1/40 (10. 09), in the Sero-prevalence of toxoplasmosis in sheep in Nepal showed that the prevalence of toxoplasma gondii was higher in sheep who more than 2 years of age (82. 35%; CI: 72. 90–89. 00%) while the lower percentage in age of 2 or less than 2 years (17. 65%; CI: 11. 00–27. 10%), this also agree with who found in Mexico by Caballero-Ortega et al. , 2008) who showed that the highest percentage of T. gondii was present at low altitude, and in serology study showed 13/50 samples tested were positive of toxoplasmosis when examined blood samples of the 50 sheep under study, the results of Polymerase Chain Reaction for investigate of B1 gene showed (7 of 80)(8. 75%) of positive samples from the sheep and (73 of 80) negative samples, moreover the B1 gene were had molecular weight of the private 399bp.
T. gondii parasite in (4) samples of the B1 gene appears, the valuable columns are represented in (1-10) as negative samples of the PCR, the column represents the (M) the Ladder with a molecular weight (100-1500bp), this results lower than what recorded by Bezerra et al. , (2014) about the frequency of 26% of positive blood samples of sheep obtained in IFA when study the occurrence of T. gondii DNA in samples collected from uterus, tubes and ovaries in naturally infected sheep slaughtered in Pernambuco city, Brazil, and the results recorded by Silva et al. (2003) 35. 3% positive percentage of seropositive sheep, as well as some studies related to spontaneous abortions in sheep caused by T. gondii, such as (10. 6%) in Germany Steuber et al. (1995) recorded (10. 6%) positive percentage, (11. 1%) with (18. 1%) recorded by Masala et al. in Italy, so (14. 3%) positive percentage in Brazil, the differences of ratios recorded in the current study and with other studies due to the difference in the number of samples examined and the uncontrolled laboratory conditions that affect the polymerase chain reaction.
