In the current research project, a pari-trypanosome polymerase chain reaction (PCR) was optimized by using 18S single sub unit ribosomal DNA amplification and restriction fragment length polymorphism (RFLP) was also optimized and evaluated for the species specific diagnosis of the trypanosomiasis in horses.
Blood samples from one hundred (100) suspected horses were collected aseptically from different localities of Lahore. Fresh blood smear was prepared from each sample. After drying and fixing with absolute methanol, the slides were stained with Giemsa stain. Microscopic examination of stained blood smears revealed 8 positive samples out of one hundred (100) suspected horses. Polymerase chain reaction (PCR) was carried out on the same trypanosomiasis suspected blood samples to evaluate its sensitivity. Genomic DNA was extracted by using Genomic DNA Purification Kit (Fermentas mci., USA). The PCR was performed in a 50 tl reaction mixture. The tubes containing PCR mix were subjected to amplification cycles in a thermocycier after adjusting the amplification conditions.
After completion of the amplification cycles, the PCR product was characterized by 1.2 % agarose gel electrophoresis along with 100 bp DNA ladder to estimate the size of the PCR product and the gel was photographed with a Polaroid camera. PCR gave a higher percentage of positive cases i.e. 21% as compared to microscopic examination. Semi-nested polymerase chain reaction was carried out on product of the first run amplification by using same reaction mixture and amplification conditions except for template DNA. In case of semi-nested PCR 1 tl of the simple PCR product was used. Semi-nested PCR gave 100% (21/21) results. Restriction fragment length polymorphism (RFLP) analysis was conducted on nested products of the positive samples. A reaction mixture of 20 1iJ was used and samples were incubated over night at 37 °C in an incubator. The restricted products were characterized by 2 % agarose gel electrophoresis along with 100 bp DNA ladder and photographed with Polaroid camera. Restriction fragment length polymorphism (RFLP) analysis of the nested products revealed that none of the species including T. congolense, T. theileri, T. brucei and T. vivax was found in all (2 1%) positive animals having trypanosoma infestation.
It can be concluded from current study that a pan-trypanosome polymerase chain reaction is a superior and sensitive test as compared to Giemsa stained blood smear examination. The test can not only be used for early diagnosis of the trypanosomiasis but it can also be used to screen out the carrier animals those act as a reservoir of the infection for the horses and other susceptible animals. The advantage of this test is its sensitivity, universal applicability and the existence various possibilities for restriction enzyme analysis of the amplified region depending on the trypanosome species.