Development of Slide Enzyme Linked Immunosorbent Assay (SELISA) for Detection of Trypanosoma evansi Infection in Bovines

The slide enzyme linked immunosorbent assay (SELISA) was standardized for detection of antibodies specific to Trypanosoma evansi and subsequently used for the screening of naturally infected bovine sera. A novel SELISA, a modification of the standard ELISA technique was used for the detection of antibodies against Trypanosoma evansi in bovines using positive and negative control sera. The test is based on immunostaining of the fixed whole Trypanosoma evansi organisms on microscopic glass slide, incubation with sera, antibovine IgG-HRPO conjugate and substrate Diaminobenzidine tetrahydrochloride (DAB). Finally the reaction was read under oil immersion of microscope. A total of 702 sera samples from bovines in Rayalaseema region of Andhra Pradesh were examined by SELISA and 192 were found positive for Trypanosoma evansi antibodies

Kloniranje, ekspresija i karakterizacija paraflagelarnog gena Rod 2 bičaša Trypanosoma evansi

Paraflagellar rod is the major structural component of the trypanosomatid flagellum and is identified as a complex lattice of filaments which runs parallel to the axoneme throughout most of the flagellar length. The present study was carried out to investigate the existence of the paraflagellar rod (PFR 2) gene in Trypanosoma evansi infecting Indian cattle. Local isolates of T. evansi collected from naturally infected cow were multiplied in Wistar rats. Complementary DNA (cDNA) was synthesized from the RNA of host cell free T. evansi parasites by reverse transcription. The gel purified PCR product (PFR 2 gene of T. evansi) was cloned into the pTZ57R/T vector system. The nucleotide sequence of the PFR 2 gene of the T. evansi S.V.V.U. isolate (Accession No. KT277497) obtained in the present study revealed 100% homology with T. evansi China isolate and 99% homology with T. evansi Izatnagar and Bikaner isolates. The recombinant protein was sub-cloned into pET 32a and expressed in the BL21 (DE3) pLysS expression system. The PFR 2 gene of T. evansi S.V.V.U. isolate was further characterized by determination of its protein profile with SDS-PAGE and western blotting. Indirect ELISA was optimized for detection of the specific antibody titre against the recombinant protein of the PFR 2 gene of T. evansi. In the kinetoplastid species the PFR 2 gene is highly conserved. Therefore the PFR 2 gene was suggested as a vaccine candidate, as well as a diagnostic antigen.Paraflagelarni štapić glavna je strukturna komponenta tripanosomskog biča i dio je kompleksa filamenaza koji teku paralelno s aksonemom duž biča. Istraživanje je provedeno kako bi se ispitalo postojanje paraflagelarnog gena Rod 2 (PFR2) u bičaša Trypanosoma evansi koji invadira goveda u Indiji. Lokalni izolat T. evansi prikupljen od prirodno invadiranih krava umnožen je u Wistar štakora. Komplementarna DNA (cDNA) sintetizirana je iz RNA obrnutom transkripcijom iz stanica neinvadiranih nositelja T. evansi parazita. Pročišćeni PCR produkt (gen PFR2 bičaša T. evansi) kloniran je u vektorski sustav pTZ57R/T. Nukleotidna sekvencija gena PFR2 bičaša T. evansi, izolat S.V.V.U. (pristupni broj KT277497) dobivena u ovom istraživanju pokazala je 100 %-tnu sličnost s izolatom T. evansi China i 99 %-tnu s izolatom T. evansi Izatnagar i Bikaner. Rekombinantni protein ponovno je kloniran u sustavu pET 32a i prikazan u sustavu BL21 (DE3) pLysS. Gen PFR2 bičaša T. evansi, izolat S.V.V.U. dalje je karakteriziran određivanjem proteinskog profila metodama SDS-PAGE i Western blotting. Indirektni test ELISA optimiziran je za dokaz titra specifičnih protutijela za rekombinantni protein gena PFR2 bičaša T. evansi. U kinetoplastida gen PFR2 izrazito je očuvan. Stoga bi se gen PFR2 mogao upotrijebiti za cjepivo te kao dijagnostički antigen

Trypanosoma evansi causes thyroxin imbalance with biochemical alterations in wistar rats

Animals affected with Trypanosoma evansi show rare serum hormonal disturbances. One of the important hormones for livestock is thyroxin, and the level of thyroxin may be reduced during the T. evansi infection. The objective of the study was to investigate thyroxin level during experimentally induced T. evansi infection in Wister albino rats. Wistar albino white rats (n=12) were challenged with the local strain of T. evansi (at 5x105 trypanosomes/animal subcutaneously). At the high parasitemia, blood was collected from the rats, and serum was separated, which was subjected for biochemical evaluation. Decreased total serum thyroxin (2.91 ±0.04 µg/dL) and free thyroxin (1.30 ±0.05 ng/dL) levels (p<0.01) were recorded in T. evansi infected rats as compared to the control group of rats. Along with lowered thyroxin levels, decreased levels of total erythrocyte count, packed cell volume, hemoglobin, total leucocyte count, total serum proteins, albumin and glucose levels were recorded. On the other hand, significant increase (p<0.01) in cholesterol, blood urea nitrogen, serum alkaline phosphatase, serum aspartate aminotransferase, and serum alanine aminotransferase levels were observed. Thus, it is concluded that trypanosomiasis induces stress on rat, which have direct effect on thyroid hormone