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Kerala Veterinary and Animal Sciences University, Wayanad

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Subject: Veterinary and Animal Sciences × Author: Jeena, K D × End date: 2016 × Start date: 2015 ×
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    ThesisItemOpen Access
    DETECTION OF RICKETTSIAL PATHOGENS OF TICKS OF WILD MAMMALS AND REPTILES
    (Kerala Veterinary and animal science university, Pookode, 2016-05-12) Jeena, K D; Reghu Ravindran
    The present study detected the rickettsial pathogens of ticks of wild mammals and reptiles from five different regions of Wayanad district, Kerala (Vaduvanchal, Vythiri, Noolpuzha, Muthanga and Meppadi). A total of 62 ticks collected from four different wild mammals and one reptile species. The collected species were included Amblyomma integrum from (wild boar, elephant calf and barking deer), Haemaphysalis bispinosa, Rhipicephalus haemaphysaloides, Haemaphysalis spinigera and Amblyomma integrum from (sambar deer) and Aponoma gerviasi from (python).The RNA was isolated from RNA was isolated from individual adult engorged female ticks, by using RNAeasy minikit (Quiagen, Germany). cDNA was synthesized from the isolated RNA samples by using Revertaid H Minus cDNA synthesis kit (fermentas, USA) based on manufacturer`s protocol. The cDNA synthesized subjected to PCR amplification using specific primers. The primers used for the PCR amplification were included, mitochondrial 16S r DNA gene of tick species which amplify a 450 bp product, a pair of oligonucleotide primer sequences which are localized within the coding V4 region in a 18S gene, Rickettsia specific citrate synthase gene (gltA) using CS2d and CSEndr primers which amplify a product of 1234 bp, and Rickettsia specific outer membrane protein A by using ompA F and R primers which amplify a product of 632 bp. The Agarose gel electrophoresis was performed on a 2.0 per cent Agarose (Biogene, USA) gel prepared in 0.5X Tris Borate EDTA buffer (Tris 2.7g, Boric Acid 1.37g, EDTA-0.5M 1ml, Aqua dist. Ad 500ml) using 80V power supply for 90 minutes. The samples were send to sequence analysis and obtained were first BLAST analyzed (www.ncbi.nlm.nih.gov/ BLAST) to confer their identity. Only Amblyomma and Rhipicephalus spp. of ticks revealed rickettsial organisms. The Rickettsia species identified in these ticks were Rickettsia africae (Amblyomma integrum), Rickettsia parkeri (Amblyomma integrum) and Rickettsia rhipicephali (Rhipicephalus sp).