MOLECULAR CHARACTERIZATION OF THEILERIAL PIROPLASMS IN CATTLE OF WAYANAD
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Date
2011
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COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR
Abstract
The objectives of the present study were to detect the presence of Theileria
like organisms in blood of cattle of Wayanad by polymerase chain reaction and to
characterize these organisms using molecular tools. A total of thirty peripheral blood
smears and heparinized blood samples were collected from five different localities of
the district. Microscopical examination of the peripheral blood smears revealed
presence of Theileria like piroplasms in 10 samples. Whole blood samples were
processed for crude DNA isolation and subjected for PCRs specific for Theileria
genus, T. annulata and T. orientalis. Theileria genus specific PCR amplifying the
1098 bp small subunit ribosomal RNA (SSU rRNA) gene revealed specific
amplification in 15 samples. T. annulata specific primers amplifying the 721 bp
merozoite surface antigen (MSA) gene did not reveal any product in any of the
samples tested. T. orientalis specific primers amplified 875 bp major piroplasm
surface protein (MPSP) gene product in all those samples which revealed positive
product using Theileria genus specific PCR. Seven PCR products (three SSU rRNA
gene viz., THG 2, 14 and 17 and four MPSP genes viz., THOR 14, 16, 17 and 20)
were cloned into pTZ57R/T and transformed using E. coli JM 107. The transformed
white colonies were selected and sent for sequencing to Sci Genome Labs, Cochin.
The sequences were submitted to GenBank and accession numbers were obtained.
The phylogenetic tree constructed based on SSU rRNA gene sequences demonstrated
that THG 14, 17 and 2 belonged to Type A (Chitose), Type C and new type (closely
related to Sable type) of Theileria sp. respectively. The phylogenetic tree constructed
based on MPSP gene sequences demonstrated that THOR 16 and 20 belonged to type
7 of T. orientalis. This type of T. orientalis is included in the Ikeda, the pathogenic
T. orientalis group. It was also observed that the isolate THOR 14 belonged to Type 1
while the THOR 17 belonged to type 3. Both belonged to Chitose group. A mixed
population of theilerial piroplasms is seen in cattle of Wayanad region. The present
study confirmed the occurrence of T. orientalis organisms in the study area using
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molecular tools. T. orientalis population in this area belonged to Chitose and Ikeda
groups. Even though T. orientalis is considered to be benign, the Ikeda group of
organisms is regarded as pathogenic. Anemia and mortality observed in animals
infected with Theileria can thus be attributed to the presence of T. orientalis Ikeda
type of organisms.
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