MOLECULAR CONFIRMATION OF TRANSOVARIAL TRANSMISSION OF PATHOGENS THROUGH TICKS INFESTING DOMESTIC CATTLE OF KERALA
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Date
2022-04-01
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COLLEGE OF VETERINARY AND ANIMAL SCIENCES POOKODE, LAKKIDI
Abstract
The tick-borne diseases cause heavy economic losses for the livestock
industry throughout the world. The transovarial transmission is one of the most
distinctive characteristics of many tick-borne pathogens. There are only very few
documented reports on the transovarial transmission of the pathogens through ticks
in India. The present study was carried out with the objectives of the detection of
parasitic pathogens in ticks, their egg masses and larvae by polymerase chain
reaction (PCR) and identification of the tick species involved in the transovarial
transmission of the pathogens in Kerala. In the present study, the molecular
technologies such as polymerase chain reaction (PCR), sequencing and
phylogenetic analysis were carried out. The male and female ticks were collected
from northern, central and southern zones of Kerala during the study. They were
morphologically identified as Rhipicephalus annulatus, R. microplus and
Haemaphysalis bispinosa. A total of 140 engorged female ticks (R. annulatus-70,
R. microplus- 40, H. bispinosa- 30) were kept for oviposition in a Biological
Oxygen Demand (BOD) incubator at 28 °C and 85 % relative humidity. Only 90
ticks laid ova and they were further used for the study. On the 15th day of
oviposition, both the female ticks and half of the eggs laid by them were stored at -
80 °C. The other half of the eggs laid by them were kept for hatching. The larvae
hatched out were also transferred to -80 °C. The genomic DNA was extracted from
the female ticks, their egg masses and larvae and utilized as the template for the
polymerase chain reactions. The PCR amplifying a ~460 bp V4 region of 18S rRNA
gene-specific for Babesia spp. and Theileria spp. revealed amplification in R.
annulatus ticks (30), egg masses (24) and larvae (24) respectively. Using them,
~758 bp rhoptry associated protein-1a (rap-1a) gene of B. bigemina was amplified
in R. annulatus ticks (19), their egg masses and larvae. The current study thus,
provided the molecular evidence for transovarial transmission of B. bigemina in R.
annulatus. Interestingly, the PCR amplification of the TRAP gene (~855 bp)
specific for B. gibsoni was also detected in R. annulatus ticks (5) engorged on cattle,
their egg masses and larvae. The ~776 bp major piroplasm surface protein (MPSP) gene specific for Theileria orientalis was amplified in engorged R. annulatus ticks
(6), however, none of their egg masses and larvae showed amplification. Similarly,
the polymerase chain reactions specific for the amplification of the apical
membrane antigen-1 (AMA-1) and β-tubulin gene of B. ovata and major merozoite
surface protein of T. annulata failed to amplify the respective products in any of the
samples. An ~854 bp product specific for major surface protein 4 (msp4) gene of
A. marginale could be amplified in R. annulatus ticks (7), their egg masses and
larvae. In addition, a ~641 bp product specific for the 16S rRNA gene of A.
phagocytophilum was amplified in H. bispinosa (8) and R. annulatus (6) ticks, their
egg masses and larvae. However, the PCR specific for the 16S rRNA gene of A.
bovis did not amplify the ~551 bp product in any of the ticks, their egg masses and
larvae. The Rickettsia spp. was detected from H. bispinosa (6) and R. annulatus (4)
ticks, their egg masses and larvae by the nested PCR amplification of ~381 bp
fragment specific for the citrate synthase (gltA) gene. A ~549 bp fragment specific
for the 17-kDa common antigen gene (htrA) of Rickettsia spp. was amplified only
from H. bispinosa ticks (6), their egg masses and larvae. The Rickettsia spp.
detected in this study was identified as closely related to the R. raoultii when the
PCR product was sequenced for phylogenetic analysis. The species of all the ticks
positive for haemoparasites were further confirmed by PCR amplification of
mitochondrial 16S rRNA, 18S rRNA and mitochondrial cytochrome oxidase 1
(COI) gene specific for ticks. The present investigation thus confirmed, the
transovarial transmission of B. bigemina, B. gibsoni, A. marginale, A.
phagocytophilum and a Rickettsia spp. closely related to R. raoultii through R.
annulatus and A. phagocytophilum and the Rickettsia spp. closely related to R.
raoultii through H. bispinosa ticks. In addition, the mixed infections with B.
bigemina and A. phagocytophilum, B. bigemina and A. marginale, B. gibsoni and
A. marginale were detected in R. annulatus ticks.
Description
Submitted in partial fulfilment of the requirement for the degree of
Master of Veterinary Science in Veterinary Parasitology