MOLECULAR CHARACTERISATION OF RHIPICEPHALUS (BOOPHILUS) ANNULATUS AND R. (B.) MICROPLUS USING MITOCHONDRIAL CYTOCHROME C OXIDASE SUBUNIT 1 (COI) GENE AND SECOND INTERNAL TRANSCRIBED SPACER (ITS2)
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Date
2018
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COLLEGE OF VETERINARY AND ANIMAL SCIENCES, POOKODE WAYANAD
Abstract
The present study was carried out to differentiate closely related
Rhipicephalus (Boophilus) species like R. (B.) microplus and R. (B.) annulatus and to
characterise these species using molecular tools. Molecular tools like polymerase
chain reaction (PCR), sequencing, phylogenetic analysis and divergence analysis
were adopted. When a total of 279 cattle were screened for presence of infestation
with hard ticks from different geographical locations of Kerala and Karnataka, 213
animals were identified as infested. In Kerala, R. (B.) annulatus was the predominant
species (54.26 per cent) followed by H. bispinosa (25.58 per cent), R. (B.) microplus
(12.40 per cent) and R. haemaphysaloides (5.42 per cent). R. (B.) microplus was the
predominant species (77.10 per cent) in Karnataka followed by H. bispinosa and R.
haemaphysaloides with 18.07 per cent and 4.81 per cent prevalence rates
respectively. Morphologically identified R. (B.) microplus (13 numbers) and R. (B.)
annulatus (14 numbers) were used for the molecular characterization using
cytochrome c oxidase subunit 1 (COI) and internal transcribed spacer (ITS2)
molecular markers. Some of the R. (B.) microplus male ticks showed presence of a
spur in the ventral surface of palpal article I similar to that seen in R. (B.) australis
males. Dorsal setae were short and medial alloscutal setae were arranged in 2-3 rows
in R. (B.) microplus female ticks. All the PCR products were sequenced and BLAST
analysis was performed for confirmation of the species. The phylogenetic analysis of
R. (B.) microplus isolates from both Kerala and Karnataka using COI classified them
into R. (B.) microplus clade C, comprising R. (B.) microplus isolates from Haryana,
India and other neighbour countries like Pakistan, Myanmar and Bangladesh. R. (B.)
annulatus isolates from Kerala clustered with other R. (B.) annulatus isolates from
Chennai, India, Iraq, Israel and Romania. The phylogenetic tree based on ITS2 failed
to distinguish closely related R. (B.) microplus complex as R. (B.) microplus and R.
(B.) annulatus isolates were clustered together. Divergence analysis was done further
to differentiate R. (B.) microplus and R. (B.) annulatus. The COI gene showed greater
value (7.9 per cent) for interspecific divergence compared to ITS2 (3.6 per cent).
Thus COI was identified as a better marker in resolving interspecific divergence in
comparison to ITS2. The intraspecific divergence for R. (B.) microplus was higher
compared to R. (B.) annulatus. When R. (B.) microplus isolates of South India were
compared with isolates of R. (B.) microplus clade A, B and C, higher divergence was
observed with clade A (11.9 per cent) followed by clade B (7.4 per cent). Least
divergence was observed against R. (B.) microplus clade C (2.3 per cent). Thus, the
results of the present study revealed that Indian isolates of R. (B.) microplus clade C
sensu lato was phylogenetically distant from true R. (B.) microplus clade A sensu
stricto.
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