MOLECULAR DETECTION OF HAEMOPROTOZOAN AND HAEMORICKETTSIAL ORGANISMS IN COMMUNITY OWNED DOGS OF KERALA
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Date
2022-02-11
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COLLEGE OF VETERINARY AND ANIMAL SCIENCES POOKODE
Abstract
India's diverse climatic zones are ideal for a wide range of vectors and
pathogens. Most of the community/stray dogs suffers from a variety of diseases due to
malnutrition and lack of basic health care. Stray dogs are thought to be suitable
reservoirs of several zoonotic parasites. The objective of the present study was to detect
haemoprotozoans and haemorickettsial organisms from community owned dogs of
Kerala. A total of (n=150) peripheral blood smears and whole blood samples were
collected from community owned dogs from different zones of Kerala. The peripheral
blood smear examination revealed prevalence of 21.33 per cent and 8 per cent for
Babesia gibsoni and B. vogeli infection by microscopy. None of the blood smears were
positive for any other haemoparasites. The genomic DNA was extracted from all the
blood samples and used as a template for detection of haemoprotozoan and
haemeorickettsial organisms. The B. gibsoni genus specific primer targeting 18S rRNA
showed an amplicon size of ~1655 bp length in 61 samples (40.67 per cent) by primary
PCR. The PCR product of primary PCR was utilized as a template for nested PCR using
a set of internal primers and amplified a ~308 bp fragment of 18S rRNA gene in 87
samples (58 per cent). The polymerase chain reaction targeting B. gibsoni TRAP gene
showed an amplification at ~855 bp size in 90 samples (60 per cent). The PCR
amplification of B. vogeli and Hepatozoon canis targeting 18S rRNA gene showed an
amplicon size of ~590 bp length in 59 samples (39.33 per cent) and 666 bp length in 42
samples (28 per cent) respectively. Trypanosoma evansi was positive in 41 samples
(27.33 per cent) using T. evansi specific RoTat 1.2 gene giving out a product size of
~205 bp. Only a single sample showed PCR amplification for Ehrlichia canis VirB9
and gp-200 genes with amplicon sizes of ~380 bp and ~1286 bp respectively.
Amplification at ~678 bp length by PCR targeting 16S rRNA gene of Aanaplasma
platys was obtained only in one sample. Among canine vector borne diseases in community owned dogs of Kerala, B. gibsoni was highly prevalent followed by B.
vogeli, H. canis, T. evansi, E. canis and A. platys. Mixed infection of B. gibsoni with
other haemoprotozoans and haemorickettsiales was recorded from all zones of
Kerala with a high co-infection of B. gibsoni and B. vogeli (32.67 per cent) followed
by B. gibsoni and H. canis (18 per cent). Selected amplicons of B. gibsoni, B. vogeli,
H. canis, T. evansi, E. canis and A. platys were sequenced. The phylogenetic
analysis based on 18S rRNA gene of B. gibsoni and H. canis, 16S rRNA gene of A.
platys and VirB9 and gp-200 gene of E. canis does not showed any genetic diversity.
However, the phylogenetic analysis of TRAP gene of B. gibsoni sequences indicated
that B. gibsoni field isolates of Kerala in the present study formed a single clade
with other Indian and Bangladesh isolates and separated away from East Asian
countries. Thus, the isolates of B. gibsoni from Indian subcontinent are genetically
unique compared to other Asian isolates showing a maximum genetic diversity. The
phylogenetic analysis based on 18S rRNA gene of B. vogeli indicated that the B.
vogeli isolates circulating in pet and community owned dogs of Kerala and other
parts of India are genetically different from other Babesia spp. isolates from other
countries, indicating two lineages of B. vogeli in the world. The phylogenetic
analysis of T. evansi using RoTat 1.2 gene showed that the isolates from dogs
formed a monophyletic clade with the T. evansi isolates from Buffalo of India and
Indonesia. The high nucleotide variation with in our sequences may be the possible
attributes for showing the high genetic diversity within T. evansi population from
other countries and different hosts. Data from the current study suggest that the tick-
borne pathogens are highly prevalent among community owned dogs of Kerala. The
hot and humid climatic conditions of Kerala is congenial for the survival of ticks
and tick- borne pathogens. The difference in prevalence in the present study from
community owned dogs and pet dogs could be due to area of sample collection,
sample size, the type of dogs screened and selection of clinically suspected samples
from pet dogs. In the present study, blood samples have been collected from
community owned dogs from Animal Birth Control Centres (ABCs) and not from
clinically suspected hospital cases for different haemoprotozoan organisms. The 129
present study formed the first and foremost prevalent study of haemoparasites in
community owned dogs of Kerala.
Description
Submitted in partial fulfillment of the requirement for the degree of Master of Veterinary Science in Veterinary Parasitology