SEROEPIDEMIOLOGY, CULTURAL AND MOLECULAR DETECTION OF BRUCELLA INFECTION IN RUMINANTS OF SOUTH SAURASHTRA REGION OF GUJARAT
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Date
2020-12
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JAU,JUANAGDH
Abstract
Brucellosis is an important zoonosis and a significant cause of economic losses to
farmers. Abortion, placentitis, epididymitis and orchitis are the most common clinical
manifestations in animals. In view of the considerable problems related to direct diagnosis of
brucellosis in animals, the present study envisaged the appraisal of seroepidemiology of
brucellosis in ruminants. For the purpose, three serological tests viz., Rose Bengal Plate Test
(RBPT), Lateral Flow Assay (LFA) and Indirect-Enzyme Linked Immunosorbent Assay
(iELISA) were employed. The Biochemical and molecular characterization of isolates was
carried out for the final confirmation.
A total of 770 serum samples from five districts (Junagadh, Rajkot, Amreli, Gir
Somnath and Porbandar) of South Saurashtra region of Gujarat were collected to detect the
brucella specific antibodies. A total of 304 clinical samples with reproductive disorders
comprised of deep vaginal swabs (125), vaginal discharge (76), placenta (33), placental
cotyledons (12), aborted foetal organs (lung-11, liver-11, heart blood-18 and stomach
contents-18) were collected from ruminants. The clinical samples were cultured on Brucella
agar medium (BAM) with selective antibiotic supplements. The Brucella isolates obtained
from positive samples were tested by using genus and species specific PCR.
Out of 770 ruminants sera screened, 116(15.06 %), 84(10.91%) and 124 (16.10 %)
sera samples were detected positive by RBPT, LFA and iELISA, respectively. Species wise
seroprevalence reported to be 18.13, 13.13 and 18.75 % in cattle, 12.09, 8.79 and 13.19% in
buffaloes, 10.71, 7.14 and 13.39% in sheep and 15.38, 11.54 and 16.03% in goats by RBPT,
LFA and iELISA, respectively. While, Sex wise seroprevalence rates of brucellosis were
14.37, 8.98 and 13.17% in males (167) and 15.26, 11.44 and 16.92% in females (167) by
RBPT, LFA and iELISA, respectively. Higher seroprevalence was observed in females than
in males. Location wise analysis revealed, seroprevalance in Junagadh district 14.33, 10.75
and 15.96 %, Rajkot district 16.88, 11.045 and 18.33 %, Amreli district 17.82, 12.61 and
14.41 %, Gir Somnath district 17.82, 11.88 and 13.86 % and in Porbandar district 10.31,
8.25 and 16.49 % by RBPT, LFA and i-ELISA, respectively. The overall higher rate of
Abstract
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seroprevalence was noticed in Rajkot district (18.33%) followed by Amreli/Gir Sonmath
districts (17.82 %) and 16.49% in Porbandar district as compared to other districts of South
Saurashtra region of Gujarat.
Overall sensitivity of RBPT and LFA was found to be of 83.06% and 65.32%, while
specificity was 97.99% and 99.54%, respectively, considering iELISA as a gold standard
test. Thus, RBPT was found to be more sensitive and less specific than that of LFA. The
overall positive and negative likelihood ratio was 41.28 & 140.66 and 0.17 & 0.35 for RBPT
and LFA respectively as compared to iELISA. The positive predictive value was 88.79% and
96.43%, while negative predictive value was 96.79%, and 93.73% for RBPT and LFA,
respectively as compared to iELISA. McNemar chi-square test for independent data revealed
non-significant difference for RBPT, while significant difference for LFA considering
iELISA as gold standard technique. The concordance of RBPT and LFA was good (k=0.832
and k=0.746, respectively) considering iELISA as gold standard test.
The clinical samples (304) subjected to cultural isolation on Brucella agar medium
(BAM) with selective antibiotic supplements. Out of these, 17(5.59%) samples (cattle-8,
buffaloes-4, goats-4 and sheep-1) yielded bacterial isolates. Which were confirmed as B.
abortus by biochemical and molecular characterization.
17 isolates were recovered from vaginal swab-3 (2.4%), vaginal discharge-3 (3.95),
placenta-6 (18.18%), placental cotyledons-3 (25%) and aborted foetal stomach content-2
(11.11%) from clinical samples of ruminants.
For confirmation of isolates, colony duplex-PCR were carried out targeting Brucella
genus specific BCSP-31 and IS-711genes to get 223bp and 350bp PCR products and all
isolates were confirmed as Brucella spp. While, all 17 isolates were identified as B. abortus
using desired amplified product of 498bp through multiplex PCR technique.
The nucleic acid sequences obtained by sequencing of studied genes (BCSP31,
IS711 and alkB) of Brucella isolates were aligned with published sequences available at
GenBank (NCBI, USA). The nucleotide sequence alignment of the BCSP31, IS711 and
alkB genes of Brucella showed as 97.9 to 100%, 98.3-100% and 95.3-100% homology
with Brucella species, respectively. It shows that either BCSP31or IS711 gene is the best
to study the genetic homogeneity in Brucella.
Phylogenetically, the BCSP31, IS711 and alkB genes of B. abortus sequences
obtained from the Junagadh isolate coming within the broader clade of different common
species of Brucella.
In conclusion, serology coupled with biochemical and molecular tests confirmed the
prevalence of B. abortus in South Saurashtra region of Gujarat.