Abstract
Brucellosis is an ancient re-emerging bacterial zoonotic disease caused by Slmies Of the Brucella genus,
affects a Wide range of domesticated and adult wildlife, and plays a significant economic impact on public
health and livestock sector. As an endemic disease species identification and biotyping of Brucella isolates
are essential for epidemiologic surveillance and investigation in newly infected regions. In this study,
strain and species of Brucella abortus were identified through MLVA (Multi Locus Variable Number
Tandem Repeats Analysis) and PCR (Polymerase Chain Reaction), respectively. This study collected 2908
samples Of 2837 sera, 63 milk, and eight aborted materials from Dhaka, Munshiganj, Manikganj, Sylhet,
and Mymensingh districts. There were 140 sera samples, 63 milk samples, and eight aborted materials With
a history Of reproductive disturbances collected from dairy cows Of selected farms for molecular diagnosis.
Again, 1925 sera samples from goats, 772 from "Ilected Where 1847 goal seru 746 sheep
sera were randomly collected from 13 Upazila district to estimatc thc annual cconomic
impact Of Brucella spp. In this study, a tote' of 78 (2" sheep, and 34 goats) samples were
seropositive screened by RBT, rapid antibody kit test, MR7 Among the positive samples, 14
high-graded seropositive sera Of cattle, goats, and and two tissues from aborted fetal
membranes Of the cow were to DNA extraction and to FLI, Germany, for molecular
diagnosis and typing. On the Other hand, the eight fetal membranes, four samples were stained
positive when screened by M(Hlified and inoculated to guinea pigs.
Ten tissue samples (spleen, lung, liver, and Of seropositive guinea pigs also positive in PCR
(Six hundred two base pair band) were sent to hboratory Of Veterinary Bacteriology (ISOI 7025), 1050
Brussels, Belgium, for genotyping. The genotyping was performed through MLVA by the use of 16
variable number tandem repeats (VNTRS). PCR With nine genes (MLSA 9) of 4 isolates (originated from
Brucella positive guinea pigs tissue culture) was done, and sequencing to compare phylogenetic position.
The multi-locus phylogenetic tree Of the concatenated data was reconstructed With reference
strains using RAxML and visualized by Tree Graph2. The prevalence of B. Spp. was found to be 20.3%
and 23.6% in serum and milk, respectively, in cattle Of dairy farms in the Dhaka district and found to
9.3% in serum in the Sylhet district. The prevalence of B. spp. was 1.56% in goats, and 1.65 % in sheep
were calculated in the subsistence management system. The prevalences were to estimate
loss over the thirteen Upazilas of Mymensingh district. For the first time, the IK'ssible annual
economic losses ofbrucellosis in small ruminants in the Mymensingh district have been estimated in this
study and shown a total of BDT 48436400 (605455USS) possible lost annually where BDT 46462900
(580786.25 USS) and BDT 1973500 (24668.75 USS) were in goaLS and sheep respectively. In this study,
Brucella abortus was identified at 223bp in (Brucella aborttts-specific bcsp31/B4B5 primer) PCR,
quantitative Multiplex Real-time PCR (Brucella genus and species-specific IS7 1 1 for Brucella abortus and
Brucella melitensis, positive at Ct value <40) and for more confirmation Bruce Ladder 2.0 PCR identified
pmsitive at 775 base pair, in FLI, Germany, where Brucella abortus was found to be the causal organism in
cattle and identified as the causative agent Of sheep (first known investigation) and goats brucellosis in
Bangladesh. Brucella abortus was isolated, characterized, and as Brucella abortus biovar 3
With both classical and molecular methods ML VA-16. The profile Of the strains from Bangladesh was
compared to B. abortus biovar 3 genotypes deposited in the Brucella aggregated database on MLVA net
(http://mIva.u-psud.fr/mlvav4/genotyping index.php), and the query strain was identical with other 20
genotypes and interpreted as similar to Asian origin strain. The multi-locus gene analysis indicated the
phylogenetic position of 4 Brucella abortus isolates from Bangladesh with well -supported value. We have
isolated biovar 3 of Brucella abortus from the cow placenta. It does indicate that Brucella abortus biovar 3
is prevalent in the study government farm. Therefore, human brucellosis is best prevented by controlling
the infection in large ruminant farms in Bangladesh as a zoonotic disease. In the absence of the BSL3
laboratory, the guineapig inoculation technique could be used as a good culture alternative for the
confirmed diagnosis of brucellosis from contaminated clinical samples like the placenta. This studys
possible annual economic impact reflected that as a non-notifiable disease, brucellosis silently constitutes
an economic loss in the country and the producers. Hence, this study's results can be used to develop
effective control and eradication strategy for brucellosis in livestock and public health. The high
prevalence of Brucella in dairy cattle suggested the research should continue to demonstrate the B.spp.
biovar in other government dairy farms and semen-prcxlucing bulls in Bangladesh.