In the present study, 42 cow raw milk samples from brucellosis-suspected cattle in the Mumbai and Pune regions were collected. Out of 42 milk samples, 24 milk samples [Pune 10/15 (66.66%), Mumbai 14/27 (51.85%)] were found positive for Brucella antibodies by MRT, indicating an overall 57.14% incidence of brucellosis in cattle. A higher incidence rate was observed in Pune than in the Mumbai region. findings are similar to the incidence rate of 44.6%, reported by Al-Shemmari et al. (2018)
A total of 42 cow serum samples having a recent history of abortion collected from different farms in and around Pune and Mumbai region were subjected to RBPT for detection of Brucella antibodies. The samples were tested using the RBPT antigen. Out of 42 serum samples tested, 19 (45.23%) were found positive for Brucella antibodies. Samples collected from the Pune region (60%) showed a higher Brucella percentage than the Mumbai region (37.03%). findings are similar to the incidence rate of 50% reported by Leishangthem et al. (2018).
Total DNA was extracted from all 110 samples containing aborted materials, fetal vitals, vaginal swabs, serum, and milk for the detection of Brucella spp by genus-specific PCR. The standard reference strain B. abortus 544 was used as a control to Brucella genus-specific BCSP-31 PCR using published B4/B5 primers. Out of the 110 samples tested 17 (15.45%) were confirmed positive for Brucella spp. They reflected the reference strain by detecting an amplification product of 223 bp (Fig. 1). Among these positive samples, five were from abomasal content, four from cotyledons, six from milk samples, one from a blood sample, and one from a fetal lymph node, all indicating the presence of Brucella spp. Interestingly, the data suggests a higher incidence of Brucella spp. in samples collected from the Pune region (24.52%) than those from Mumbai (7.01%)
The results of the efficacy of BCSP31 PCR and size of amplicons generated in detecting genus Brucella are in agreement with the findings of Thorat et al. (2019), Patel et al. (2007), Satei et al. (2020), Guarino et al. (2000), Cortez et al. (2001), Kanani (2007) found similar efficacy of the BCSP31 B4/B5 primers in detecting Brucella organism at the genus level.
Using species-specific IS711/AB primer pairs, 17 samples positive by genus-specific PCR were analysed; only 5 of the samples had positive 498 bp revealed for B. abortus species in the IS711/AB PCR assay (Fig. 2). Two samples from the Mumbai region, one from lymph node and one from cotyledon, and 3 samples from the Pune region with 1 sample of cotyledon and 2 samples of abomasal content, out of 5, had positive results for Brucella abortus. The incidence rate of B. abortus recorded was 29.41%. (5/17). According to the present study, samples taken from the Mumbai region (50%) had a greater incidence of Brucella spp than the Pune region 23.03%. The results of IS711 AB PCR assays recorded in the present investigation agree with the findings of Hamdy and Amin (2002), Londhe (2009), Khan et al. (2017), Ridhae and Hussein et al.(2021) Luna et al. (2016) They employed IS711/AB PCR assays to identify Brucella abortus and found it to be effective.
Brucella abortus specific virulent gene (virB, bvfA) PCR
All 17 Brucella genus positives were subjected to Brucella virulent gene-specific PCR assay targeting virB and bvfA genes. Out of 17 Brucella genus positive samples, together with reference strain B. abortus 544. 5 samples (29.41%) were found positive for specific virB and bvfA genes, which were also positive for Brucella abortus the IS711/AB PCR test by generating an amplification product of 881 bp and 1281 bp respectively are presented in (Fig. 3), (Fig. 4) along with the reference strain and 5 samples. The overall percentage of positive showed by both the virulent genes was 29.41%. 23. Abo-Ksour, MF et al. (2018) studied 51 blood samples from animals suffering abortions, finding the bvfA gene in 70% of B. abortus, 66.6% of B. ovis, and 50% of B. canis. Hashemifar et al. (2017) reported that nearly all B. abortus strains in Iranian patients and livestock were positive for the virB5 and virB2 genes. Fathy et al. (2023) found that among 33 B. abortus isolates from cattle, the virB gene had the highest incidence (97%), followed by bvfA (93.9%). Esmaeel R J (2019) detected bvfA and virB genes in 22.2% and 55.6% of 35 human blood and 50 bovine milk samples, respectively. Papaparaskevas et al. (2023) showed that all B. abortus clinical isolates contained eight virulence genes, including virB. Aman et al. (2020) found virB, bvfA, and Ure genes in 88%, 79%, and 74% of 200 bulk milk samples.
Sequencing and Phylogenetic Analysis of PCR products of Brucella abortus (IS711) gene using Bioinformatics tools.
PCR product of 5 samples positive for species-specific IS711/AB region (498 bp) of the only B. abortus was subjected to partial sequenced. The partial nucleotide sequence data obtained after sequencing was. The 5 sample sequences obtained were further edited by Cluster method in Molecular Evolutionary Genetics Analysis (MEGA5) software and compared with representative partial Brucella abortus sequence submitted in Genebank.
The results of the Maximum Likelihood showed three lineages for the IS711 region sequence (Fig. 5). The Brucella abortus sequence under study was clustered in lineages 1 and 2. The maximum Likelihood method (MLT) seeks to identify evolutionary history on the base of evidence and reasoning. In the present study, the MLT analysis of the Brucella abortus sequence under study revealed that the sequence originated from India. The isolate under study was grouped under Lineage-1 and 2. All the lineages belonged to a single Arctic lineage. Lineage 1 exhibits the Brucella abortus sequence of GenBank from India, Pakistan, China, Saudi Arabia, and Egypt. Lineage 2 contained the Brucella abortus sequence from the Mumbai region in India. Lineage 3 contained the Brucella abortus sequence from the Mumbai region in India. Under the study, the four Brucella abortus sequences (B02, C03, A01, and E04) fell in linage 2 and showed a close relationship with the Brucella abortus sequence previously submitted from the Mumbai region in India. The previously submitted Brucella abortus sequence with accession numbers GU433108.1, OR344908.1 and OR339879.1 were isolated from Brucella abortus positive cattle tissue and vaginal swab. Hence indicates that the four Brucella abortus sequences (B02, C03, A01, and E04) under study belong to the same geographical areas.
The MRT and RBPT finding shows the prevalence of brucellosis in the dairy cattle in the study group, which is essential for public health issues. The BCSP- 31 PCR assay, unique to the Brucella genus, successfully detects Brucella from direct specimens, including milk, blood, cotyledons, abomasal fluid, and fetal lymph nodes in dairy calves. Brucella abortus from direct specimens in dairy cattle was successfully detected using the species-specific IS711/AB PCR test.Sequencing analysis confirmed that all five samples positive by Brucella species-specific IS711/AB PCR belong to Brucella abortus. Phylogenetic analysis indicated that the isolates belong to some geographical region of India and match the sequence of Brucella abortus submitted previously from the same area.
The precise molecular characterization of Brucella spp. obtained directly from dairy cow specimens entails identifying and analyzing their genetic makeup using diverse molecular biology methodologies. By scrutinizing the expression of virulent genes in Brucella spp., we gain insights into their role in pathogenicity and how these bacteria interact with hosts, facilitating disease transmission. This understanding is paramount for devising effective control strategies against brucellosis in both human and animal populations. Furthermore, research on virulent gene expression holds promise for developing highly accurate diagnostic tools to detect Brucella infections with greater precision in both human and animal hosts.