Brucella melitensis: Divergence Among Indian Strains and Genetic Characterization of a Strain Isolated from Cattle.

Brucella melitensis primarily affects sheep, goats and is associated with brucellosis in humans, which is one of the world's most widespread neglected zoonotic disease. The current study attempted the determination of genetic diversity through comparative genome analysis of B. melitensis strains reported from India with other countries. The study also reports the isolation and identification of B. melitensis BMNDDB8664 from a cow with a history of abortion, whole-genome sequencing (WGS), determination of virulence factors, genotyping, and comparative genome analysis. Multilocus sequence typing, Multiple locus variable number of tandem repeats analysis (MLVA), and WGS based phylogeny revealed the predominance of ST-8 and genotypes (116 and II respectively) that clustered to the East Mediterranean lineage. Identification of hitherto unreported genotypes by MLVA also indicated the existence and circulation of West Mediterranean and American lineages in India. Though the AMOS-PCR results suggest the BMNDDB8664 isolate as Brucella abortus, the outcomes from multiplex PCR, ribosomal multilocus sequence typing, and WGS analysis confirmed it as B. melitensis. The analysis revealed the presence of adeF gene (aids conferring resistance to fluoro-quinolone and tetracyclines). The isolate lacked two important T4SS genes virB2 and virB7 genes (roles in infection and rifampicin resistance respectively) and also lacked the Brucella suis mprF gene that aids intracellular survival. Further, BMNDDB8664 lacked some of the genes associated with LPS synthesis (wbkB, wbkC) and transport (wzm, wzt) and hence, is most likely a rough strain. WGS-based phylogenetic analysis revealed close genetic relatedness of this BMNDDB8664 with a sheep isolate and two human isolates. The results prompt systematic, broad-based epidemiological studies on brucella infection at the species level. For effective control of human brucellosis, a concerted One Health approach with studies encircling the identification of aetiology at species, strain level to find their prevalence, spread, and inter-host transmission patterns need to be understood, for better design and implementation of effective control strategies in India and other endemic regions. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-023-01081-w.

Evaluation of commercial ELISA kits for diagnosis of brucellosis in cattle and buffaloes in different epidemiological scenarios.

Seven ELISA kits were evaluated for the fitness of purpose in diagnosing brucellosis among cattle and buffaloes in the endemic scenarios of India. The sera (675 numbers) for the study were sourced from brucellosis-free as well as infected herds. The diagnostic sensitivity (dsn) and specificity (dsp) of the kits were determined by three approaches: based on the results of the Rose Bengal test, history of the animals (sera from infected or naïve animals), and based on the results obtained from the 'majority of the tests'. The dsn and dsp ranged from 65.10% to 98.66%, and 98.04% to 100% respectively. The results and suitability of the kits for diagnostic application in various epidemiological situations were discussed.

Detection and genetic characterization indicates circulation of a possible new Theileria species (Theileria sp. Yokoyama) in India.

Bovine tropical theileriosis, a tick-borne disease, causes huge economic loss to the Indian dairy industry. Theileriosis in India is mainly caused by Theileria annulata, although the presence of T. orientalis has also been reported. The present study was undertaken to investigate the deaths of cross-bred Holstein Friesen (CBHF) cows on a farm in the state of Telangana, India. Deceased animals had recently calved and prior to death had developed high fever (107 °F) and anaemia. Infected cows were infested with ticks (Hyalomma species). Theileria piroplasms were noticed in the Giemsa stained blood smears. PCR assays further confirmed the presence of Theileria in the blood samples of the infected cows. Partial Tams1 gene sequences from the infected animals shared 99.87% to 100% identity scores with the sequences of Sri Lankan isolates recently proposed as a novel Theileria species (provisionally designated as Theileria sp. Yokoyama). To the best of our knowledge, this is the first report of the novel species of Theileria from India. Infected animals were effectively treated with buparvaquone and oxytetracycline. The introduction of new animals into the farm without risk assessment was found to be a major cause of the outbreak.

Infectious bovine abortions: observations from an organized dairy herd.

Abortions in dairy animals can be caused by several infectious agents. Identification of the actual causal agent(s) is important for formulating suitable control strategies. A 3-year (2016-2018) longitudinal study was conducted in a dairy farm following an abortion storm in the mid- to late gestations. The investigation focused on the seven major infectious abortifacient in cattle, viz. bovine alphaherpesvirus-1 (BoHV-1), bovine viral diarrhoea virus (BVDV), Neospora caninum, Brucella abortus, Coxiella burnetii, Leptospira Hardjo, and Listeria monocytogenes. High seroprevalence was observed for BVDV (79.4%), Leptospira (70.5%), BoHV-1 (53.5%), and Brucella (45.0%) at the beginning of the investigation (August 2016). The incidence proportion increased for BVDV, Leptospira, and Brucella in the following years of the investigation. A strong association of Brucella seropositivity with history of abortion (OR = 3.27) was recorded. Incidence of BoHV-1 reduced during the period of study coincident with systematic IBR inactivated marker vaccination of the herd. Sixty-four abortion cases were investigated for the identification of causative agent(s) by microbial culture, serological (ELISA), and molecular detection (PCR/ real-time PCR). Antibodies to BVDV, Brucella, BoHV-1, Leptospira, Neospora, and Coxiella were detected in 63, 61, 56, 35, 5, and 6 aborting cattle, respectively. Real-time PCR/PCR of clinical specimens detected DNA of Brucella, BoHV-1, Coxiella, Leptospira, and Listeria in 34, 13, 12, 9, and 4 abortion cases, respectively. BVDV and Neospora were not detected in any specimen samples. Brucella abortus isolated from the farm was determined as ST1 by multi-locus sequence typing (MLST). DNA of multiple agents were detected in 21 of the 64 cases (43.75%). Overall, the data suggests, Brucella was the major causative agent, although multiple causative agents circulated in the farm.

Shedding of bovine alphaherpesvirus-1 in bovine extended frozen semen in Indian semen stations: A longitudinal analysis.

Infectious bovine rhinotracheitis (IBR) caused by bovine alphaherpesvirus 1 (BoHV-1) is an economically important disease of cattle and buffaloes. Following acute infection, the virus usually attains latency in the sensory neurons. Stress-induced reactivation of latency can cause the infected animals to intermittently shed the virus in body secretions including semen. A longitudinal analysis was carried out to study BoHV-1 shedding in the semen of IBR seropositive cattle and buffaloes. The study involved data generated from the screening of 119,850 extended frozen semen (EFS) batches, collected from 1,229 IBR seropositive bulls, over a period of four years (April 2015 to March 2019). A TaqMan based real-time PCR assay was employed to detect the gB gene BoHV-1 DNA in the EFS batch samples. Each sample was tested in duplicate and amplification in any of the replicates at or below the threshold cycle (Ct ≤ 40) was considered positive. The overall positivity of BoHV-1 in EFS batches was 1.18%. About 41% of the bulls (509 of 1,229) were found to have excreted the virus in semen at least once during the study period. The frequency of viral shedding in buffaloes (0.96%) was significantly lower than that of cattle (1.3%) (p < 0.001). No significant difference was noted in the rate of shedding between the first and the second ejaculates collected on the same day (p = 0.607). The rate of shedding also did not vary among various breeds of cattle (p = 0.454) or with the age of the bulls (p = 0.054). No significant variation in the shedding rate was observed in cattle across different seasons (p = 0.101); while in buffaloes, the rate was higher in autumn (1.2%) than in winter (0.7%) (p = 0.037). The difference in positivity among semen stations was statistically significant (p < 0.001). Analysis of data revealed that ≥100 EFS batch samples/bull were screened from 361 of the 1,229 bulls included in the study. None of the EFS batches screened from 39 of these 361 bulls were found positive during the four years, suggesting they were non-shedders. Further research is warranted to delineate the underlying features of the seropositive non-shedders; following which an adequate risk assessment may be made for the maintenance of infected but non-shedding bulls in semen production.

Development and laboratory validation of duplex real-time PCR for simultaneous detection of Brucella and bovine alphaherpesvirus from clinical specimens.

A duplex real­time PCR was developed and validated for the simultaneous detection of Brucella and bovine alphaherpesvirus­1 (BoHV­1) from bovine clinical specimens. The bcsp31 gene of Brucella and gB gene of BoHV­1 were used as targets in the assay. The limit of detection for BoHV­1 was 0.03 TCID50 of virus and 10 plasmid copies containing the target gene while for Brucella it was 4.1 × 101 CFUs. Intra­assay and inter­assay values showed high repeatability and reproducibility of the assay. The diagnostic sensitivity (dsn) and diagnostic specificity (dsp) of the duplex assay were determined by screening 443 clinical specimens and comparing the results with the respective individual assays. The dsn and dsp for detection of Brucella were found to be 95.24% and 95.65%, respectively whereas for BoHV­1, the dsn (100%) and dsp (99.47%) were slightly higher. The duplex assay had a very good degree of agreement with the respective individual real­time PCR test {kappa value 0.97 for Brucella and 0.95 for BoHV­1}. The results of the current study suggest that the duplex assay would be a cost­effective and time­saving alternative for the individual real­time PCR assay for the detection of Brucella and BoHV­1.

Evaluation of a specialized filter-paper matrix for transportation of extended bovine semen to screen for bovine herpesvirus-1 by real-time PCR.

The extended frozen semen (EFS) batches produced from infectious bovine rhinotracheitis (IBR) sero-positive cattle and buffalo bulls housed in various semen stations in India are transported to the testing laboratory in liquid nitrogen (LN2) for screening bovine herpesvirus-1 (BoHV-1). This procedure is laborious and poses LN2 related hazards. An alternative logistics for transportation of samples was investigated. Use of Flinders Technology Associates (FTA®) elute card was evaluated for transportation of extended bovine semen to screen BoHV-1 DNA by real-time PCR targeting gB gene and the method was compared with the OIE approved Chelex resin based method. A protocol for extraction of BoHV-1 DNA from FTA® card spotted with extended semen was optimized. The viral DNA was found to be stable on FTA® card for at least 28 days when the cards are stored at 4°-37 °C. The analytical sensitivity for the assay was determined using variable dilutions of BoHV-1 spiked semen and positive plasmid harbouring gB gene (97bp) spotted onto FTA® card and it was found to be 100.8 TCID50/ml or 100 copies respectively in real-time PCR. The test could detect as low as 100.008 TCID50/ml or 1 copy of positive plasmid when more number of replicates (n = 6) of the same sample were tested. This sensitivity was found to be comparable to Chelex method and both the methods demonstrated a very strong correlation (r = 0.9774; 95% CI: 0.9620-0.9860) in terms of Ct value (p < 0.0001). The diagnostic sensitivity and specificity of the FTA method in comparison to the Chelex method was 83.08% (95% CI: 71.73%-91.24%) and 93.23% (95% CI: 89.38%-96.01%) respectively when 316 samples were screened by both the methods. The degree of agreement between these two tests was good (Kappa value: 0.738; 95% CI: 0.646-0.829). The method was found to be robust and highly repeatable in inter-assay and intra-assay precision testing. The result suggests that the FTA® card holds promise as an alternative system for transportation of EFS for downstream screening of BoHV-1 DNA.