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.

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.