Sero-diagnostic efficacy of various ELISA kits for diagnosis of infectious bovine rhinotracheitis (IBR) in cattle and buffaloes in India.

Bovine alphaherpesvirus-1 (BoHV-1), the causative agent of infectious bovine rhinotracheitis (IBR), is an economically important viral pathogen affecting cattle and buffaloes. Serological assays are mostly used for detection of the antibodies, but variation has been detected in the diagnostic performances of the individual assay. In the present study, four commercially available ELISA kits {two indirect ELISA (kits A and B) and two blocking ELISA (kits C and D)} were evaluated for the detection of antibodies against BoHV-1 in Indian cattle and buffaloes (fitness of purpose). The diagnostic sensitivity (dsn) and specificity (dsp) of these kits were determined by three ways; considering virus neutralization test (VNT) as gold standard test, using pre-test information of the samples, and majority of tests. Screening of 200 known negative sera (124 cattle, 76 buffaloes) sourced from IBR free farms revealed gB based ELISA kits are more specific than the indirect ELISA kits. Testing of 125 known positive sera (81 cattle, 44 buffaloes) suggests kit B be most sensitive followed by kit C, A and D. Interestingly, kit D was found to be most sensitive for detection of vaccination-induced BoHV-1 antibodies followed by kit B. Similar trend were also observed in the limit of dilution experiment performed using known infected and vaccinated sera. VNT was found to be the most specific test and its use as the gold standard test revealed all kits to have more than 99 % sensitivity. All the ELISA kits could detect BoHV-1 specific antibodies in the IBR vaccinated calves as early as 11 days post-vaccination. In Kappa statistics, an almost perfect agreement between the ELISA kits was recorded. The overall performance of the kits in serodiagnosis of IBR as determined by the area under curve in ROC analysis was good.

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.

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.