The majority of tuberculosis cases in ruminants are caused by Mycobacterium bovis (MB). However, in this study, the authors reported the isolation of Mycobacterium tuberculosis (MT) from bovine milk, nasal swabs and post-mortem tissue samples (n = 841) collected from cattle and buffaloes in the states of Telangana, Maharashtra and Gujarat in India in the period from 2010 to 2015. The isolates (n = 7) were confirmed as Mycobacterium due to their growth characteristics and colony morphology in a commercial liquid medium Mycobacteria Growth Indicator Tube (MGIT)™ employing the BD BACTEC™ MGIT™ 960 system and the Lowenstein Jensen (LJ) medium supplemented with glycerol but not with sodium pyruvate, and BD-DIFCO™ Middlebrook 7H10 agar containing oleic albumin dextrose catalase (OADC). These isolates were initially identified as members of the Mycobacterium tuberculosis complex (MTC) using a commercial nested polymerase chain reaction (PCR) kit based on the IS6110 MTC specific nucleotide sequence. The isolates were confirmed as MT using three commercial line probe assay kits, were further genotyped, and the spoligotypes identified were of East African Indian (EAI) 3_IND, EAI5, Central-Asian (CAS) 1_DELHI, U and T1 lineages. Two MT isolates from one antelope (Antelope cervipara) and one gazelle (Gazelle bennettii) from Gujarat, which were identified previously, were spoligotyped during this study and identified as belonging to EAI3_IND and EAI5 lineages, respectively. The epidemiological significance and zoonotic implications of regional presence and documentation of the same or two different spoligotypes in different species within the family Bovidae as well as humans is discussed.

Tuberculosis (TB) in domestic and wild ruminants is mostly caused by Mycobacterium bovis (MB). However, the present paper describes the first report of TB of antelopes (Antelope cervicapra) and chinkara (Gazella bennettii) due to Mycobacterium tuberculosis (MT) in India. These wild hosts may represent a vehicle for the dissemination of MT infection to domestic livestock or human. MT was isolated by culture employing the MGIT TM BACTEC TM 960 (Becton Dickinson, BD) system and Middlebrook 7H10 Agar enriched with oleic albumin dextrose catalase (OADC) and Lowenstein-Jensen (LJ) medium supplemented with glycerol, but not sodium pyruvate. The isolates were confirmed as a member of the Mycobacterium tuberculosis complex (MTC) by using a commercial nested PCR that targeted the IS6110 sequence. Further confirmation of the isolates as MT strains was achieved by employing commercial line probe assay genotyping kits (Hain Lifescience, Germany) that specifically identifies MT within the MTC group.

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| A diagnostic Real time PCR (qPCR) for detection of Mycobacterium avium subspecies paratuberculosis (MAP) was optimized using the ISMav2 sequence specific for MAP. The analytical sensitivity of the assay was 500 fg, and it was able to detect approximately 7 copies of the positive control plasmid construct. The qPCR detected up to 1x10 3 , 1x10 4 and 1x 10 5 MAP cells per reaction from spiked bovine semen, milk and feces respectively. The assay was reliable, reproducible and could be completed in 87 minutes. Comparative quantification for MAP copy number was established by utilizing normalized Cq values. The ISMav2 qPCR specifically detected 8 MAP strains, but was unable to amplify DNA from any other strains of Mycobacterium (n=5) or non-Mycobacterium strains (n=10). Several factors were tested to study their impact on the validation of the assay under field conditions. It was observed that sample size, number of sampling time points and test methods adopted for accepting the infection status were strongly correlated. The best return of validation estimates were obtained when sample-wise results of the qPCR were compared to the combined status of culture and acid fast bacilli (AFB) staining. The diagnostic sensitivity and specificity, positive and negative predictive values were estimated to be 100% (95% CI |63.06 -100.0|) and 99.9% (95% CI |99.44 100.0|), 88.89% (CI |51.75 -99.72|) and 100% (95%CI |99.75 -100.0|), respectively. Also, the qPCR and the MAP identification test conditions were strongly associated (κ = 0.941 (95% CI |0.825 -1.00|). The validation estimates were true only when the sample size was large (n=1008) and sample collection was based on a single time point sampling strategy. Thus it appears that the assay could be considered as a reliable and rapid diagnostic test for field application. Keywords

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