An 8-year-old Kachchi breed of dromedary camel was presented with abortion after the 9th month of gestation during her 2nd pregnancy. This camel belonged to a herd comprised of 350 dromedary camels in the Thar desert of Rajasthan, India. The herd is maintained in a semi-intensive husbandry system, mostly in outdoor facilities, and fed with a mix of pellet feed, hay, and ad libitum water. A history of sporadic occurrence of TB has been reported in this herd. Clinically, the aborted she-camel appeared healthy with a good body condition score and normal appetite and did not show any respiratory symptoms and fever at the time of the abortion. The she-camel was tested negative for brucellosis on Rose Bengal Plate Test. However, she tested positive in a single intradermal tuberculin test performed a day after abortion, showing marked swelling and a 2-fold increase in skin thickness at the injection site.
After the abortion, the placenta and aborted fetus were examined for gross lesions. The expelled placenta was thickened, edematous, and multifocal to coalescing ecchymotic hemorrhages on the allantoic surface (Figure 1A). The fetus showed generalized subcutaneous edema, congestion, and a moderate amount of sero-hemorrhagic fluid in the abdominal and thoracic cavity along with generalized congestion of all internal organs, which is likely due to autolysis and hemoglobin imbibition. The striking finding in the fetus was the presence of multiple, white-yellow, solid nodules scattered over the lung, the pleura, and the liver which measured from 2 to 15 mm in diameter (Figure 1B). The lung was collapsed, severely congested, and had multiple small white-yellow tubercle nodules scattered on all lobes. These nodules were also found attached to the pleura and inner surface of the rib cage. The liver was found enlarged considerably, congested, and had multiple tubercle nodules (Figure 1C). The other organs viz., the heart, the spleen, the kidney, and the intestines showed severe generalized congestion without any evidence of tuberculous lesions.
The tissue samples suspected to have TB lesions, such as the lung, the liver, and the placenta, were collected in 10% neutral-buffered formalin for histopathology, as well as in sterile vials for DNA extraction. For histopathology, tissues after fixation were embedded in paraffin, cut into 4-mum-thick sections and stained with hematoxylin and eosin. Selected sections were also subjected to Ziehl-Neelsen (ZN) staining. On histology, the lung and liver sections showed typical granulomatous lesions characterized by a central area of caseous necrosis and mineralization surrounded by scattered lymphocytes, macrophages, and/or occasional giant cells, and concentric layers of fibrosis (Figures 2A,B, 3A). Scarce acid-fast bacilli were observed on ZN-stained sections of the lung and the liver (Figure 3B). Histopathology of the placenta showed normal physiologic and/or autolytic changes of mineralization of the chorionic epithelium, necrosis of villous stroma, and hyperemic blood vessels. However, granulomatous inflammation and acid-fast bacilli were not observed.
Tissues (placenta, fetal lung, and liver) were processed for DNA extraction using the PureLink  Genomic DNA Mini Kit (Invitrogen). The DNA was subjected to PCR amplification of a 245bp region of IS6110 sequence specific for the MTBC, using primer pairs INS1 (5'-CGTGAGGGCATCGAGGTGGC-3') and INS2 (5'-GCGTAGGCGTCGGTGACAAA-3'). Briefly, a 25 mul reaction was prepared using 12.5 mul Gotaq  green master mix (Promega), 1 mul each primer (10 picomoles), 5 mul of DNA, and 5.5 mul of nuclease-free water. The cycling conditions used were initial denaturation at 94 C for 5 min, followed by 35 cycles of denaturation at 94 C for 1 min, annealing at 65 C for 1 min, and extension at 72 C for 1 min. This was followed by a final extension step of 72 C for 10 min. The PCR-amplified products were visualized in 2% agarose gel and the purified PCR products were subjected to nucleotide sequencing for the IS6110 gene using Sanger sequencing based on the chain-terminating dideoxynucleosides method (Eurofins, India). These sequences were deposited in NCBI GenBank (accession numbers: MW393780 and OL436218) and aligned with the published sequences for phylogenetic analysis using the ClustalW tool and the Maximum Composite Likelihood method. This analysis involved 30 nucleotide sequences, and the evolutionary analyses were conducted in MEGA X. The DNA extracted from the lung and the liver showed amplification of the IS6110 region of MTBC by PCR (Supplementary Figure 1). The sequencing and phylogenetic analysis revealed that sequences in this study clustered with M. tuberculosis.
On a managemental aspect, this she-camel was isolated and maintained away from the herd after abortion. After 1 year of isolation, the she-camel exhibited the clinical signs of chronic infection, including poor appetite, weakness, and progressive emaciation. Eventually, this she-camel died after 7 months of exhibiting symptoms. Considering the clinical history and the safety of the personnel, neither necropsy nor tissue evaluation was performed on this she-camel. Instead, the carcass was immediately disposed of by deep burying.