Int.J.Curr.Microbiol.App.Sci (2020) 9(6): 844-851

International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 9 Number 6 (2020) Journal homepage: http://www.ijcmas.com

Original Research Article https://doi.org/10.20546/ijcmas.2020.906.107

Microbiological and Molecular Diagnosis of Mycobacterium tuberculosis Infection in Spotted (Axis axis) of an Indian Zoo

Sourabh Ranjan Hota1, Sarat Kumar Sahu2*, Bikash Kumar Behera1, Avishek Pahari1 and Niranjana Sahoo1

1Centre for Wildlife Health, College of Veterinary Science & Husbandry, Odisha University of Agriculture and Technology, Bhubaneswar, Odisha, -751003 2Nandankanan Zoological Park, Bhubaneswar, Odisha, India- 754005

*Corresponding author

ABSTRACT

A detailed laboratory investigation was undertaken to correlate the cause of death in two spotted deer (Axis axis) of Nandankanan Zoological Park,

Odisha, India, suspected due to Tuberculosis based on necropsy findings. K e yw or ds The pus samples collected from lungs nodules were inoculated to

Mycobacterium Lowenstein‒Jensen medium with glycerol which showed growth of buff tuberculosis , 16S colored smooth to bread-crump like colonies after 6‒10 weeks of rRNA sequencing, ° RT PCR, Axis axis incubation at 37 C. The culture positive samples were subjected to 16SrRNA PCR that amplified both the isolates producing an amplicon of

Article Info 500bp size upon gel electrophoresis. Sequencing of this product yielded a

Accepted: gene sequence of 520 nucleotides which upon blast, showed 99% similarity 18 May 2020 with Mycobacterium tuberculosis (NCBI accession no. MH393632 and Available Online: MH393637). A chip-based real-time PCR tests showing a peak Ct value of 10 June 2020 19.5 confirmed this . Identification of human type TB amongst spotted deer warranted implementation of effective managemental practices

in the zoo.

Introduction (Chakraborty et al., 1993; Thakuria, 1996) Chhatbir Zoo, Punjab (Singh and Gupta, Tuberculosis (TB) is a widely prevalent 1988), Nandankanan Zoological Park zoonotic disease that affects both domestic (NKZP), Bhubaneswar (Rao and Acharjyo, and wild animal across the globe. Many 1996) have reported tuberculosis among many zoological gardens of India viz., National species of cervids like spotted deer (Axis Zoo, Delhi (Arora, 2000), Kanpur Zoo axis), brow-antlered deer ( eldii), (Gairola, 1986), Assam State Zoo hog deer (Axis porcinus), swamp deer

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(Rucervus duvaucelii), barking deer supernatant. The processed sample was (Muntiacus muntjak), mouse deer (Moschiola inoculated onto the slopes of Lowenstein- indica) and antelopes like (Boselaphus Jensen slants, one with glycerol and another tragocamelus), (Gazella bennettii), with sodium pyruvate, in McCartney tubes. black buck (Antilope cervicapra) and four The tubes were incubated at 370C in an horned antelope (Tetracerus quadricornis). inclined position overnight and vertically for Human type TB caused by M. tuberculosis 8–12 weeks with weekly examination starting has also been isolated earlier from hog deer, from three days post inoculation. black buck, spotted deer, and sambar (Upadhyay et al., 1986; Chakraborty et al., Acid-fast and fluorescent staining 1993; Bhowmik, 1999; Arora, 2000). Present study describes microbiological and Two thin smears were prepared from the molecular identification of M. tuberculosis colonies on L‒J medium, on glass slide and from pus samples of lungs collected during stained by BD TB Ziehl‒Neelsen stain kit necropsy of two spotted deer in NKZP that (Becton, Dickinson and Company, Becton died in quick succession of 3 days. Drive, Franklin Lakes, NJ) and HiMedia fluorescent stain kit (HiMedia Laboratories Materials and Methods Pvt. Limited, Mumbai, India) with Auramine O dye as per the manufacturer’s instructions Sample collection and examined under oil-immersion lens of optical microscope and fluorescent Postmortem examination of the two spotted microscope, respectively for the presence of deer died during March 2018 revealed Mycobacterium. presence of extensive areas of caseous and abscess-like lesions in the lungs and adjoining Molecular identification lymph nodes with accumulated pus discharges in the pleural cavity (Figure 1).The pus Genomic DNA was extracted from the samples from the pleural cavity were sampled positive culture by using DNeasy blood and aseptically in sterile screw-capped containers tissue kit (QIAGEN GmbH – Germany) and for microbiological and molecular checked for purity and quantity. The target examination. DNA used for amplification was the first 500 bp fragment of 16SrRNA gene used for Isolation of Mycobacterium sp identification of unknown bacterial isolates. PCR amplification was carried out in 25 ml Approximately 5.0 ml of pus sample was reaction mixtures containing 1.0 ml of DNA taken and equal volume of 4% NaOH was template, 20pM of 1.0 ml each of forward and added to it for initial decontamination reverse primer, 12.5 ml of 2X red dye PCR (Modified Petroff’s method; Vestal, 1977). master mix (Amplicon) and 9.5 ml of The mixture was allowed to stand for 15 nuclease free water. The reaction mixture was minutes at room temperature. Following subjected to initial denaturation at 94oC for centrifugation at 3000g for 15 minutes, the 10mins followed by 30 cycles of supernatant was carefully removed and 15 ml amplification consisting of denaturation at of sterile distilled water was added to 940C for 1 min, annealing at 54ºC for 1 min resuspend the sediment. The mixture was re- and extension at 720C for 1 min with a final centrifuged at 3000g for 15 minutes and a extension at 720C for 5 min. The final PCR pellet was extracted after decanting off the amplified product was separated by 1.5%

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agarose gel electrophoresis and gel slants with sodium pyruvate. This shows documented. The primer sequence used are: presence of M. tuberculosis and absence of M. 0008F (5’-AGAGTTTGATCCTGGCTCAG- bovis in both the samples. Both the bacterial 3’) and 0532R (5’-TACCGCGGCTG colonies, extracted from L-J slants, were CTGGCAC-3’). Following PCR, the found positive for presence of Acid Fast product was purified from gel using Bacilli (AFB) based on Z-N staining method QIAquick gel extraction kit (QIAGEN GmbH (Figure 3). In fluorescent dye (auramine) – Germany) and subjected to DNA stained slides, distinct fluorescent bacilli were sequencing. observed under high power magnification of fluorescent microscope that confirmed Pus samples found positive for presence of Mycobacterial AFB (Figure 4). Mycobacterium tuberculosis were further analyzed for confirmation of species-specific Sequencing of 16SrRNA PCR amplified pathogen using a chip-based real-time PCR products yielded a gene sequence of 520 test ‘Truenat MTB’ (Molbio Diagnostics Pvt. nucleotides (Figure 5) which upon blast, Ltd., Verna Industrial Estate, Verna, , showed 99% similarity with Mycobacterium India). tuberculosis. The said gene sequences has been submitted in the NCBI gene bank with Results and Discussion accession no. MH393632 and MH393637. Further analysis of pus samples in Truenat In this study, pus samples from both the deer (chip-based) real-time PCR test revealed showed growth of buff colored smooth to presence of Mycobacterium tuberculosis that bread-crump like colonies in Lowenstein- produced a peak at Ct value of 19.5 (Figure Jensen medium with glycerol after 6 - 10 6). It double checked the findings of sequence weeks of incubation at 37oC (Figure 2). results. However, no growth was observed in the L-J

Figure.1 Cut surface of lungs showing extensive areas of caseous and abscess-like lesions

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Figure.2 Buff colored bread-crump like colonies in Lowenstein-Jensen medium with glycerol after 6 - 10 weeks of incubation at 37oC

Figure.3 Acid Fast Bacilli (AFB) based on Z-N staining under oil immersion lens (100x)

Figure.4 Fluorescent bacilli under high power magnification (100x)

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Figure.5 PCR showing 500bp fragment of 16SrRNA of the bacterial isolate. Lane 1-1000bp gene marker, Lane 2- Negative control, Lane 3- Test sample and Lane 4 - Positive control

Figure.6 Truenat (chip-based) real-time PCR test revealed presence of Mycobacterium tuberculosis that produced a peak at Ct value of 19.5

Bovine TB predominantly caused by M. bovis against the common notion of M. bovis as the and seldom M. tuberculosis, continues to be a common cause of TB associated mortality in matter of concern for its potential zoonotic herbivores including deer (Kaneene et al., risks. South Asia consisting of eight 2010). countries, namely, Afghanistan, Bangladesh, Bhutan, Maldives, Nepal, Pakistan, Sri Lanka, Severe outbreaks of tuberculosis in deer are and India has emerged as a focal spot for uncommon though in captive settings a large tuberculosis witnessing one of the highest number of are likely to be infected incidence rates sharing more than one-third sub-clinically (Waters et al., 2011). In deer, (34%) of the world’s human TB burden tuberculosis is a sub-acute to chronic disease (Thapa et al., 2017). In the similar scale and even the critically infected ones may not tuberculosis is of paramount importance in demonstrate obvious clinical signs before captive wildlife across zoological collections death (Palmer et al., 2015). Absence of globally (Montali et al., 2001). Isolation and clinical signs in both the spotted deer in our identification of Mycobacterium tuberculosis study supports this statement. This may be the from both the spotted deer at NKZP, runs result of one or more animals acting as

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‘shedders’, efficiently transmitting disease to Zoological Parks with parallel increase in herd mates. visitors’ entry.

NKZP harbors more than 1200 no. of cervids Exposure and infection of captive wildlife in a with spotted deer acquiring the major share of high TB prevalent country like India, is the pie. Therefore, detection of TB in this therefore a growing concern not only for species significantly elevates the possibility of wildlife bio-diversity conservation but also the existence of this infection in neighboring for human health. This further escalates the susceptible hosts. There is a common concern of public health hazard of retrograde perception that TB can spread uncontrollably transmission of TB from wild animals to in captive deer herds (Essey and Koller, humans through potential spillback. In this 1994). This message was reinforced by Parra context, Barron et al., (2013) described the et al., (2005), who observed that the deer emerging role of deer in TB epidemiology as involved in a TB outbreak, possibly can a persistent source of infection to humans. In maintain the transmission cycles over long addition to a string of reports available on periods of time, lasting at least up to 3 years wild deer acting as a reservoir of infection, in the same area. Palmer et al., (2012) reported ‘spilling back’ of infection from wild to domestic animals. Presence of M. tuberculosis and absence of M. Chu et al., (2012) demonstrated that deer may bovis in both the positive samples in our be the reservoir for M. tuberculosis, posing as findings raised the possibility of human-to- a potential public health threat. animal transmission and possible adaptation of this organism in animal tissues. As per Present finding would be helpful at Thoen and Steele (1995), humans suffering formulating a strategic approach towards from active TB are the most probable source prevention of TB not only for captive wild of M. tuberculosis infection for animals. A animals of the zoo but also for the human number of researchers have reported the resources that work in close contact with existence of M. tuberculosis in domestic and these animals. wild animals, most frequently living in close contact with humans (Montali et al., 2001; It can be concluded that the death in two Sternberg et al., 2002; Alfonso et al., 2004). spotted deer in quick succession of three days Most reports of M. tuberculosis infection in and the confirmation of M. tuberculosis animals are confined to developing countries infection through molecular diagnosis, raises with a high prevalence of human TB (Epstein concern regarding the possibility of existence and Price, 2009). Hutchings and Harris (1997) of infection among other deer species in the stressed that inhalation of bacilli from grass neighbouring herbivore enclosures. The contaminated with infected human urine, zoonotic potential of the pathogen warranted faeces, or sputum may be the most important immediate implementation of a route of transmission of TB from human to comprehensive preventive protocol that animals. Poor hygiene, overcrowding, would combat the disease in the zoo. unsuitable environments and close contact to visitors may also act as an aggravating factor Acknowledgement in transmission of TB (Kohn, 1994). It is not unwise to speculate that this shift is likely to The authors express their deep sense of thanks lead to a net increase in the incidence of M. and gratitude to the Director, Nandankanan tuberculosis in captive animal populations of Biological Park, Forest and Environment

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How to cite this article:

Sourabh Ranjan Hota, Sarat Kumar Sahu, Bikash Kumar Behera, Avishek Pahari and Niranjana Sahoo. 2020. Microbiological and Molecular Diagnosis of Mycobacterium tuberculosis Infection in Spotted Deer (Axis axis) of an Indian Zoo. Int.J.Curr.Microbiol.App.Sci. 9(06): 844-851. doi: https://doi.org/10.20546/ijcmas.2020.906.107

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