Mycobacterium bovis induced human tuberculosis in India: Current status, challenges & opportunities

Tuberculosis (TB) continues to be an important disease both for humans and animals since the origin of human civilization. Evidence of TB exists in 3000 year old Egyptian mummies and about 17,000 year old fossilized bison1-3. TB affects both humans and animals (domestic and wild ruminants), and Mycobacterium tuberculosis is the primary cause of human TB. M. tuberculosis survives under extreme adverse conditions in the host4, infects nearly one-third human population5 and is responsible for about 1.6 million deaths per year (including 0.3 million deaths in HIV-positive patients)6. Mycobacterium bovis has 99.5 per cent genomic similarity with M. tuberculosis7, and the proportion of human TB cases caused by this bacillus in the world still remains to be defined. Although M. bovis is primarily of bovine origin, the bacilli can infect almost all the species of mammals including domestic livestock reared for food production8,9. In most of the species, M. bovis causes a slow debilitation of health, although it may remain asymptomatic for many years in infected animals10.

The diagnosis of M. bovis infection in animals [bovine TB (bTB)] is difficult and there is a general belief that none of the single diagnostic tests can detect all infected animals in every stage of M. bovis infection11,12. The accurate diagnosis of bTB mainly relies on the clinical manifestation of disease, tuberculin skin test (TST) and isolation of M. bovis on artificial medium13,14. TST is the most widely used method for the diagnosis, but this assay has the limitations of sensitivity and specificity and requires cautious administration and expertise in the interpretation of results. The issues associated with TST were minimized by interferon-gamma release assays, but high cost and difficulties in test standardization15 may limit its scope in middle- and lower-income countries including India. Currently, the microbiological culture of M. bovis on pyruvate-containing media is considered as the “gold standard test” for diagnosis of bTB. However, it also has certain limitations including a long turnaround time to get the result and lower sensitivity16. The use of molecular techniques to detect M. bovis provides a rapid alternative to culture. A number of polymerase chain reaction (PCR)-based methods have been developed and proposed for the rapid diagnosis of bTB in animals17. Shah et al18 developed a multiplex PCR assay for the single-step detection and differentiation of M. bovis and M. tuberculosis. Other molecular assays, especially PCR-RFLP targeting 16S-23S rRNA, the insertion sequences IS6110 and IS1081 as well as genes coding for proteins specific for M. tuberculosis complex such as MPB70, MPB64 and hsp65 have been developed and used for the diagnosis of bTB in animals14,17,19-23. Molecular typing techniques, such as spoligotyping and pulsed-field gel electrophoresis, have also been applied directly on clinical samples for rapid diagnosis of M. bovis in animals24,25. Molecular typing assays have the advantages of simultaneous detection of disease and transmission dynamics of infection in animal herds25-27. However, the use of molecular typing tools on paucibacillary samples has limitations regarding the sensitivity of the test12,25.

Transmission of M. bovis from animals to humans (zoonosis) constitutes a major public health concern worldwide13,28. Infection primarily occurs through close contact with infected animals or by consumption of contaminated food of animal origin (viz., unpasteurized milk and undercooked meat)29. Although the exact underlying mechanism of M. bovis infection in humans is not well understood, it is believed that the alimentary route of infection leads to extra-pulmonary TB30, whereas the aerosol route of M. bovis infection during close contact with animals results in pulmonary disease in humans31. Macrophages are among the first cells to encounter invading mycobacteria, and the fate of the disease mainly depends on the response of these macrophages to the incoming threat32. It has been established that M. bovis can alter the antimicrobial pathways of the macrophage and cause disease by replicating inside the host cells33-36. Recently, Mendum et al37 identified a few novel genes of M. bovis BCG which are responsible for the specific interaction of the bacilli with the host’s immune system for long-term survival in the host. M. bovis causes zoonotic bTB which is indistinguishable from TB caused by M. tuberculosis based on clinical and radiological features38. In developed countries, cases of zoonotic TB are uncommon. This may be due to the implementation of bTB control programmes and practices of pasteurization of raw milk used for making dairy products. Therefore, majority of cases of zoonotic bTB have been frequently reported from resource-limited developing countries where pasteurization is often inadequately done or control programmes are not in place20,28.

Historically, bTB has been considered to be a rare disease in India39. However, in recent years, bTB has been frequently reported from domestic livestock species located in different parts of the country40-49. A recent meta-analysis revealed a moderate (7.3%) prevalence of bTB in domestic livestock in India49. Several researchers have reported the animal breed variation in resistance to bTB in animals48,50. In India, Das et al48 reported a significantly higher prevalence of bTB in exotic crossbreed animals (P<0.001) as compared to indigenous cattle in West Bengal, India. The presence of M. bovis has also been reported in samples from the environment (soil and water) and animal-derived milk in India43,51,52. In view of the inadequate and sporadic control measures, cases of zoonotic transmission have been frequently reported in India52,53. The human population continues to be at risk of exposure to M. bovis through contact and food products. The present review is based on published information and an attempt to discuss the status of M. bovis infection in the human population and highlights key challenges in minimizing cases of M. bovis-derived human TB in India.

Status of Mycobacterium bovis-derived zoonotic tuberculosis (TB)

Infection of M. bovis in humans results in the disease identical to TB caused by M. tuberculosis. It has been estimated that M. bovis accounts only for one per cent of all cases of human TB in developed countries as compared to 10 per cent in the developing world54. The predominance of M. bovis infection has been reported in cases of extra-pulmonary TB as compared to pulmonary TB. As per the estimation of the World Health Organization (WHO)6, about 1,47,000 people fell ill and around 12,500 people died due to zoonotic TB in 2016, mostly from African and South-East Asian countries. Cases of M. bovis-derived human TB are usually underreported due to clinical, radiographical and histopathological similarities between the cases of human TB caused by M. tuberculosis and M. bovis55.

India has the highest number of TB patients in the world. The cases of M. bovis-induced human TB in the country were documented in the early years of the last century14,56. However, the disease has not received much attention and the country lacks data on the national prevalence of M. bovis-derived human TB. This may be due to the difficulties in differential diagnosis and the limited availability of laboratory infrastructure to isolate and differentiate M. bovis strains from M. tuberculosis. Species identification is important in guiding treatment, as the members of M. tuberculosis complex have important differences in response to anti-TB treatment57-59. The failure of distinction of M. bovis from M. tuberculosis complex can have fatal consequences in the management of TB patients60.

In recent years, sporadic studies have reported variable prevalence of M. bovis infection in suspected TB cases from different geographical regions of India (Table). Recently, Bapat et al65, using duplex PCR, studied the prevalence of M. bovis in three groups of high-risk human population from Central India – group 1: farmers, dairy workers and livestock keepers; group 2: zookeepers and animal handlers and group 3: residents of high-TB burden endemic area. They reported a higher prevalence (12.6%) in blood samples of participants belonging to group 3, followed by group 1 (11.4%) and group 2 (8.9%). The study also reported consumption of raw milk and previous contact with active cases of TB as important determinants for zoonotic TB in the human population residing in the area. Jain61 screened 300 patients of extra-pulmonary TB (tubercular meningitis, tubercular ascites and tubercular lymphadenitis) using PCR targeting hupB gene and reported 85.7, 9.5 and 4.7 per cent of PCR samples positive for hupB gene for M. tuberculosis and M. bovis and co-infection of both, respectively. Kohli et al62 screened 100 infertile women for tuberculous endometritis using PCR targeting hupB gene and reported 13 per cent as positive for extra-pulmonary TB. Of the 13 PCR-positive cases, 38.4 per cent (5/13) were positive for M. tuberculosis, 23.07 per cent (3/13) for M. bovis and 38.4 per cent (5/13) showed co-infection with both the Mycobacterium species62.

Most of the data available on the presence of M. bovis-induced TB in India are based on observational studies conducted on a small number of suspected TB patients. Therefore, these studies may not be truly indicative of the general population and may under report the burden of M.bovis-induced TB in the country65. These studies indicate that M. bovis-induced cases of human TB may account for about 10-15 per cent of all the cases of extra-pulmonary TB in India deserving, therefore, more attention of public health officials in the country. Real-time prevalence frequency and clinical manifestations of M. bovis-induced TB and cases of co-infection with M. tuberculosis or non-tuberculous mycobacteria need to be investigated on priority for better management and planning for control of TB in the human population of the country.

Key challenges for effective management of zoonotic tuberculosis (TB)

The most formidable challenges for the control of zoonotic TB in India are as follows:

Limitations of routine diagnostic tests and limited laboratory infrastructure for the diagnosis of zoonotic tuberculosis (TB): The most commonly used laboratory tests for the diagnosis of TB do not differentiate M. tuberculosis from M. bovis and may potentially overlook zoonotic TB cases. Accurate diagnosis of M. bovis-induced zoonotic TB is complex and requires isolation and characterization of the bacilli from clinical samples. For this purpose, an equipped laboratory infrastructure is needed and currently, the country has 80 Revised National Tuberculosis Control Programme [renamed as National Tuberculosis Elimination Programme (NTEP)] certified culture and DST laboratories for the diagnosis of TB66. India has a huge number of TB patients (around 2.74 million), accounting for about 27 per cent of newly diagnosed TB cases in the world6. The NTEP mainly focuses on early detection and initiation of treatment with an aim to control the spread of TB in the community. Due to the huge burden of pulmonary TB, most of the NTEP-culture laboratories are over-burdened and use non-pyruvate culture media for the isolation of mycobacterial species rather than pyruvate-containing media which are considered the media of choice for primary isolation of M. bovis from clinical samples67. Therefore, the strengthening of laboratory infrastructure and improved laboratory diagnostic tests are essential for better detection and control of zoonotic TB in India.

Lack of information on effective treatment and recovery of patients: Treatment of M. bovis-induced TB differs from the pulmonary TB by M. tuberculosis and is a major challenge for the recovery and effective management of the patient. M. bovis has been found to be intrinsically resistant to pyrazinamide, one of the most important first-line anti-TB drugs used in the treatment regimen68,69. In India, most of the healthcare providers initiate treatment of TB based on the presence of acid-fast bacilli in the clinical samples, without knowing the actual Mycobacterium species involved. With universal DST in place, treatment is modified based on the results of cartridge-based nucleic acid amplification test (CBNAAT or GeneXpert) which, however, cannot differentiate M. tuberculosis from M. bovis as both belong to M. tuberculosis complex. In such situations, patients with M. bovis-induced TB receive inadequate treatment which may lead to adverse consequences on the management of TB patients60 and may even induce the emergence of drug resistance to anti-TB drugs. Hence, the differential diagnosis of M. bovis from M. tuberculosis and systematic checking of resistance to pyrazinamide for patients suspected for zoonotic TB should be ensured. Previous studies also reported co-infection of M. bovis and M. tuberculosis63,70. Therefore, clinical variations and treatment outcomes of co-infection need to be investigated for better management of patients in our country and in countries where bTB is endemic.

Population density and close interaction between humans and animals: India possesses about 2.4 per cent of the world’s geographical area71. The country has the highest number of animals and ranks second where the human population is concerned. In the countryside, animals and humans live in close proximity to each other. bTB has been well documented in herds and flocks of domestic livestock and wild animals from all parts of the country40-49. The majority of livestock are owned by farmers in India, and more than 68.0 per cent of the Indian workforce depends on farming that is in close contact with domestic animals and poultry to earn their livelihood in India72. Aerosol route and ingestion of contaminated milk/milk products are two important means of transmission of M. bovis from animals to humans73,74. The humans can also acquire the M. bovis infection from direct contact with the wound75. Studies reported a higher risk of exposure to M. bovis in individuals working or living with infected animals76. TB patients may also transmit M. bovis or M. tuberculosis to animals or to the in-contact human population through aerosol route77,78. Infection of M. tuberculosis has been reported in domestic or wild animals, living in prolonged contact with humans in captive settings79-84. Prasad et al53 reported mixed infection of M. tuberculosis and M. bovis in humans and animals, and highlighted the risk of transmission of two pathogens from humans to animals (reverse zoonosis) and vice versa (zoonosis). Ultimate elimination of human TB will first require control of bTB in animals. The strategies for the control of bTB in animals may include the development of facilities for animal quarantine of TB-infected animals and implementing legislation to control the marketing and movement of TB-infected animals in the country. Recent studies have revealed that the knowledge in context to TB among Indian farmers is mainly limited to human TB and majority of farmers are not aware about the transmission of TB from animals to humans or vice versa85,86. Therefore, awareness and educational campaigns for human TB would also need to highlight the risk factors and public health implications of zoonotic TB in India.

Infection of the immune-compromised individuals: Suppressed immune system is one of the most important risk factors for M. bovis infection in humans. Previous studies have shown that HIV-positive individuals are more susceptible to M. bovis infection and it influences mortality rate and clinical presentation of the disease69,87. Suppressed immunity also promotes interpersonal transmission of M. bovis as reported amongst immune-suppressed and even immune-competent persons in the UK88. Park et al89 retrospectively reviewed 86 cases of HIV and TB co-infection and reported that M. bovis infection was present in 34.9 per cent of cases in San Diego, USA. India has a huge burden of HIV/AIDS (2.14 million)90 and other immune-compromised (diabetes, organ transplant recipients, cancer, drugs user, etc.) patients91. Since, human beings acquire M. bovis infection mainly through ingestion of unpasteurized milk and dairy products72,73,; the presence of M. bovis in these products in India43,51, poses a great risk of exposure to M. bovis in immune-compromised patients in India. Presently in India, the status of M. bovis infection in HIV or other immune-compromised patients is not known, and therefore, this aspect needs to be investigated. In the current scenario, precautionary measures are needed to restrict the transmission of live M. bovis to humans. Counselling of immune-compromised patients about zoonotic TB and risk factors for the acquisition of M. bovis infection should be made compulsory at the healthcare facilities in India.

Sociocultural and religious values: In India, cultural diversity in day-to-day life has led to stratification of the society with respect to religious beliefs and practices and specific nutritional and healthcare requirements. In few communities, raw cattle milk has been considered healthy and is therefore a staple diet of some families and societies. An interesting outbreak of TB among the cattle and people on the dairy farm due to consumption of raw milk has been reported in 200993. Hence, the traditional practice of boiling of milk before consumption, as practiced in our society since time immemorial, should be revived and propagated by increasing awareness in the rural and tribal communities. Recently, Srinivasan et al49 estimated that 6.3 per cent of cattle in the country are infected with bTB. As infected cows can transmit M. bovis infection to other animals and to human beings, these are likely to increase the bioburden of M. bovis in India. Development of quarantine facilities for infected cattle and initiation of bTB control programme will require joint efforts of the State and Central Governments in India.

Conclusions

Despite being declared a priority disease by the WHO as early as in the 1950s, zoonotic TB has not received due attention in India. This may be due to several reasons including a lack of exhaustive surveys on bTB in animals and zoonotic TB in humans. The available evidence sufficiently indicated that bTB is an ‘insidious problem’ among domestic livestock species in India and the human population carries the risk of exposure to M. bovis. A surveillance programme is urgently needed to estimate the prevalence and economic losses caused by M. bovis-induced TB. This information as well as other specific steps (Box) can be crucial for the strengthening of control strategies of bTB and for minimizing the transmission of M. bovis infection to the human population in India. The key international health organizations (WHO, World Organization for Animal Health, the Food and Agricultural Organization and the International Union against Tuberculosis and Lung Disease) have advocated for the ‘One Health’ approach to combat zoonotic infections in animals and humans94. The spillover of TB from animals to humans or vice versa may jeopardize the efforts of TB eradication programmes in high-endemic countries like India. The existing TB elimination programme in the country (NTEP) can adopt comprehensive policies to tackle human as well as zoonotic TB under the ‘One Health’ umbrella. The implementation of this approach will be helpful towards the speedy reduction of human TB and achieving the ambitious goal of TB elimination in India by 2025.