A one health-focused literature review on bovine and zoonotic tuberculosis in Pakistan from the past two decades: challenges and way forward for control

Bovine tuberculosis (bTB), caused by Mycobacterium bovis (M. bovis), is a globally prevalent zoonotic infectious disease. World Organization for Animal Health (WOAH) estimates indicate that up to 10% of the total human TB cases in developing countries are attributed to M. bovis. Pakistan ranks 4th in global milk production with a livestock population of over 212 million animals. Over 8 million families are involved in raising these animals as a means of livelihood. To date, there is an absence of national-level data on the prevalence of bTB and an effective control program is still lacking. The multifaceted impacts and substantial economic losses render addressing bTB a daunting, but highly important challenge. In this review, we summarise all the freely available literature on M. bovis infection from Pakistan using Google scholar and PubMed databases. A total of 40 animal studies were identified using search terms: “bovine tuberculosis in Pakistan, bTB, Pakistan, Mycobacterium bovis in Pakistan, M. bovis in Pakistan”; while seven human studies were identified using the terms: zoonotic tuberculosis in Pakistan’, ‘M. bovis in humans Pakistan’, ‘zTB in TB patients in Pakistan”. We have summarized all these studies to identify critical risk factors involved in transmission of bTB among animals and humans. Despite lack of comprehensive and geographically representative studies, the literature suggests a varying prevalence of bTB in animals, ranging from as low as 2% to as high as 19%. Regarding zTB prevalence in humans, estimates range from 1.5% to 13% in high-risk group of farm and abattoir workers, with notably higher percentages in extra-pulmonary TB cases. The review also addresses the challenges that Pakistan faces in formulating an effective policy for the control and eradication of bTB. We conclude with one-health based recommendations as a way forward for controlling TB caused by M. bovis in cattle and humans.

Although M. tb primarily causes TB in humans; however, it is also occasionally isolated from diseased cattle and buffaloes in several countries of Asia and Africa [6][7][8][9][10].M. bovis also exhibits an extensive range of hosts, encompassing domestic animals, livestock, wildlife, and humans [11].Transmission among animals is thought to mostly occur through the respiratory route; however, vertical transmission via the digestive route from an infected mother to calf has also been reported [12,13].This disease prompts substantial financial losses in animals worldwide.The financial losses are estimated to reach 3 billion USD each year worldwide due to loss of production, culling of animals and trade impediments [14,15].
M. bovis is zoonotic in nature causing zoonotic TB (zTB) in humans.The transmission of the bacteria is linked to human-animal proximity, lack of sanitation, consumption of unpasteurized milk and uncooked meat [16][17][18].This makes it an important public health threat for lowand-middle income countries (LMICs).In Pakistan, 63% of the population lives in rural areas [19] and 62% of these people are either directly or indirectly involved with livestock.It has been reported that M. bovis can contribute up to 15% of human TB cases in LMICs, compared to <1% in high income countries [4,18,[20][21][22].Concurrently, Pakistan is ranked 5th among human TB high-burden countries worldwide and alone accounts for 61% of the TB burden in the WHO Eastern Mediterranean Region [23].Each year over 500,000 new human TB cases are reported in Pakistan [23].However, national-level data on the prevalence of bTB and the contribution of M. bovis to the incidence of human TB is unavailable.The symptoms and tissue lesions caused by M. bovis infection in humans cannot be distinguished from those caused by M. tuberculosis [4,24,25].Moreover, zTB is frequently extra-pulmonary, which complicates accurate diagnosis using conventional human TB diagnostic tests; leading to misdiagnosis or missed diagnosis of zTB [26].Despite its immense economic and public health importance, there are currently no surveillance or control programs in place against bTB in Pakistan.In this manuscript, we seek to fill this paucity of knowledge by providing an upto-date overview of bTB disease status in animals and its contribution to human TB cases in Pakistan.The review in addition, highlights the challenges and proposes a way forward for control.

Current status of bTB in Pakistan
Bovine TB is endemic in Pakistan and was first reported in 1969 in district Faisalabad with a prevalence of 6.72% in dairy animals [27].For this review, a literature search was conducted for all the studies described until 2023 on bTB in Pakistan.The strategy (presented as flow chart, Fig. 1) involved selecting all the published literature, in English language, from Google scholar and PubMed databases with the search terms: "bovine tuberculosis in Pakistan, bTB, Pakistan, Mycobacterium bovis in Pakistan, M. bovis in Pakistan".
The search led to the identification of 40 studies (all of them were summarized and have been presented in literature review matrix in Table 1).The studies available online were conducted over a period of two decades, even though research on bTB had been conducted earlier but was not available on online search portals.These 40 studies reported data from overlapping district locations accounting to a total of districts, with most studies being conducted in Lahore (n = 11), followed closely by Faisalabad (n = 8), Tando Allahyar (n = 4), Peshawar (n = 4) and Okara (n = 4) (supplementary fig.1).The 19 districts covered testing sites.On mapping the study sites (Fig. 2, left), three main clusters were formed (Fig. 2, right), one in Khyber Pakhtunkhwa (KPK), the second in Northern Punjab and the third in Sindh, leaving behind Southern Punjab, Baluchistan, Gilgit Baltistan as well as Azad Jammu and Kashmir.The clustering patterns and the affiliations of the corresponding authors in those clusters suggests that the majority of studies were performed near veterinary educational institutes that had academic expertise as well as laboratory facilities to conduct the required research.The data points were further parsed based on species tested in each study (Fig. 3).The data set from Pakistan showed that out of testing sites, 19 sites tested buffaloes only, 13 targeted cattle, 19 sites included cattle as well as buffaloes, two sites tested cattle, buffalo, goat (or) sheep, and three sites tested captive wild animals (including deer, chinkara, antelope, black buck, gazelle, goral, nilgai, urial and zebra) (Fig. 3).
Table 1 summarizes all studies conducted on bTB in Pakistan along with their study designs, sampling/approaches used and major conclusions drawn by the authors.Most studies adopt a similar approach, reporting prevalence estimates using a variety of diagnostic approaches.While we understand the funding constraints for the local researchers, many studies lack a proper study design and comparison to goldstandard mycobacterial culture for confirmation.Nonetheless, these studies suggest that the prevalence of bTB is <10% in many areas, while exceeding 10% in other locations, with estimated rates varying from 2% to 19.3%.Differences in bTB prevalence arise from diverse factors, including geographical variations influenced by farm management conditions [67].Natural susceptibility levels also vary among species and breeds, as highlighted in two studies [46,68].Additionally, the variation in bTB prevalence is influenced by topography, climate, weather patterns, and the presence of diverse animal species including wildlife.Notably, these estimations often stem from limited studies rather than comprehensive, geographically representative investigations [69].The listed studies also suggest that the prevalence of Fig. 1.Flow chart depicting the structure of this review.Aim and the data search strategy has been described with the categories of information summarized from all the identified studies.Comprehensive analysis of risk factors, current challenges and control recommendations have also been included.bTB is increasing at well-established government production farms, which is alarming [46,53].Different risk factors identified in studies conducted in Pakistan include the age of the animal, close animal sheltering, number of calving, origin of animals (brought in by purchasing from other locations) and herd size.
Studies conducted in Pakistan have also shown that bTB is present in captive wild animals.A study conducted recently proved that bTB is present in zoo animals with prevalence range of 3.78% to 8.1% depending on diagnostic tool employed [28].Similarly, results of a 2019 study on captive antelopes, which belong to the Bovidae family, demonstrated an M. bovis infection incidence of around 30%, suggesting a high affinity for bTB in these animals [36].In 2015, a study conducted on zoo animals belonging to bovidae, cervidae and equidae family indicated an overall prevalence estimate of 3.3% [57].These studies suggest that bTB is present in captive wildlife as well as zoo animals, which poses immense challenge of spread of bTB not only to animals but also people working at these facilities and visiting as tourists.
The studies reported in the current review used a variety of TB diagnostic tools such as the single intra-dermal tuberculin test (SITT; n = 7), comparative intra-dermal tuberculin test (CITT; n = 23), PCR (n = 14), rapid bovine antibody test (n = 3), ELISA (n = 5), Ziehl-Neelsen (ZN) staining (n = 10), culture (n = 7), and gross examination of visceral organs especially lungs and liver (n = 1) (supplementary table 1).Of the 40 studies, 20 studies employed only one form of test while the remaining 19 studies used a combination of tests to determine the status of bTB in animals.On further exploration of the publications, only seven studies used mycobacterial culture to confirm the presence of live bacteria (supplementary table 1).In addition, molecular tests were only employed in few research studies where sophisticated laboratories were accessible [33].Therefore, variable prevalence of bTB was reported by different studies: making it difficult to ascertain the true picture of bTB in Pakistan.The situation is further complicated by the fact that most studies utilized convenience sampling that can only provide an estimate rather than the true prevalence of the disease.Nonetheless, the studies provide a starting point for the government agencies to initiate an integrated surveillance study to ascertain the true prevalence of bTB.Overall, it is not possible using the currently available data to accurately assess the impact of bTB on either the bovine or human population (next sections) in Pakistan.

Status of zoonotic tuberculosis due to M. bovis infection in Pakistan
Research articles that determined causative contribution of M. bovis in human TB cases were searched using Google scholar and PubMed with the following inputs: 'zoonotic tuberculosis in Pakistan', 'M.bovis

Risks factors for zTB in Pakistan
There are many factors which exacerbate the spread of M. bovis to humans and M. tb from human to animals (reverse zoonosis) in LMICs [78].In their studies, Desta et al., [79] and Kouengoua et al., [80] identified several direct and indirect factors that can influence the spread of tuberculosis between animals and humans.These include demographics like age and gender, along with dietary habits such as consuming unpasteurized milk and meat.Close contact with livestock also plays a role, whether through shared living spaces, common water sources, or direct interactions.The presence of tuberculosis within a household, either human or animal, further increases the risk.Practices like spitting indoors and using manure in animal feed can also contribute to transmission.Furthermore, poor ventilation in homes and a lack of awareness about TB transmission modes can hinder control efforts.
In Pakistan, people in rural areas often have traditional livestock housing systems with close contact between humans and animals, thereby increasing the risk of zTB transmission from infected animals [81].Similarly, sale and purchase of infected animals in the market is found to be a significant risk factor [31].Co-sleeping in poorly ventilated areas with animals and consumption of unpasteurized milk and dairy products in rural Pakistani settings is quite common [74,81].In fact, most people in rural areas who are already at high risk due to close contact with infected animals prefer drinking raw milk, which further increases the risk of M. bovis transmission [56,82].Yet there is no mandatory pasteurization law in Pakistan to prevent transmission of M. bovis through contaminated milk.Currently only 6% of total milk produced is pasteurized [83].In 2018, Punjab Food Department enacted a law called 'Minimum Pasteurization Law' in Punjab, which to date has not been implemented in the country [84].
Another important risk factor for transmission of M. bovis to humans is through handling of contaminated carcasses at abattoirs [85].Unfortunately, the awareness level in slaughterhouse workers and animal owners regarding the risk of zoonosis is very low in Pakistan: as demonstrated by a study conducted at abattoirs in Karachi reporting that only 15% workers and herdsmen knew about bTB and only 30% workers were taking some precautions such as wearing of personal protection equipment and washing their hands [81].These risk factors have been observed globally: in Mexico 11.8% of sputum samples from cattle farm    workers were positive for M. bovis [86], while in Argentina, 65% of M. bovis-infected patients were found to have occupational exposure [87].
The role of National Tuberculosis Control Program (NTP) in humans becomes important under current conditions.Yet the NTP in Pakistan has not even acknowledged the existence of zTB and its contribution to the TB burden in a largely agrarian country [88].The absence of legislation mandating pasteurization, coupled with indoor animal housing practices and insufficient awareness within susceptible populations, has afforded M. bovis unchecked transmission to humans, thereby posing enormous health challenges to public health.

Challenges in control of bTB and zTB in Pakistan
Developed nations (global north) have successful programs that have reduced infections in animals to <1% at the herd level.Unfortunately, many LMICs lack similar efforts since it requires knowledge of data on prevalence and associated risk factors in each country.Absence of systematic surveillance and incomplete data on disease incidence and prevalence results in lack of attention and resources from policymakers [24,82,[89][90][91].Furthermore, compromised field performance of the available diagnostic tests for bTB i.e., low sensitivity and specificity, is also a challenge [92][93][94].Bovine TB prevalence is primarily concentrated in specific regions where poverty intersects with high population density, a predictable outcome for an illness that thrives in areas with limited resources to prevent bTB transmission [95,96].
Pakistan is among the LMICs that have yet to develop a surveillance program to effectively determine the prevalence of bTB in animals [30,75].The veterinary education establishments such as University of Veterinary Animal Science Lahore and University of Agriculture Faisalabad have conducted small scale studies to determine the prevalence of bTB (Table 1).Yet, the Ministry of National Food Security and Research of Pakistan does not have programs to conduct active surveillance of bTB even though they have the required infrastructure in place that was previously developed during the eradication of Rinderpest and maintained for the current control efforts being targeted towards Foot-and-Mouth Disease (FMD) control.
Majority of the bTB studies have been conducted around metropolitan cities like Karachi and Lahore; where animals have been removed from city centres and gathered in the peri-urban areas in dense dairy colony structures.This facilitates the transport of milk directly to consumers within a short frame of time.The forced clustering of animals in small areas has likely led to ideal conditions for the increased transmission of M. bovis in animals.Indeed, two studies have demonstrated significantly high prevalence of bTB in both animals and milk samples [41,52].This is worrisome since the dairy animals that are brought to the colonies get infected and at the end of the lactation-cycle are either sent to the slaughterhouse or back to their original villages, facilitating transmission of bacteria to different parts of the country.
A significant challenge in control of bTB in animals is the lack of knowledge regarding the dynamics of M. bovis infection in wildlife.Wildlife hosts such as white-tailed deer in Michigan (USA), Eurasian badgers in the UK, brush-tailed possums in New Zealand etc. are considered complicating factors in control of bTB in developed countries [97].In Pakistan, no study till date has assessed the prevalence of bTB in wildlife species while only a handful studies have reported prevalence of bTB in captive wildlife (Table 1).Lack of bTB data from both captive and wildlife species also makes it difficult to assess their potential role (if any) in transmission of bTB to farm animals.
Another important challenge is the non-existence of government policies for the control of bTB.Policies such as those related to animal movement across borders, insufficient funding for culling, lack of quarantine facilities, shortages of meat inspectors and veterinarians in slaughterhouses; all contribute to a hostile environment for bTB control [98].Pakistan has porous borders with Afghanistan and Iran leading to frequent cross-border animal and human movement, which is very serious risk factor for spread of infectious diseases like bTB [38,70].This, combined with lack of a surveillance program and a coordinated effort between veterinary research and educational establishments in the country to conduct a thorough research are major hurdles in initiating control of bTB.
So far, the challenges described above were specific to control of bTB in animals in Pakistan.Yet the zoonotic nature of this disease with ~ 10% contribution to human TB cases in LMICs [22], makes it imperative that we highlight the challenges faced in preventing transmission of the bacteria from infected animals to the human population, since the clinical presentation of tuberculosis caused by M. bovis and M. tuberculosis is indistinguishable.The African region has the highest cases of zTB (72700), followed by Southeast Asian region (46700); yet they lack sufficient resources, laboratory infrastructure as well as technical expertise for differentiating M. bovis from M. tuberculosis at essentially all healthcare levels [99][100][101].Additionally, there is absence of training and lack of knowledge among occupationally exposed groups about the zoonotic potential and spread of M. bovis infection to humans [75].According to studies conducted to evaluate knowledge and awareness of different exposed high risk groups, it was found that none of the abattoir workers received formal training and majority of them do not practice any precaution such as wearing PPE and washing of their hands [74,75].In another study, it was established that abattoir workers are more affected by zTB due to direct contact and continuous exposure [74].The condition is worsened because M. bovis inherently exhibits resistance to pyrazinamide [102].In a Peshawar-based study, 12 patients were diagnosed with M. bovis infection and 11 of those patients' demonstrated resistance to pyrazinamide treatment.This underscores a significant challenge posed by antimicrobial resistance associated with zTB [70].
It is crucial that bTB in animals is controlled to prevent its transmission to humans.On the human health side, M. bovis is neither considered a risk factor for human TB, nor are there any guidelines for its control in the newly formed "National Guidelines for the Control of Tuberculosis in Pakistan-2019" [88].This is irrespective of the numerous studies having reported the impact of M. bovis on human TB (zTB) cases in veterinarians, livestock workers, butchers, abattoir workers and other high risk groups Table 2: List of studies conducted in Pakistan to access contribution of M. bovis in human TB cases (zTB) [70].Lack of concentrated efforts by animal and human health departments so far has led to a precarious situation that needs immediate attention.

Future perspectives for bTB and zTB control in Pakistan
The way forward for Pakistan to control bTB is to carry out comprehensive surveillance to determine incidence and prevalence of M. bovis infection in cattle and other hosts, along with risk assessment of zoonosis and reverse zoonosis.The data regarding prevalence of bTB and share of zTB in human TB should be compiled.This will be helpful in efficient allocation and management of resources [69] because the lack of data is the main hurdle in seeking policymakers' attention and provision of funding to fight the problem [103,104].
There is a need to identify and address research gaps in zoonotic and bovine TB to determine the true burden of TB caused by M. bovis and to unravel the epidemiology of disease at the human, animal, and environmental interface [26].This can be achieved through meaningful collaboration between local researchers and experts from developed world and international organizations working on TB control.This will also offer capacity building opportunity for the local researchers, enabling them to better design future research studies.Additionally, in endemic countries like Pakistan, epidemiological studies to determine which animal species, both domestic and wild, serve as hosts or reservoirs for M. bovis can be very informative.This process can identify areas of high prevalence, enabling the allocation of additional funds and efforts to those specific regions [105,106].
Routine surveillance of bTB in live animals through skin testing and blood tests can play vital role in control of disease.Animal owners should be incentivized for screening their animals for bTB and maintenance of disease free herds [104].Additionally, inspection of the animals prior to slaughtering and detailed post-mortem inspection (following the protocols implemented in UK and the EU) should be ensured to stop entry of meat of any infected animal into the food chain [107,108].This should be accompanied by tracing back to the herd to which the diseased animal belonged [101].
The effective control of zoonotic diseases is only possible by adopting multi-sectorial, one health approach [109,110].There is need to design a national control program with integrated 'One Health' approach through collaboration between public health and animal health departments with aim to develop control measures while considering human, animals, wildlife and environmental fronts [104,111].The success of the TB control program in humans in the USA is commendable, owing to collaborative efforts from states, businesses, and the federal government to control and eradicate the disease [104].Pakistan can establish a similar program through collaboration between the federal and provincial governments on both animal and human health sector.The program should emphasize surveillance to determine the prevalence of bTB and identify risk factors.Integrated and collective control efforts are imperative: including awareness among masses, necessary legislation, and its implementation along with uplifting socioeconomic status of the people.
The surveillance campaign to detect cases of human TB due to M. bovis should be strengthened by improved laboratory facilities, including polymerase chain reaction (PCR) and gene sequencing after culturing the isolates from patients [77].
Pasteurization and food safety laws should be introduced with strict implementation by adopting WHO food safety measures to break the cycle of transmission of M. bovis to the masses.This will require a public awareness campaign to educate people about the importance of pasteurizing milk and other dairy products [26].The control strategies can be made successful by educating the masses, especially the farmers about the economic and public health significance.
Field veterinarians and para-veterinary staff should be aware of the mode of transmission, range of hosts, clinical signs, and diagnosis of the disease [29,74].There is also need for implementation of standard operating procedures (SOPs), creating a national database, training of technical and non-technical staff for ante-mortem and post mortem examination along with educating animal handlers to break the cycle of zTB transmission [75,112].

Conclusion
The absence of comprehensive data for bTB and zTB in Pakistan has resulted in failure to develop a structured control and eradication program.Recognizing this critical gap, it is imperative to develop an effective policy that prioritizes the control and eradication of bTB and zTB.A One-Health approach is advocated, calling for collaboration among various departments such as livestock and dairy development, ministry of national health services, public health, agriculture, and food security to collectively achieve targeted goals.Furthermore, there is a pressing need for active surveillance to accurately determine the prevalence of M. bovis and its contribution to the human TB burden.This data is crucial not only for understanding the disease's national status but also for identifying pertinent risk factors.Such insights are integral in the formulation of a comprehensive control program against the disease and for efficient resource allocation.In conclusion, representative data is pivotal for addressing the substantial challenge posed by bTB to both livestock and human population.The establishment of a robust control program, informed by accurate prevalence data and a One-Health approach, is imperative for mitigating the impact of this infectious disease in Pakistan.

Fig. 2 .
Fig. 2. The maps demarcate the political borders of Pakistan.Left): The blue location icons represent the cities in which the 40 studies were conducted.Bovine tuberculosis prevalence studies were performed in 19 districts, with most districts targetted multiple times over the course of two decades.Right): Data points of 40 studies.Each location icon is represented by either a cow or a deer icon with different background colours.Blue colour represents studies that tested only cattle, black colour represents studies that only tested buffalos, pink colour represents studies that tested buffaloes, cattle, goat and (or) sheep, orange colour represents studies that tested both buffaloes and cattle.Green colour deer icon represents studies that tested different wildlife species.The large circles represent the three clusters of studies identified in this review paper.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Fig. 3 .
Fig. 3. Zoomed images of the three clusters created by the 40 studies performed in Pakistan to evaluate prevalence of bovine tuberculosis.Cluster 1: Top left, Cluster 2: Top right, Cluster 3: Bottom left.Note that the location represented on the maps by each icon is that of the city and not the exact farm where the animals were tested.

The prevalence of M. bovis in milk samples was found to be 3.73, 4.04 and 5.29% by ZN staining, culture and PCR respectively
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