Bovine Tuberculosis ( bTB ) : Prevalence and Associated Risk Factors in Large Ruminants in the Central Zone of Khyber Pakhtunkhwa , Pakistan

Asad Ullah1, Umar Sadique Khattak2, Sultan Ayaz1, Muhammad Subhan Qureshi2, Imad Khan1, Ibad Ullah Jan2, Irfan Khattak1, Raheela Taj3, Sahar Nigar4, Naimat Ullah Khan1, Mumtaz Ali Khan 5 and Muhammad Luqman Sohail6,* 1College of Veterinary Sciences and Animal Husbandry, Abdul Wali Khan University, Mardan 2Department of Animal Health, The University of Agriculture, Peshawar, KPK 3Institute of Chemical Sciences, The University of Peshawar, Peshawar 4Centre of Biotechnology and Microbiology, The University of Peshawar, Peshawar 5University of Veterinary and Animal Sciences, Lahore 6University College of Veterinary and Animal Sciences, The Islamia University of Bahawalpur, Bahawalpur Article Information Received 25 February 2018 Revised 28 March 2018 Accepted 05 June 2018 Available online 19 November 2018


INTRODUCTION
B ovine tuberculosis (bTB) is an infectious zoonotic disease caused by Mycobacterium bovis (M.bovis), having a wide range of hosts (Okeke et al., 2016;Batool et al., 2017).In an area where bTB is endemic, uncontrolled or partially controlled, aerosol is the key route of transmission for the humans along with ingestion of contaminated water, meat and milk (Awah-Ndukum et al., 2016).Workers handling carcasses of infected animals get infection through skin and mucous membranes (de la Rua-Domenech, 2006;Michel et al., 2010).Bovine TB outbreaks trigger large economic losses to agricultural economies by curtailing international trade of animals and animal products, causing reduction in milk and meat production, compromising fertility, massive control and eradication strategies, and increased public health risks (Dejene et al., 2016).Although some countries managed to eradicate bTB through regular testing and removal of infected animals, the occurrence of bTB is higher in developing countries since the control and eradication policies are not sufficiently executed (Amanfu, 2006;de la Rua-Domenech, 2006).More than 125 countries reported prevalence of M. bovis infection in cattle herds to OIE, during 2005to 2008(Michel et al., 2010).Literature reported higher prevalence of disease with increasing age (Ameni et al., 2007), while at herd level, herd size and movement of animals were associated with the bTB (Kazwala et al., 2001).New animals must be intro-

A B S T R A C T
duced from non-endemic areas to minimize the risk of new infection in the herd (Reilly and Courtenay, 2007).
Endemicity of bovine TB among animals has been attempted to be controlled using test and slaughter strategy.For past decades, infected or carrier subjects are identified using the comparative cervical intradermal tuberculin test (CCIT), also termed as the tuberculin skin test (TST).This test is based on delayed-type hypersensitivity reaction, evoked by purified protein of Mycobacterium tuberculosis called tuberculin (Monaghan et al., 1994).Despite certain limitations, the importance of CCIT in control of TB can never be ignored.It is simple and does not demand extensive training and special laboratory aids for its procedure and interpretation of results (Mohamed, 2017).
Extensive struggle has been made around the globe to eradicate bTB because of its zoonotic and economic consequences.In developing countries, precise data on prevalence of infection is needed to improved surveillance programs and control of bTB (Moiane et al., 2014).Pakistan faces a serious dearth of epidemiological data epidemiology on bTB, rendering the surveillance and eradication targets of M. bovis infection, unaccomplished.National Tuberculosis Control Program, Pakistan (NTCP) doesn't address the transmission of bTB to humans, adding to the difficulty.
This study was designed and conducted to determine prevalence and associated risk factors of bTB caused by M. bovis in most populated and centrally located districts of Khyber Pakhtunkhwa in Pakistan.

Study design and area
A cross-sectional epidemiological study was conducted to determine the prevalence of M. bovis and risk factors associated with presence of infection in large ruminants of urban and rural areas of central zone of Khyber Pakhtunkhwa, Pakistan.This study was carried out in the central zone (districts Peshawar, Nowshera, Charsadda, Mardan and Swabi) of Khyber Pakhtunkhwa, Pakistan.A total of 2400 asymptomatic large ruminants (1225 cattle and 1175 buffalo) were tested by convenience sampling approach (Tyrer and Heyman, 2016) using CCIT from January 2016 to December 2016.

Data collection
To identify the associated risk factors, semistructured interviews were conducted after formal written consent from herd owners and livestock workers.A structured questionnaire was designed to gather animal and herd level information.Animal level data included information regarding location of the animal, species, age, gender and lactation status of the animal, while herd level data included information regarding herd size, procedure of animal procurement in the herd, breeding of animals, housing and feeding management practices and presence of various animal species in the vicinity of tested animals.Trained local government officials, conversant in local and the national language assisted with data collection.Information regarding contact of tested animals with various other species of the animals was gathered from herders.Presence of wild animal species in and around the grazing area and water source was described as contact with wild animals.The questionnaire template used, can be obtained from the corresponding author on request.

Comparative cervical intradermal tuberculin test
Comparative cervical intradermal tuberculin test was performed, briefly 0.1ml (25,000 IU) avian purified protein derivative (a-PPD) and 0.1ml (20,000 IU) bovine purified protein derivative (b-PPD) were injected at 12 cm distant points on left lateral side of neck.After 72 h of injecting a-PPD and b-PPD, the thickness of skin-fold at each injection site was measured using digital calipers.Animals were considered positive if the fold to the skin was thickened >4 mm at b-PPD injection site as compared to a-PPD injection site.While, if skin thickness at a-PPD injection site is 4mm higher than that at b-PPD injection site, the animal was considered a reactor.

Statistical analysis
The collected data was entered using the Microsoft Excel version 2013, and then exported to the Statistical Product and Service Solutions (SPSS) version 20.0 to establish associations between positive animals and the risk factors at 95% level of confidence.Summary statistics, Pearson's chi-square test and multivariable logistic regression were done to identify associations between selected risk factors and CCIT positivity.The prevalence was calculated by dividing the number of positive animals by the total number of tested animals.Variables with a p-value of <0.05 on Pearson's chi-square test (χ 2 test) were included in multivariable models.Finally, logistic regression was used to analyze the effect of potential risk factors.
Respondents were also interviewed to determine their knowledge about bovine tuberculosis.Around 33.68% herd owners mentioned about persistent cough (>3 weeks) as an indication of tuberculosis.Similarly, 75.26% herd owners believed weight loss a symptom of tuberculosis while 23.68% herd owners mentioned coughing up sputum or blood as an indication of tuberculosis.On the other hand, many respondents inaccurately linked some symptoms such as vomiting (7.37%) and diarrhea (10%) with TB.Livestock owners identified chest pain, fever and chills as signs and symptoms of tuberculosis (23.16%, 80% and 47.89%, respectively).Some animal handlers identified general weakness and loss of appetite (76.32% and 80.53%, respectively) as indications of TB.All three primary signs (persistent cough, weight loss and fever) of TB were mentioned only by 7.1% of respondents showing poor awareness among farmers and animal handlers (Fig. 1).
In this study, 190 livestock farmer were interviewed about the zoonosis of bTB.Almost 97.9% herd owners had some kind of knowledge regarding tuberculosis while 2.1% had not heard of the infection.Less than one third (30.1%) of the farmers were aware of the zoonotic nature of the disease.Unpasteurized milk was thought to be the source of infection by 37.4% of the dairy farmers.Similarly, 17.4% and 11.1% knew about the air and raw meat, respectively as the source of infection (Table IV).

DISCUSSION
Pakistan is a sub-tropical country and Central zone of Khyber Pakhtunkhwa (KP) is located in continental grasslands and has five administrative units/districts i.e.; district Peshawar, Nowshera, Charsadda, Mardan and district Swabi lying at the porous afghan border, with frequent movement of animals and humans across the border.Dearth of data on epidemiological dynamics of bTB in region can surge serious menace of infection among subjects in the central zone of KPK.Constant investigation of M. bovis infection and associated risk factors in large ruminants in the region is mandatory to design effective control measures.During the current investigation, the overall prevalence for M. bovis infection was 5.88% in large ruminants.Within Pakistan, several studies reported variation in prevalence i.e. 3% (Javed et al., 2010), 2.2% (Javed et al., 2009), 1.7% (Ifrahim, 2001), 9.6% (Mumtaz et al., 2008), 10.6% (Khan et al., 2008), 11.3% (Javed et al., 2012) and 12.72% (Khan and Khan, 2007).This variation in the prevalence of infection is attributed to differences in husbandry practices and higher numbers of crossbreds as well as exotic animals at the dairy farms (Memon et al., 2017).No significant difference was found in the prevalence among the different study regions because of similarity in animal husbandry and management practices.
Outcomes of multivariable statistical assessment revealed that 5 years or older animals are at higher odds of getting infection.Similar findings were reported by previous studies conducted in similar socio-economic settings (Javed et al., 2006(Javed et al., , 2009(Javed et al., , 2012;;Khan and Khan, 2007;Moiane et al., 2014).Prevalence increased with the age of livestock animals because of the lengthier exposure to the etiological agent over time among older animals (Dejene et al., 2016).In endemic scenarios, older animals were exposed to infectious agent for longer periods of time, increasing the prevalence with increasing age (Mahmud et al., 2014).Cleaveland et al. (2007) proposed reactivation of latent infection of tuberculosis in older age to be the cause of greater proportions of human tuberculosis but this is yet to be confirmed in animals.Larger herd size was found to be significant risk factor associated with bTB, which might be because of presence of higher number of animals in larger herds (Proaño-Perez et al., 2009).Dairy farmers improve the herd size to increase farm yield which can lead to overcrowding causing increased animal-toanimal transmission (Ghebremariam et al., 2016).This study indicated that the positivity of bTB was meaningfully linked with buying and induction of new animals into the farm.These conclusions are in line with the findings of several studies as piloted in England (Gopal et al., 2006) and Uganda (Oloya et al., 2007;Kazoora et al., 2014), which can be attributed due to their interaction with various animals during their stay at different dairy farms or at live animal markets.The animals housed indoor during the night were found at higher risk of being CCIT positive as compared to those who were housed outdoor during night.These outcomes may be due to the aerosol route of transmission among closely tied animals.Hence, extra risk of close contact of susceptible animals might be the main cause behind greater ratio of CCIT positive animals when housed indoor.Our results are in line with the findings of Katale et al. (2012) who reported substandard ventilation as the cause of infection transmission.
The learning rate is relatively low in emerging nations including Pakistan (Mangesho et al., 2007;Proaño-Perez et al., 2009;McGeary, 2008).Different seminars, wakefulness promotions, learning sessions and use of print and electronic media must be an important part of TB control programs.

CONCLUSIONS
The conclusions of current research provide evidence about the prevalence and possible risk factors linked with positivity of M. bovis infection in large ruminants (cattle and buffalo) in Central Zone of Khyber Pakhtunkhwa, which need to include M. bovis infection in NTCP.

Fig. 1 .
Fig. 1.Distribution of signs and symptoms of bTB known to the livestock farmers in the Central Zone of Khyber Pakhtunkhwa, Pakistan.