Prevalence of tuberculosis among southern Zambian cattle and isolation of Mycobacterium bovis in raw milk obtained from tuberculin positive cows

Aim: To determine the prevalence of tuberculosis among traditional milking cows in southern Zambia and isolation of Mycobacterium spp from the tuberculin positive cow's milk. Materials and Methods: A total of 1,025 cows originating from traditional cattle sector, in Mapepe, Magoye, Monze, Batoka, GARTBatoka, Kalomo areas of Zambia, supplying major quantity of milk to milk processors, were tested for bovine tuberculosis using single comparative intra-dermal tuberculin test during 2011 and 2012. Milk samples obtained from 16 bovine tuberculin reactor cows were cultured for isolation of Mycobacterium spp and those showing growth of Mycobacterium spp. were identified through biochemical tests of the culture. Further confirmation and species differentiation of the Mycobacterium spp. isolates was done using the loop-mediated isothermal amplification system and multiplexpolymerase chain reaction Results: 27 (2.6%) of the cows tested were found tuberculin reactors, 9 cows (0.87 %) gave inconclusive reaction and 989 (96.48%) were non-reactors. Three milk samples (18.7%) out of the 16 tuberculin reactor cow's milk when cultured and upon molecular analysis, were found positive for presence of M. bovis indicating these positive cows were shedding M. bovis in their milk. Conclusion: The isolation of M. bovis in freshly drawn milk from the tuberculin positive reactor cows is being reported for the first time in Zambia. Bovine tuberculosis is an animal and human health risk in the traditional dairy herds supplying milk to the Zambian population especially in the informal market and needs attention of the public health and veterinary authorities.


Introduction
has drastically reduced and in some cases the transmission of BTB to humans is controlled [6].In developing Zoonotic tuberculosis is a chronic, infectious, contacountries particularly in low income group, bovine gious, debilitating disease caused by Mycobacterium tuberculosis is still prevalent and is responsible for bovis, that has become a resurgent problem in animals significant economical loss in animal production and humans in a number of developing countries.
through reduced milk yields and low reproductive Tuberculosis has been reported to be endemic in both performance [7].The global prevalence of human domestic and wild animals in Zambia [1,2,3].Bovine tuberculosis due to M. bovis has been estimated at 3.1% tuberculosis (BTB) a major zoonotic disease with of all human tuberculosis cases [8].In countries where worldwide distribution especially in developing majority of poor rural pastoral communities depend on countries where disease is endemic, is an important for cattle for their livelihood, BTB is reported to be a both economic and public health reasons [4].In cattle, constant threat due to lack of pasteurization of milk and the disease is mainly caused by the bacteria M. bovis.
tradition and culture of consuming raw milk and its Mycobacterium tuberculosis in cattle accounts for a other preparations.In most African countries, consumsmall proportion of usually sub-clinical cases [5].
ption of unpasteurized milk is a regular practice [9].Bovine tuberculosis used to be a serious zoonosis and Exact data on the prevalence of human disease due to represented a tremendous public health problem in M. bovis in Zambia and other developing countries is developed countries mainly through consumption of absent or limited owing to technical problems posed by contaminated unpasteurized milk.However after the identification of this species such as trained personnel disease was controlled in cattle through designed test and laboratory facilities.This lack of information on and slaughter schemes together with milk pasteuri-BTB in many African countries including Zambia has zation and public awareness and education, incidence lead to reduced attention to this infectious agent (M.bovis) in terms of prioritizing resources devoted to its research capacity, control of animal diseases and public education.Mwachalimba et al [10] in his recent

Prevalence of tuberculosis among southern Zambian cattle and isolation of Mycobacterium bovis in raw milk obtained from tuberculin positive cows
publication from Zambia reported that using test and cows aged between the range of 3 -10 years originating slaughter of livestock and promotion of milk pasteuri-from Mapepe, Magoye, Monze, Batoka, GART-Batoka zation will reduce the zoonotic transmission of BTB and Kalomo areas of southern Zambia, supplying and reduce the cost of treatment among humans.
major quantity of milk to processors originating from Bovine tuberculosis is known to be a major cause of traditional cattle sector, were tested for bovine human extra pulmonary tuberculosis and is of particular tuberculosis.For the determination of the prevalence of concern in developing countries where milk is often BTB in cattle, the single comparative intra-dermal not pasteurized or boiled before use.into sterile pre-cooled McCartney bottles after cleaning, and in all these areas people consume mostly raw milk washing and disinfecting the udder to avoid contaor sour milk made from raw milk without boiling.mination from environment.Samples were taken to Zambian human population also carries the public health laboratory of The School of Veterinary tuberculosis burden in Africa.The emergence of drug Medicine at University of Zambia in a cooler box with resistant strains of Mycobacterium spp., the rise and 0 ice and kept refrigerated at 4 C until analysis within 24 synergism of HIV/AIDS infection with tuberculosis, hours.Milk samples were centrifuged at 3,000 rpm for poverty and neglect of bovine tuberculosis control 15 minutes and the supernatant was discarded.The programs have contributed immensely to the sediments were suspended in 2 ml of sterilized resurgence of TB [16].While M. bovis is a major cause physiological saline solution.To the suspension, equal of pulmonary tuberculosis in cattle, it is also the volume of sterilized 4-N sodium hydroxide solutions primary cause of extra-pulmonary tuberculosis in humans and one drop of 0.05% phenol red indicator were added where milk is consumed fresh and unpasteurized [9and the mixture was incubated for 30 minutes at 37ºC.11].The clinical signs of this infection in humans are Finally the samples were neutralized with sterilized 4hardly differentiated from those caused by the classical N hydrochloric acid solution and centrifuged at 3,000 human type and are often difficult to assess clinically.rpm for 15 minutes, and sediment was used for micro-The most common practice and way of diagnosing scopic and cultural examination.Among 27 tuberculin tuberculosis is by radiology and sputum examination, positive cows, milk was obtained from only 16 cows and both cannot confirm what type of tuberculosis that were in lactation, for milk smear examination and bacterium is involved in the case.Despite this bacterial culture.resurgence of TB in animals and man, and the Pre-culture microscopic examination of the sediment: consumption of large quantities of raw and sour milk in From sediments of each milk sample, two smears were Zambia, no effort has been made to estimate the gravity prepared, dried, slightly fixed over flame and stained of the risk and its threat to public health.
with Acid Fast Stain (Ziehl-Neelsen Stain).The stained There has been no attempt towards isolation of smears were examined under oil-immersion lens of the Mycobacterium species from raw milk in Zambia.
microscope for demonstration of Acid Fast Bacilli Therefore, this study was carried out to determine the (AFB) which stain as bright/rose red rods with a blue prevalence of tuberculosis among traditional cattle background.used for milk production.Furthermore isolation of

Culture of the sediment and species differentiation:
Mycobacterium species in the milk obtained from One ml aliquots of the sediments from each milk tuberculin positive cattle was carried out, with a view sample were spread on the surface of each of the LJ to highlight the risk of consuming raw milk.medium slants with glycerol and LJ medium slants This growth also included the sample that was positive These isolates were further subjected to biochemical for AFB (sample MG-2) on pre culture sediment.The tests for characterization and identification as number of colonies on the LJ-P media varied from 1-3. described by Kent and Kubica [19].Growth on LJ-All three post culture smears were positive for AFB.LJ pyruvate is suggestive of M. bovis while those on LJ slants containing glycerol did not show any growth at th with glycerol is suggestive of M. tuberculosis, although the end of 8 week of culturing.All the 3 isolates were there was no any growth on LJ-G slants.Positive Nitrate reduction negative and Niacine paper strip test controls with LJ-glycerol and LJ-pyruvate with known production negative suggesting that isolates were most inoculated strain of H37Rv and negative controls on probably M. bovis (Table -2).LJ-glycerol and LJ pyruvate media without inoculation Only 1/16 (6.2%) of the tuberculin test positive were also maintained for comparison.Confirmation milk samples demonstrated AFB in the milk sample and species differentiation of the Mycobacterium spp.
while 3/16 (18.7%) of the tuberculin test positive milk isolates was done using the loop-mediated isothermal samples cultured, showed growth of M. bovis.Thirteen amplification system (LAMP) and multiplex-polymilk samples (81.2 %) from tuberculin positive cows merase chain reaction (PCR) respectively according to did not show any growth suggesting that these were Hang'ombe et al [20] on cultures showing growth.
probably not shedding TB bacteria in their milk.

Briefly, the genomic DNA from Mycobacterium
To confirm the Mycobacterium grouping of the bacterial cultures was prepared from colonies using isolates, the LAMP system was used for rapid detection DNAzol reagent (Invitrogen, Carlsbad, CA, USA).A of the Mycobacterium tuberculosis complex (MTC) loopful of colonies was suspended in DNAzol group.In this study, all the 3 cultures were confirmed as followed by mechanical disruption as described by belonging to the MTC group (Figure -2).On further Suzuki et al [21].The DNA was extracted according to analysis using the multiplex polymerase chain reaction the manufacturer's instructions and then dissolved in (PCR) for Mycobacterium species differentiation, the 50 µL TE buffer (10 mM Tris/HCL (pH 8.0) and 1 mM isolates were identified as Mycobacterium bovis EDTA).The extracted DNA was then subjected to the following the amplification of a 786 bp DNA fragment LAMP test and multiplex-PCR as described earlier by of the cfp32 region (Figure -3).Hang'ombe et al [20].

Discussion Results
Our estimated overall prevalence of bovine A total of 27 (2.6%) of the 1,025 cows tested were tuberculosis in the study area (southern Zambia) was found tuberculin reactors, 9 of them (0.87%) gave 2.6% which is lower than those reported by Malawi [22], Tanzania [23] and Uganda [24].The (1.4 %) TB reactos.Batoka and GART -Batoka did not reason for lower prevalence rate than those previously record any TB reactor.Out of the 16 milk samples reported in Zambia could be due to annual TB testing obtained from tuberculin positive cows and cultured, programme in place in our study area of southern one of them (MG-2) showed acid fast bacilli on pre-Zambia and elimination of reactors from milk supply culture sediment smear, while 15 did not show any acid chain as legally required.Monze had the highest fast bacilli.Of all the 16 milk samples sediment prevalence rate (5.1 %) followed by Kalomo (3.8%), .It is common practice among herdsmen GART-Batoka did not record any TB reactor.Regular to depend upon the raw milk from cows as their main testing and elimination of TB reactors, from herds food while staying in the flood plains and many times supplying milk for public consumption should be a they suckle milk directly from the udder of the cows.In regular practice supported by government and processors.
countries like Zambia where milk is not usually boiled We report for the first time isolation of M. bovis in rural areas before use, tuberculosis due to M. bovis from freshly drawn milk from 3 tuberculin reactor may possibly be the major cause of extra pulmonary cows from Zambia based on cultural, biochemical and tuberculosis in humans.In this study M. bovis was molecular tests.The definitive identification of the detected in freshly drawn cow's milk from three TB species of M. bovis is largely based on biochemical and reactors cows out of the 16 TB reactors and therefore molecular criteria which targets three genetic regions the chances for human infection are high since cow's that are specific to species belonging to the MTC [25, milk is regarded as one of the important foods in native 26].Similar reports of isolation of M. bovis from milk population of Zambia.In addition, people living in of cows has been published from elsewhere in Nigeria Africa are comparatively at high risk due to close [27], Turkey [28], India [29],Tanzania [30], Tunisia contact with animals and high incidence of HIV/AIDS [31], Iraq [32]and Brazil [33,34].
[35].Our study, for the first time confirms the presence From the study, it is quite clear that out of the six of zoonotic M. bovis in the milk of tuberculin reactor study areas in Zambia, Monze has higher prevalence cows in Zambia.This further suggests that the three TB rate of tuberculosis among cattle (5.1 %) (Table -1 autopsy examination demonstrating TB resembling bovis infection accounted for 1.6 % of the cases of lesions as high as 24.0 % [1] and 24.3 % [36].This tuberculosis in HIV patients globally, the detection of could be one of the possible reasons for comparatively this pathogen in cow's milk is worrisome as milk from high prevalence and isolation rate of M. bovis among cows forms the bulk of animal protein that is normally cattle originating from Monze area in our study as recommended for immune-compromised persons [11].
compared to other places studied.The presence of the zoonotic pathogen poses serious The study also shows that bovine tuberculosis is public health hazard to the herdsmen and other animal and human health risk in the traditional dairy consumers of raw milk and milk products made from herds supplying milk to the Zambian population, Materials andMethods with pyruvate.Cultures were incubated aerobically at 0 Tuberculin test: During 2011-2012, a total of 1,025 growth.The produced bacterial growth in slants with inoculated on LJ-P and LJ-G media, only 3 (18.7% ) th pyruvate that showed small, moist, creamy to showed growth on LJ-P media (one on 6 week of th yellowish, smooth, flat colonies after 4 weeks of culturing and two on 7 week of culturing) (Figure-1).incubation, were presumed to be M. bovis positive [18].
) and reactor cows, in our study were shedding viable M. culture positive cases were two (33.4%)out of the total bovis bacteria in their milk.The population consuming culture positive cases of three (66.6 %) from Monze raw milk and HIV/AIDs affected individuals are at only.Cook et al [12] reported prevalence of positive high risk of contracting the disease since the most reactors among cattle as 7.4 % from a study in Monze common form of milk consumption is raw fresh and alone.A good number of cattle population from Monze sour milk made from raw milk in rural areas of Zambia.30 -45% move to Kafue flats for 6 months during dry Sitima et al [15] in an experimental study in Zambia season (June-November) in search of water and reported the viability of M. bovis in traditionally made grazing and in the process mingle with Kafue lechwe sour milk.Young children in rural areas and the babies (Kobus leche kafuensis) an antelope with concentrated born from HIV/AIDS positive mothers are encouraged population of about 40,000 at one place in Lochinvar to be given other forms of milk including cow's milk to Nantional Park [1].These Kafue lechwe (Kobus leche avoid mother to child transmission of HIV/AIDS kafuensis) had tuberculosis prevalence based on through breast feeding.Considering the report that M.

Figure- 1 .Figure- 2 .
Figure-1.Growth of yellowish to creamish Mycobacterium spp colony on LJ-P media Figure-2.LAMP product observation under the UV light.Left to Right: Tube 1 and 2 are positive and negative controls respectively, while tubes 3, 4 and 5 are the Mycobacterium bovis isolates from raw milk.
It is a legal requirement from the of human tuberculosis is 707/100,000 [14].The processors that farmers should supply milk only from prevalence of BTB in Zambia among traditional cattle, TB and brucellosis negative cows. in selected cattle rearing areas has been reported to be Milk samples: 10 ml of milk from 16 tuberculin 6.8 % by Munyeme et al [2], 7.4 % by Cook et al [12], positive cows was obtained from udder while milking, 7.8 % by Sitima et al [15], 6.3 % by Muma et al [16],

Table - 2
. Results of milk smear staining and cultural characteristics showing growth of Mycobacterium spp.