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Bovine tuberculosis is more prevalent in cattle owned by farmers with active tuberculosis in central Ethiopia

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A case control study was conducted between October 2004 and April 2005 to determine the prevalence of bovine tuberculosis (BTB) in cattle in central Ethiopia relative to the tuberculosis status of their owners. A total of 174 farmers (87 with active
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  Bovine tuberculosis is more prevalent in cattle owned by farmerswith active tuberculosis in central Ethiopia Alemayehu Regassa  d , Girmay Medhin  a , Gobena Ameni  a,b,c,* a Aklilu Lemma Institute of Pathobiology, Addis Ababa University, P.O. Box 1176, Addis Ababa, Ethiopia b Armauer Hansen Research Institute, P.O. Box 1005, Addis Ababa, Ethiopia c Faculty of Veterinary Medicine, Addis Ababa University, P.O. Box 34, Debre Zeit, Ethiopia d Faculty of Veterinary Medicine, University of Hawassa, P.O. Box 05, Awassa, Ethiopia Accepted 25 June 2007 Abstract A case control study was conducted between October 2004 and April 2005 to determine the prevalence of bovine tuberculosis (BTB) incattle in central Ethiopia relative to the tuberculosis status of their owners. A total of 174 farmers (87 with active tuberculosis and 87 withno active tuberculosis), and 1041 cattle (506 owned by farmers with active tuberculosis and 535 by farmers without active tuberculosis)were included. The comparative intradermal cervical tuberculin test was used in cattle while clinical symptoms, chest X-ray and Ziehl-Neelsen staining were used for the diagnosis of tuberculosis in the farmers. In addition, mycobacterial culture, biochemical tests, anddrug susceptibility tests were performed for the identification  Mycobacterium  spp. from both humans and cattle.The prevalence of BTB was threefold higher (odds ratio [OR] = 4.2, 95% confidence interval [CI] = 2.79–6.2) in cattle owned by farm-ers with active tuberculosis (24.3%) than in those owned by farmers who did not have active tuberculosis (8.6%). Cattle owned by farmerswith active tuberculosis were four times more likely to have tuberculosis than cattle owned by farmers with no active tuberculosis. Fur-thermore, cattle owners who consumed raw milk were at greater risk ( v 2 = 14.1,  P   < 0.001, OR = 3.34) of having active tuberculosis thanthose who consumed boiled milk. Of the 42 human isolates, 31 (74%) were  Mycobacterium tuberculosis , seven (16%) were  Mycobacteriumbovis  while four (10%) were considered a typical mycobacteria on the basis of biochemical and drug sensitivity tests. Of the 11 cattleisolates, two (18%) were  Mycobacterium tuberculosis,  five (46%)  Mycobacterium bovis , and four (36%) were atypical mycobacteria.The prevalence of tuberculosis was higher in cattle owned by farmers with active tuberculosis than in cattle owned by farmers whodid not have active tuberculosis, which could suggest possible transmission of   Mycobacterium  spp. between cattle and their owners.   2007 Elsevier Ltd. All rights reserved. Keywords:  Bovine; Farmer;  Mycobacterium ; Prevalence, Risk factor; Tuberculosis Introduction Tuberculosis (TB) is an infectious disease caused bymycobacteria that have been a major health risk to humansand animals for more than a century. It is widely distrib-uted throughout the world affecting all age groups of humans and animals. Human TB of animal srcin, partic-ularly  Mycobacterium bovis  is becoming increasinglyimportant in developing countries (Cosivi et al., 1998). M. bovis  is one of the etiological agents of mammalianTB that is antigenically inter-related and is grouped inthe  M. tuberculosis  complex (Barwinnek and Taylor, 1996).In countries where bovine tuberculosis (BTB) is stillcommon and pasteurisation of milk is not practised, anestimated 10–15% of human TB is caused by  M. bovis (Ashford et al., 2001). Human infection due to  M. bovis is thought to be contracted mainly through drinking rawmilk (Kazwala et al., 1998); however, cases of pulmonary TB have also been reported particularly in patients from 1090-0233/$ - see front matter    2007 Elsevier Ltd. All rights reserved.doi:10.1016/j.tvjl.2007.06.019 * Corresponding author. Address: Aklilu Lemma Institute of Pathobi-ology, Addis Ababa University, P.O. Box 1176, Addis Ababa, Ethiopia.Tel.: +251 111 3763091; fax: +251 111 3755296. E-mail address:  gobenaameni@yahoo.com (G. Ameni). www.elsevier.com/locate/tvjl The Veterinary Journal xxx (2007) xxx–xxx  The Veterinary Journal ARTICLE IN PRESS Please cite this article in press as: Regassa A., et al., Bovine tuberculosis is more prevalent in cattle owned by farmers ..., The Veter-inary Journal (2007), doi:10.1016/j.tvjl.2007.06.019  rural areas who live in close contact with cattle. Transmis-sion of TB caused by  M. bovis  or  M. tuberculosis  fromhumans to cattle may also be by contamination of pasturefrom urogenital TB cases (O’Reilly and Daborn, 1995).Ethiopia is a country where the impact of BTB is partic-ularly important and it is amongst the three African coun-tries with the highest burden of human TB cases (WHO,1997). Similarly, the prevalence of BTB in Ethiopia is alsohigh, ranging from 3.4% in smallholder production systemsthat keep Zebu cattle to 50% in peri-urban (intensive) dairyproduction systems (Ameni and Roger, 1998; Kiros, 1998;Ameni et al., 2001; Bogale et al., 2001). The high preva-lence of TB in cattle, the close contact of cattle and humansin rural areas, the habit within the community of consum-ing raw milk and the increasing prevalence of humanimmunodeficiency virus (HIV) may all increase the poten-tial for transmission of   M. tuberculosis  and other mycobac-teria between cattle and their owners. HIV infection isspreading from towns to the countryside among the farm-ers (MOH, 1997). As HIV infection suppresses the immunesystem, if farmers are infected with mycobacteria, they willbe at an increased risk of developing TB, which in turn canlead to the transmission of TB from farmers to their cattle.The present study was formulated to assess the risk factorsthat could promote the transmission of   M. tuberculosis complex between cattle and their owners in either direction. Materials and methods Study subjects and sampling  The study was conducted on human TB cases and non-TB patients(control group) as well as on cattle owned by both groups. A human TBcase was defined as a patient farmer diagnosed at Fitche Hospital TB clinicby a physician on the basis of clinical findings as a new case of activepulmonary or extrapulmonary TB. Confirmation of pulmonary humanactive TB case was made by a positive chest X-ray examination and Ziehl-Neelsen staining of sputum, or chest X-ray and clinical symptoms forsmear-negative cases. An extrapulmonary TB case was defined in thisstudy as a farmer with lymphadenitis or TB peritonitis as confirmed bydetection acid fast bacilli from the lymph node aspirate and ascites. Acontrol was a patient farmer who visited Fitche Hospital for diseases otherthan TB and in whom active TB was excluded clinically by a physician.Laboratory methods were also used to exclude these cases as not being TB.The tests employed to rule-out infection with mycobacteria were Ziehl-Neelsen staining of sputum and chest X-ray.During the study period (October 2004–March 2005), 200 patientswith TB visited the hospital for diagnosis and treatment follow up. Of these patients, only 87 who owned cattle participated voluntarily, andwere considered as cases. An equal number of controls who owned cattleand volunteered to participate in the study were included. All cattle above6 months of age owned by cases and controls (1041) were included. Diagnosis of tuberculosis in cattle owners The farmers were approached through their health worker to assesstheir willingness to participate in this study. Consent was requested fromthe eligible farmers to use their samples in the study. Samples were col-lected from 87 consenting human TB cases as part of the routine diag-nostic procedure. The specimens were collected before the antimicrobialtherapy was commenced using sterile, leak proof, disposable plasticmaterials labelled with the patient’s code number, type of specimen anddate of collection. In the cases of TB lymphadenitis, fine needle aspiration(FNA) (OIE, 2000) was used for collecting samples for isolation andidentification of mycobacterial species. The samples were kept at 4   C untiltransported to the laboratory within a maximum of 4 days. A total of 111specimens (87 sputum, 21 FNA and three ascitic fluid) from 87 human TBpatients (63 pulmonary and 24 extra pulmonary TB cases) were collectedand processed in the laboratory.In addition, the farmers were interviewed about the degree of theirassociation with cattle, habit of consumption of meat and milk and otherrelevant information related to TB in their household.Sputum samples were stained with Ziehl-Neelsen stain and examinedunder a microscope with a 100 ·  oil immersion lens. Culture for myco-bacteria was carried out according to WHO (1998) guidelines. Briefly, thesputum samples were decontaminated using 2% NaOH (1:3 ratio), agi-tated in a vortex mixer for 15 min at room temperature and centrifuged at1310  g   for 15 min at 4   C. The supernatant was removed and the sedimentsuspended in 2 mL of sterile physiological saline solution. One to twodrops of 0.05% phenol red indicator was added to indicate pH change andthen neutralized using concentrated HCl until the colour changed to yel-low. The sediment was inoculated onto two slants of Lowenstein-Jensenmedia, one with pyruvate to enhance growth of   M. bovis  and the otherwith glycerol to suppress growth of   M. bovis  and stimulate other myco-bacteria. Twenty-one FNA and three ascitic fluid samples were collectedfrom 21 TB lymphadenitis and three TB peritonitis cases, respectively, andprocessed in a similar way as sputum samples, and then inoculated ontothe same media. Comparative intradermal tuberculin test Within a few weeks of the identification of a human TB case, the cattleowned by the patient were identified and tested for TB with the compar-ative intradermal tuberculin test. A study team including a veterinarianand laboratory technicians administered the intradermal comparativetuberculin test, interviewed farmers, and collected milk samples fromreactive cows in the herds. A total of 1041 cattle (506 cattle from humanTB cases and 535 cattle from the control group) above 6 months of agewere tested by comparative intradermal tuberculin test. Avian purifiedprotein derivative (PPD) was used to exclude mycobacteria which are notmembers of   M. tuberculosis  complex. Two sites on the skin of the mid-neck of the study animals, 12 cm apart, were shaved and skin thicknesswas measured in millimetres with digital callipers before the injection of tuberculin. Aliquots of 0.1 mL of 20,000 IU/mL bovine PPD (VeterinaryLaboratories Agency), and 0.1 mL of 25,000 IU/mL avian PPD (Veteri-nary Laboratories Agency) were injected into the dermis at these sites. Acorrect injection was confirmed by palpating a small pea-like swelling ateach site of injection. After 72 h, the thickness of the skin at the injectionsites was again measured. The interpretation of the result was made on thebasis of the difference in skin indurations at bovine PPD (B) and avianPPD (A) injection sites. A difference (B  A) of 4 mm or above wasconsidered as a positive while a difference of less than 2 mm was consid-ered as a negative tuberculin test. Readings of a difference between 2 and4 mm were considered suspect. Milk culture Approximately 30 mL of the last few streams of milk were collected bythe owners at milking into sterile universal bottles from each quarter of TB-positive dairy animals. Milk samples were cultured according toKazwala et al. (1998). Briefly, samples were centrifuged at 1310  g   for15 min and the supernatant discarded. The sediments were suspended in2 mL of sterile physiological saline solution and decontaminated withequal volume of sterilized 4% NaOH solution. One or two drops of 0.05%phenol red indicator were added and then neutralized using concentratedHCl. The suspension was centrifuged at 1310  g   for 15 min at 4   C and thesediment was inoculated onto two slants of Lowenstein-Jensen media asindicated above. Growth for Mycobacteria was checked every week.2  A. Regassa et al. / The Veterinary Journal xxx (2007) xxx–xxx ARTICLE IN PRESS Please cite this article in press as: Regassa A., et al., Bovine tuberculosis is more prevalent in cattle owned by farmers ..., The Veter-inary Journal (2007), doi:10.1016/j.tvjl.2007.06.019  Positive cultures were subcultured onto another set of media and incu-bated for another 3–4 weeks for further identification. Differential tests Identification of mycobacterial isolates was based on colony mor-phology, nitrate reduction test, pyrazinamidase test and the thiophene-2-carboxylic acid hydrozide susceptibility test. The detail procedures for theisolation and identification of species of mycobacteria were undertakenaccording to Vestal (1981). Data analysis First, study participants were classified as farmers with TB and thosewithout TB as judged by a physician on the basis of clinical examination,chest X-ray and presence of acid-fast bacilli, or clinical examination andchest X-ray. Demographic information of each study participant wasrecorded. In addition, the farmers were interviewed on the extent to whichthey consumed milk and milk products. This was followed by tuberculintesting of their cattle.Tuberculin reactivity of the test animals across different categories of categorical variables such as sex, age, breed, physical condition of animals,the TB status of the animal owner, the size of the herd from which thatparticular animal was selected were all summarized using percentages.Further, the tuberculin test result was considered as a dependent variableand the effect of selected risk factors (age with four categories, sex, breedwith three categories, physical condition of an animal with three catego-ries, TB status of an owner with two categories, and herd size with threecategories) on the tuberculin test result were analysed using bivariate,multivariate and stepwise logistic regression using both STATA Version 7(STATA Corporation) and SPSS version 11. This modelling was per-formed twice (a) considering suspects as negatives and (b) consideringsuspects as positives. In stepwise logistic regression the fit of the model wasevaluated using the likelihood ratio test. The influence of removing eachvariable included in the final model was also evaluated using change inlog-likelihood. As a measure of effect odds ratio (OR) and 95% confidenceinterval (CI) around the OR were used.  P  -values <0.05 were used to decideon statistical significance of a given estimate. Results Tuberculosis in cattle owner farmers and associated risk  factors Of 87 patients with TB, 63 (72%) and 24 (28%) werediagnosed as pulmonary and extrapulmonary TB, respec-tively. Of all the TB patients, about 84% (73/87) were con-suming raw milk and milk products, while only 7% (6/87)were consuming boiled milk regularly; the remaining 9%(8/87) reported that they did not consume milk at all. Clas-sification of the cases on the basis of age indicated that 56%of the patients were between the ages of 15 and 39 while20% and 24% were <15 and >39 years, respectively. Classi-fication of the patients on the basis of residence showedthat 76% of them were rural dwellers and 79% of extrapul-monary TB cases were also from rural areas.Most of the assessed risk factors (sex, age, physical con-tact and the srcin of cattle owners) did not differ betweenfarmers who had TB and those who did not have TB. How-ever, a significant ( v 2 = 14;  P   < 0.001) difference wasobserved between the milk consumption habit of the twogroups. Farmers who consumed raw milk were at higherrisk (OR = 3.3) of infection with TB than those who con-sumed boiled milk. Herd prevalence The herd prevalence was significantly ( v 2 = 19.5, P   < 0.001) higher in herds owned by farmers who had TBthan in those owned by farmers who did not have TB.When suspect herds were considered as positive, herd pos-itivity was more likely (OR = 3.1;  P   < 0.001) in cattleowned by farmers who had TB as compared to thosewho did not have TB (Table 1). Animal prevalence The result of the intradermal tuberculin test in cattleowned by farmers who had TB and those who did not haveTB is given in Table 2. The prevalence of BTB was found to Table 1Tuberculin test status of herds owned by farmers with active tuberculosis and farmers without active tuberculosis in central EthiopiaOwner status Tuberculin test status of herdNo. examined Positive Suspect NegativeNo. % No. % No. %TB cases a 87 54 62 9 10 24 28Non-TB patients b 87 25 29 15 17 47 54Total 174 79 45 24 14 71 41 a Farmers diagnosed with TB clinically and bacteriologically and under treatment during the study period. b Farmers who had no clinical symptoms suggestive of TB but visited the health facility during the study period for other illness.Table 2Tuberculin reactivity of individual cattle owned by farmers with activetuberculosis and farmers without tuberculosis in central EthiopiaOwner status Tuberculin reactivity of animalNo. examined Positive Suspect NegativeNo. % No. % No. %TB positive a 506 123 24 35 7 348 69TB negative b 535 46 9 55 10 434 81Total 1041 169 16 90 9 782 75 a Farmers diagnosed with TB clinically and bacteriologically and undertreatment during the study period. b Farmers who had no clinical symptoms suggestive of TB but visited thehealth facility during the study period for other illness. A. Regassa et al. / The Veterinary Journal xxx (2007) xxx–xxx  3 ARTICLE IN PRESS Please cite this article in press as: Regassa A., et al., Bovine tuberculosis is more prevalent in cattle owned by farmers ..., The Veter-inary Journal (2007), doi:10.1016/j.tvjl.2007.06.019  Table 3Association between selected risk factors and tuberculin reactivity of cattle owned by farmers with active tuberculosis and farmers without activetuberculosis in central EthiopiaNumber (%) positive a Crude OR and 95% CI Adjusted OR and 95% CI Adjusted OR and 95% CI fromstepwise logistic regression Age <2 5 (6) 1 1 12–4 23 (9) 1.4 (0.52,3.88) 1.7 (0.59,4.76) 1.7 (0.59,4.65)4–7 55 (22) 4.2 (1.63,10.97) 4.9 (1.78,13.22) 4.7 (1.74,12.78)>7 86 (20) 3.8 (1.50,9.71) 5.0 (1.88,13.51) 4.8 (1.82,12.87) Sex Male 68 (16) 1 1Female 101 (17) 1.1 (0.75,1.46) 1.1 (0.76,1.64) Breed  Local 49 (10) 1 1 1Cross 54 (14) 1.4 (0.95,2.16) 1.9 (1.17,2.91) 1.9 (1.21,2.92)Holstein 66 (40) 5.9 (3.83,9.02) 7.0 (4.37,11.21) 6.9 (4.34,10.93) Body condition Poor 43 (12) 1 1 1Medium 107 (17) 1.5 (1.04,2.22) 1.4 (0.94,2.20) 1.4(0.94,2.16)Good 19 (30) 3.2 (1.69,5.89) 2.4 (1.21,4.85) 2.3 (1.16,4.62) Case-control  Control 47(9) 1 1 1Case 122 (24) 3.3 (2.28,4.71) 4.0 (2.70, 5.94) 3.9 (2.62, 5.73) Herd size <5 39 (41) 1 16–10 29 (48) 1.0 (0.68, 1.44) 0.7 (0.49, 1.12)>10 12 (65) 0.9 (0.53, 1.37) 0.8 (0.49, 1.39) a Suspects were considered as negatives.Table 4Association between selected risk factors and tuberculin reactivity of cattle owned by farmers with active tuberculosis and farmers without activetuberculosis in central EthiopiaNumber (%) positive a Crude OR and 95% CI Adjusted OR and 95% CI OR and 95% CI fromstepwise logistic regression Age <2 12 (15) 1 12–4 37 (14) 0.9 (0.46,1.87) 1.0 (0.49,2.18) 1.1 (0.51,2.17)4–7 77 (30) 2.5 (1.29,4.92) 2.7 (1.32,5.56) 2.8 (1.41,5.64)>7 133 (31) 2.6 (1.36,4.93) 3.0 (1.48,5.97) 2.9 (1.50,5.77) Sex Male 105 (25) 1 1Female 154 (25) 1.0 (0.78,1.37) 1.2 (0.89,1.70) Breed  Local 105 (22) 1 1Cross 72 (19) 0.8 (0.59,1.15) 0.9 (0.64,1.33)Holstein 82 (49) 3.5 (2.43,5.13) 4.0 (2.69,6.04) Body condition Poor 73 (20) 1 1Medium 163 (26) 1.4 (1.01,1.89) 1.3 (0.90,1.80) 1.0 (0.67,1.35)Good 23 (37) 2.2 (1.26,3.97) 1.6 (0.84,2.93) 3.9 (2.59,5.65) Case and control  Control 102 (19) 1 1Case 157 (31) 1.9 (1.42,2.52) 2.3 (1.58,2.95) 2.0 (1.46,2.67) Herd size <5 52 (54) 1 16–10 41 (67) 0.6 (0.42,0.78) 0.4 (0.32,0.63)>10 12 (65) 0.5 (0.34,0.76) 0.5 (0.31,0.75) a Suspects were considered as positives.4  A. Regassa et al. / The Veterinary Journal xxx (2007) xxx–xxx ARTICLE IN PRESS Please cite this article in press as: Regassa A., et al., Bovine tuberculosis is more prevalent in cattle owned by farmers ..., The Veter-inary Journal (2007), doi:10.1016/j.tvjl.2007.06.019  be three times higher in cattle owned by patients withtuberculosis (24.3%) than in those owned by patients whodid not have tuberculosis (8.6%). The overall prevalence,considering suspect reactors as negative was 16% (169/1041) while it was 25% (259/1041) when the suspects wereconsidered as positives. Association of risk factors with tuberculin positivity(reactivity) of animals Table 3 shows the association of tuberculin positivityand different risk factors when suspects are considered asnegative reactors (Model I). Similarly, the result of multi-variable logistic regression analysis of risk factors andtuberculin positivity (suspects taken as positives, ModelII) is presented in Table 4. Bacteriological findings Table 5 shows mycobacterial isolates from the sputumof human TB patients and milk samples of tuberculin posi-tive cows. The result of speciation of the isolates on thebasis of biochemical and drug sensitivity tests is presentedin Table 6. Discussion Both individual and herd prevalence of BTB were higherin the cattle/herds owned by TB-affected farmers as com-pared to those owned by TB-free farmers, suggesting theexistence of transmission of mycobacterial species betweenthe human TB patients and their cattle. This transmissionis possible in both directions.The disease transmission may be cyclical: cow-to-man-to-cow (reviewed in Cosivi et al., 1998), underlying the exis-tence of higher risk of dissemination of mycobacteriaamong the cattle and human populations. Similar findingswere reported by Cook et al. (1996) and Ameni et al.(2001). In addition, Andersen (1997) indicated that humans acquire the infection primarily by ingesting the agent inraw milk and milk products, and secondly by inhaling itwhen there is close physical contact between the ownerand his/her cattle, especially at night since in some casesthey share shelters with their animals. Cosivi et al. (1998)indicated that pulmonary as well as extra pulmonary casesof human TB of animal srcin will continue to be of publichealth importance especially in areas where the prevalenceof infection is high in cattle and where raw milk or its prod-ucts are commonly consumed.In support of the results of this study, Ashford et al.(2001) indicated that milk is still regarded as the principalvehicle for transmission of   M. bovis  to humans in countrieswhere BTB is not controlled. Ingestion of contaminatedmilk or other dairy products is more often associated withscrofula, abdominal TB and other extrapulmonary formsof the disease due to  M. bovis  (Ashford et al., 2001).Consumption of food of animal srcin including rawmilk, and the isolation of   M. tuberculosis  and  M. bovis from milk of reactor cows, was a risk factor for acquiringinfection. Kazwala et al. (1998) suggested that  M. bovis infection is acquired mainly through consumption of con-taminated milk. Furthermore, Sinha (1994) estimated that M. bovis  accounts for 30% of TB in children and up to 10%of the extra pulmonary form of TB in humans as whole inthe border area between Mexico and USA. Ashford et al.(2001) also stated that in countries where BTB is still com-mon and pasteurisation of milk is rare, an estimated 10– 15% of human cases of TB are caused by  M. bovis  but inareas where milk is routinely pasteurised or boiled beforeconsumption,  M. bovis  infection is responsible for 1–6%of human TB cases. Although contaminated milk is theusual source of infection for the town dwellers, farm work-ers can also acquire lung disease directly by inhalation(O’Reilly and Daborn, 1995).Analysis for the effect of risk factors revealed that theanimal prevalence of BTB increased with age up to theage of 7 years, and was then observed to decrease slightly.This finding is consistent with other reports (Cook et al.,1996; O’Reilly and Daborn, 1995). O’Reilly and Daborn(1995) reported that the reaction to tuberculin test in cattleincreases uniformly by 7.5% for every year of life reaching40% at 6–7 years old. As explained by other workers (Bar-winnek and Taylor, 1996), this could be because as the ageincreases the probability of acquiring TB infection alsoincreases. On the other hand, the decrease in prevalenceobserved in old age may not reflect the actual disease statusof the animal. The probable reason may be that reaction to Table 5Mycobacterial growth from human and cattle specimens in centralEthiopiaType of specimen Growth on Lowenstein-Jensen mediumNo. examined PositiveNo. %Sputum 87 36 41FNA a 21 6 29Peritoneal fluid 3 0 0Milk 60 11 18Total 171 53 31 a Fine needle aspirate.Table 6Species of mycobacteria from human and cattle specimens in centralEthiopia as confirmed by biochemical testsType of specimen Total isolates  M. tuberculosis M. bovis  OthersNo. % No. % No %Sputum 36 29 81 4 11 3 8FNA a 6 2 33 3 50 1 17Peritoneal fluid 0 0 0 0 0 0 0Milk 11 2 18 5 46 4 36Total 53 33 62 12 23 8 15 a Fine needle aspirate. A. Regassa et al. / The Veterinary Journal xxx (2007) xxx–xxx  5 ARTICLE IN PRESS Please cite this article in press as: Regassa A., et al., Bovine tuberculosis is more prevalent in cattle owned by farmers ..., The Veter-inary Journal (2007), doi:10.1016/j.tvjl.2007.06.019
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