A cross-sectional study was conducted from November 2007 to April 2008 to estimate the prevalence of bovine tuberculosis using the comparative intradermal tuberculin test in 510 heads of cattle in intensive, semi-intensive and small holder dairy farms of Mekelle and its surrounding. In addition, a questionnaire survey was conducted in 230 people to assess the public health implication of the disease. In addition, milk samples were taken from 39 tuberculin positive cows for the identification of pathogen. The overall prevalence of bovine tuberculosis was recorded 13.9 % in dairy farms. Herd prevalence was noticed 54 % in the area. Using confidence interval of 95%, statistically significant variables was observed among age (x2=19.4, P <0.001), herd size (x2=8.0, P<0.001), breed (x2=19.4, P<0.001), management (x2=8.8, P<0.003) and body condition (x2=53.0, P<0.001). The people in study area have poor awareness on bovine tuberculosis. About 50% of respondents were aware that cattle are affected by tuberculosis. Also, 40% of the people know that raw milk is a vehicle of transmission of disease. Cattle owners and non cattle owners had 5%, 3% tuberculosis patients in their house, respectively. The organism was isolated from 15.4% of milk samples on Lowenstein-Jensen medium. It is deduced that sharing of the same households by tuberculosis patient and reactor cattle suggest transmission of Mycobacterium species between cattle and their owners.
Human population is growing in Sub-Saharan countries leading to increasing demands on animal products. These require introduction of intensification of animal production. However, bovine tuberculosis is becoming the main constraint for such strategies. It causes great economic losses as a result of slaughter of tuberculin positive cattle and condemnation of infected carcasses.
On the other hand, HIV/AIDS and Mycobacterium bovis transmission are high in Afirca with 90% of the population of Africa living in area where neither pasteurization nor bovine tuberculosis (BTB) control programs occur; and up to one in ten adults living with HIV/AIDS. The association between the two diseases is of particular concern on this continent (Collins and Grange, 1985). In developing countries where bovine tuberculosis is present, knowledge on the prevalence and risk factors associated with disease is mandatory.
In Mekelle town, keeping of Holstein cattle and Holstein’s cross-breed with zebu is becoming more common. However, the extent of tuberculosis has not been known before in this area of Ethiopia. The objective of the research was to study the prevalence of bovine tuberculosis and potential risk factors associated with disease and public health implications in the area.
Materials And Methods
The study was carried out from November 2007 to April 2008 in and around Mekelle town located about 780 km North of Addis Ababa with 33024/30// to 13036 /52// latitude and 390 25/ 30// to 39038/33// longitude.
Intradermal Tuberculin Test, Questionnaire, Sample Collection and Procedures
The sample size of cattle was determined according to Thursfield (1995). A total of 41 households owning about 510 cattle were included in the study. From the 7 local administrations, 4 of them were selected randomly. List of the household owning dairy cattle was obtained from town’s administration. All animals above 6 month age were tested; and age, herd size, body condition, management and sex were considered as risk factors.
Comparative Intradermal Tuberculin Test
This was used to differentiate Mycobacterium bovis and Mycobactrium tuberculosis from other Mycobacterial genera. The skin of animal was shaved at two sites (12 cm apart) on the right hand side of the mid neck area. The skin thickness was measured with caliber and recorded before the tuberculin was injected. A liquots of 0.1 ml of 2,500 international units (IU) per milliliter (ml) of bovine purified protein derivative (PPD) (Veterinary Laboratories Agency, Addlestone, United Kingdom), and 0.1 ml of 2,500 IU /ml of avian PPD were injected into the dermis at these sites using semi automatic syringe on the posterior and anterior parts, respectively. After 72 hrs, thickness of skin was interpreted in accordance with recommendations of OIE (2000). When change in skin thickness was greater at the avian PPD injection site, it was considered positive for Mycobacterium species other than the mammalians types. However, when increase in thickness was observed at both sites, the difference was considered. Thus if the increase in thickness at injection site for bovine PPD (B) was greater than the avian PPD (A), and if B minus A was less than 2 mm, it was considered as negative for mammalian type. But when B minus A was between 2 mm and 4 mm, and 4 mm and above, it was considered as suspected and positive, respectively.
Assessing Risk Factors
Age, herd size, breed, management, body condition and sex were among the risk factors expected to have the influence on the extent of the disease. Dairy cattle selected for the CIT-test were grouped by age less or equal one year, animals between one year and less or equal to three and animals between four and less or equal to six, with age of greater than six years were considered. Similarly, herd size was assessed by grouping number of animals in a herd less than or equal to five, between five and less or equal to fifteen and with herd size of greater or equal to sixteen numbers. Considering that the genetic makeup of the animals have an effect on the prevalence of the disease, it was grouped as zebu, cross and Holstein dairy cattle. To observe role of confinement, management of the farm was consider as good and poor. According the body conditions, the animals were grouped into lean, medium and fat body score (Nicholson and Butterworth, 1986). Sexes of animals and farming system were also considered.
A risk assessment questionnaire survey was administered randomly to some of the cattle owners. The people were interviewed using questionnaire to assess the role of various risk factors in the occurrence and spread of bovine TB among cattle, from cattle to people and vice versa. They were asked questions focused on their awareness on the disease and transmission of BTB and their habit of consumption of raw milk and meat. They were also asked if resent history of TB cases were in their family.
Milk Culture and Niacin Paper Strip Test
A 25 ml milk of the last few strips was collected into sterile universal bottles from each quarter of 39 tuberculin positive cows. It was transported to Akelilu Lemma Institute of Pathobiology, Addis Ababa using ice box. The culture was done from tuberculin positive animals to isolate M. bovis and M. tuberculosis. The specimen was processed according to Kazwala et al. (1998). The milk sample added into 15 ml centrifuge test tubes was centrifugeted at 3000 rmp for 15 minutes at room temperature. The cream was separated with sterile spatula, the supernatant was decanted and sediment was decontaminated by equal volume of 4% NaOH. It was centrifuged again in the same condition and the supernatant was decanted. Three drops of phenol red was used to monitor neutralization. Then HCl was added drop by drop till the color changed from deep purple to yellow. After the supernatant was decanted, the sediment was then inoculated on to Lowenstein Jensen media, one with pyruvate to enhance the growth of M. bovis and the other with glycerol to facilitate the growth of M. tuberculosis. The cultures were incubated at 37oC in slant position for a week and upright position for 7-10 weeks with weekly observation.
Niacin Paper Strip Test
Three to four weeks old cultures were extracted and prepared by adding 1ml of sterile saline. The tube was placed horizontally so the fluid covers the entire surface of the medium and allows 30 minutes for the extraction of niacin. The slant was raised upright for 5 minutes to allow the fluid to drain the bottom and 0.5 ml of the fluid extract was removed to a clean screw caped tube. The strip was inserted with identification end up and immediately the tube was sealed, and then left at room temperature for 15-20 minutes. When the color of the strip changed from white to yellow, the colony was considered as positive of niacin test (WHO, 1998).
Individual animal prevalence was defined as number of positive reactors per 100 animals tested. The herd prevalence was calculated as number of herds with at least one reactor animal per 100 herds tested. Logistic regression analysis was used to assess the association between prevalence and animal risk factors using STATA statistical software (STATA Corporation, 4905 Lakeway Drive, College Station, Texas 77845 USA). A p-value <0.05 was considered to be statistically significant with 95% confidence interval. The effects of different risk factors on prevalence were analyzed using Pearson chi-square (χ2) test. Odds ratio was calculated to assess strength of association of factors to the prevalence of bovine tuberculosis.
Prevalence of bovine tuberculosis at animal level using the comparative intra dermal tuberculin test was 13.9% (71/510) when doubtful was considered as negative. At herd level, the prevalence using the similar method was registered as 54% (22/41) taking doubtful as negative. The prevalence of bovine tuberculosis in the area is presented in Table-1.
Table-1: Result of comparative intradermal tuberculin test
|CIT test||Frequency||Percentage (%)|
Risk factors of bovine tuberculosis – The prevalence was affected by age. It has statistically significant variation (X2 = 52.5; P<0.001). Cattle with age between three and six years were 8 times more reactive to the test than those which were in the age less than 1 years (odds ratio (OR)=8.2; 95% CI=5.9,11.5) (Table-2).
Table-2: Effect of age on the prevalence of bovine tuberculosis
|No. of animals tested||Percentage of positivity||
(x=age in years)
Similarly, herd size was found statistically significant (X2=8.0; P<0.019). Herd size > 15 in number are 3 times more reactive than a herd size < 6 animals (odds ratio (OR)=3; 95% CI=2.3,4.3) ( Table-3).
Table-3: Effect of herd size in bovine tuberculosis.
Herd size( in number)
|No. of animals tested||Percentage of positivity||
Exotic breeds were 6 times more exposed to the disease than local breeds (odds ratio (OR) =6; 95% CI=3.8, 9.4). It has statistically significant association with the results (X2=19.4; P<0.001) (Table-4).
Table-4: Effect of breed on the prevalence of bovine tuberculosis.
|No. of animals tested||Percentage of positivity||
Management (X2=8.8; P<0.003) has a significant effect on the prevalence of the disease. The difference in reactivity test in different body condition was also statistically significant (X2=53.0; P<0.001). However, the prevalence of the disease was not affected by sex (X2=2.2; P=0.142) (Table-5).
Table-5: Effect of management, body condition and sex on bovine tuberculosis
|Variables||No of animals tested||Percentage of positivity||X2||P Value||OR||CI|
Farming system also affects the prevalence of bovine tuberculosis. Animals in intensive farming system were almost 4 time exposed to the disease than animals in small holder farming system (odds ratio (OR) = 3.8; 95% CI=2.7, 5.2). The result have statistically significant variation(X2=9.7; P<0.01) (Table-6).
Tale-6: Effect of farming system on bovine tuberculosis
|Farming system||No of herd
of animals tested
|Positive||Negative||Percentage of positives|
|Small holder farming||21||90||7||83||7.8|
Two hundred thirty people (100 cattle owners and 130 none cattle owners) were asked to assess their awareness on bovine tuberculosis and its transmission from cattle to human and vice versa. About 50% of the respondents were not aware as cattle are affected by tuberculosis. The survey indicated that 40% of the people drank raw milk and almost 36% consumed uncooked meat. Out of the 100 cattle owners, 5% had recent tuberculosis patient in their family and 3% of the non cattle owners had recent tuberculosis patients in their house.
Growth of Mycobacteria was observed in 15.4 % of the milk cultured. Out of these, 5 grew in Lowenstein-Jensen media enriched with pyruvate and the other one in glycerol. These which grew in pyruvate were negative of niacin strip test. Where as one which grew in glycerol, was niacin strip test positive suggesting M. tuberculosis.
The overall prevalence of bovine tuberculosis recorded in this study (13.9%) was higher as compared to Laval and Ameni (2004) who reported 5%. This could be because of the breed and management effects. Similar results to our study were reported by Regassa (1999) in Wolaita Soddo, Southern Ethiopia, where cattle farming is related to the highlands of Ethiopia with a prevalence of 14.2%. In addition, Ameni et al. (2007) mentioned a prevalence of 13.5% in two areas, namely, West Shewa (Holeta area) and North West Shewa (Selalle). In contrast, higher prevalence (46.8%) was found in 12 intensive dairy farms which had Holstein and cross breed cattle (Ameni et al., 2003b).
In accordance with finding of Cook et al. (1996), as herd size increases, prevalence of bovine tuberculosis also increases.
The finding of current study showed that prevalence of bovine TB was significantly higher in Holsteins as compared to cross breeds or in Zebus kept under similar husbandry. This simulates with the observation of Yehualashet (1995). In agreement with the work of Cook co-investigators (1996), the prevalence of bovine tuberculosis was higher in animals with better body conditions. Few reactors were recorded in younger age groups and reactivity to the test increased with age, up to six years of age, after which it declined. This goes parallel with finding of Cook et al. (1996).The infection may not become established in young animals but, as they get older, their chance of acquiring infection also increases. Higher prevalence in intensive (18.8%) than semi-intensive (12.4%) and small holder (7.7%) farming systems were registered in our study area. However, higher (79.46 %,) prevalence was noticed by Ameni et al. (2003a) in Dessie intensive dairy farm. On the contrary, lower prevalence of 3.4% in Asela was reported by Redi (2003). The occurrence of bovine tuberculosis in the dairy farm in Mekelle and its adjoining area was confirmed by isolation of M. bovis from milk of intradermal tuberculin test positive animals using the niacin strip test. It is emphasized that further studies on the characterization of M. bovis strains by employing molecular tools may be rewarding.
We wish to thank Dr. Gobena Ameni, Addis Ababa University, Institute of Pathobiology, for providing laboratory facilities.
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