Available online at www.pelagiaresearchlibrary.com
Pelagia Research Library
European Journal of Experimental Biology, 2013, 3(6):443-447
ISSN: 2248 –9215
CODEN (USA): EJEBAU
Evaluation of bovine tuberculosis by the milk secretions of cattle and
buffaloes from some dairies of Quetta city
Mohammad Baquir1, Afroz R.Khan2, Nizam. U. Baloch3, Muhammad Anwar4, Safiullah1
and Muhammad Javed Khan5*
1
Department of Microbiology, University of Balochistan, Quetta, Pakistan
Department of Botany, Sardar bahadur khan university, Quetta, Pakistan
3
Department of Chemistry, University of Balochistan, Quetta, Pakistan
4
Pharmacy department, University of Balochistan, Quetta, Pakistan
5
Institute of Biochemistry, University of Balochistan, Quetta, Pakistan
2
_____________________________________________________________________________________________
ABSTRACT
Bovine tuberculosis (BTB) is widespread but poorly controlled in Quetta city and M. bovis is posing threats to
human health. The aim of the present study deals with the evaluation of Bovine tuberculosis in the milk secretions of
cattle and buffaloes. Total hundred milk samples were taken. Sixty (60) were the cattle and forty (40) were the
buffaloes. In 60 samples of cattle only one case was positive. Thus, the percentage of bovine tuberculosis was [1]67
% in the cattle of those dairy farms and 98.33 % they were free from the M.bovis while in 40 samples of buffalo no
positive case came on front therefore, the percentage for positivity in buffaloes remained 0 %. Hence, the current
study reveals that the overall prevalence of Bovine tuberculosis is only 01 % in the selected area.
Key words: Bovine Tuberculosis, cattle and buffaloes, dairy milk.
_____________________________________________________________________________________________
INTRODUCTION
Meat and milk and are significant sources of protein and other nutrients but can be infected by pathogenic agents.
The likelihood exists for the spread of tuberculosis and other mycobacterial infections from animals to humans,
probably by ingestion of infected raw (unpasteurized) dairy products or meat [1, 2, and 3]. Milk itself possesses
small number of micro-organism when it leaves the normal udder however, it may get contaminated from manure,
water, soil, milker’s hands, utensil and flies [4]. Due to high prices of packed milk as compared to raw milk, the
majority of Pakistanis purchase raw milk from milk-men and milk shops [5]. Bovine Tuberculosis caused in animals
is mostly present in the milk of that infected animal which is a chronic contagious respiratory disease of cattle
spreading horizontally within and between species by the aerosol and ingestion .The etiology of B.Tuberculosis is
the infection caused by Mycobacterium bovis, a Gram positive, acid-fast bacterium in the Mycobacterium
tuberculosis complex of the family Mycobacteriaceae [6, 7]. Although respiratory route is considered the most
important for transmission of tuberculous bacilli. The alimentary route of infection plays an important role in
spreading this disease because milk carries microorganisms farther into urban areas if the population still drinks raw
milk [8]. M.Bovis can survive for several months in the environment, particularly in cold, dark and moist conditions.
443
Pelagia Research Library
Muhammad Javed Khan et al
Euro. J. Exp. Bio., 2013, 3(6):443-447
______________________________________________________________________________
At 12-24 Centigrade (54-75 Fahrenheit), the survival time varies from 18 to 332 days, depending on the exposure to
sunlight [9]. Bovine Tuberculosis is occurring in almost all developed and developing nations of world .The
incidence of disease is not only higher in the developing nations but in the absence of any national control and
eradication programmes is also increasing worldwide particularly in the Asian , African and Latin American
countries [10]. In humans the Mycobacterium Bovis is the major cause of extra-pulmonary tuberculosis like
tuberculosis of cervical and mesenteric lymph nodes, the peritoneum and the genito- urinary tract. In countries
where bovine milk is not pasteurized before use, Bovine Tuberculosis has emerged as the single major cause of
extra-pulmonary human tuberculosis [11]. Animal and human tuberculosis (TB), caused by pathogenic bacteria of
the Mycobacterium tuberculosis complex, M. bovis, and M. tuberculosis emerging or reemerging and [12] are
prevalent and affecting the human health in Africa and animal industries [13-19]. The transmission of M.Bovis to
humans via milk and its products are eliminated by pasteurization of milk [20]. Tuberculosis lesions can affect any
part of the body but generally affect the lungs and lymph nodes and the chest cavity. The normally smooth internal
lining of the chest cavity may be marred by lesions. Production losses in cattle occur by earlyculling, ill-thrift, and
decreased milk production. Carried out a research in determining the contribution of Mycobacterium bovis to active
tuberculosis in the Australian population during mid eighties to mid ninties, and also anylzed the demographic data
from bacteriologically proven cases [21]. The Present study was conducted in Center for Advanced Study in
Vaccinology and Biotechnology (CASVAB), Quetta for the prevalence of Bovine Tuberculosis. The study was
performed by the processing of milk samples of cattle (i.e.) buffaloes and cows obtained from The Military Milk
Dairy Form Quetta, Cantonment and from the some dairies of Sariab Road Quetta.
MATERIALS AND METHODS
The Present study was conducted in Center for Advanced Study in Vaccinology and Biotechnology (CASVAB),
Quetta for the prevalence of Bovine Tuberculosis. The study was performed by processing the milk samples of
cattle (i.e.) buffaloes and cows obtained from the Military Milk Dairy Form Quetta, Cantonment and some dairies of
Sariab Road Quetta.
Collection of milk samples
Total 100 milk samples of cattle i.e. buffaloes and cows, were randomly collected from the dairy farms of Quetta
City, Pakistan .Some samples were collected from few dairy farms of Sariab Road, Quetta and half of them from
Milk Dairy Farm Quetta Cantonment. All the possible hygienic measures were adopted during collection and
transportation of milk samples. Before sampling each teat was scrubbed with a pledged of cotton moistened with
70% ethyl alcohol. A separate pledge was used for each teat. After discarding the first few streams, about 5-10ml of
milk was collected. The collected samples were immediately transferred to laboratory refrigerator at 4˚C and not for
more than 24 hours.
Processing of samples
The samples were analyzed and processed with the procedure of centrifugating each sample at 3000 RPM for 15
minutes and the supernatant was discarded. The pellet was suspended in 2ml of sterilized physiological saline
solution. To the suspension was added equal volume of sterilized 4-N sodium hydroxide solutions and one drop of
0.05% Phenol red indicator and the mixture incubated for 30 minutes at 37˚C. Finally the samples were neutralized
with sterilized 4-N hydrochloric acid solution and were centrifuged at 3000 RPM for 15 minutes, and sediment was
used for microscopic and cultural examination.
Acid Fast Staining:
The characteristic difference between Mycobacteria and other microbes is the presence of a thick, waxy (Lipoid)
wall that makes the penetration of simple stain (i.e. Gram Stain) very difficult [11].
Microscopic examination:
From pellets of each sample two smears were prepared, dried, slightly fixed over flame and stained with Acid Fast
stain (Ziehl-Neelsen stain). The stained smears were examined under oil-immersion lens for Acid Fast Bacilli
(AFB). Acid Fast bacteria stain bright /rose red with a blue background.
444
Pelagia Research Library
Muhammad Javed Khan et al
Euro. J. Exp. Bio., 2013, 3(6):443-447
______________________________________________________________________________
Test Protocol:
The samples were stained with acid fast staining. Owing to the fact that they can’t be stained with other ordinary
stains because of high lipid content of acid-fast cell walls; in particular, mycolic acid—a group of branched chain
hydroxyl lipids—appears responsible for acid fastness.
Thus, they were stained by a harsher treatment: heating with a mixture of basic fuchsin and phenol (the ZiehlNeelsen Method).when basic fuchsin got penetrated with the aid of heat and phenol then, the acid fast cells did not
easily decolorize by an acid-alcohol washing hence, the positive samples remained red [22].
RESULTS AND DISCUSSION
During this study the Acid fast bacillus (AFB) was detected microscopically and isolated from the milk sample of
only one cattle out of 60 cattle, while from 40 buffaloes no AFB was visualized after microscopy. Therefore, only
one out of 100 dairy animals was secreened for Acid Fast Bacilli (AFB). Rest of the animals were remained
exempted from the discharge of Mycobacterium bovis in their milk in the chosen area of research. Of the 60 cattle,
01 (1.67%) gave positive result for Mycobacterium bovis while other 40 buffaloes showed no positive results. The
complete research work depicted 1 % prevalence of Bovine tuberculosis in the targeted area.
Table- 1: Result of positive and negative cases after performing Acid Fast Staining
Samples
Buffalo
Cattle
40
60
Positive cases
Negative cases
Buffalo Cattle Buffalo
Cattle
00
01
40
59
Result in percentage
0%
1.67%
100%
98.33%
Total samples =100
Table- 2: Areas of sample collection from where cattle and buffalo milk samples were taken
Species
Cattle
Buffalo
Areas of sample collection
LODHI DAIRY FARM, SARYAB ROAD QUETTA MILLITARY DAIRY FARM QUETTA, CANTANOMENT
…….
60
40
………
Total samples = 100
Graph 1:The graphical representation of milk samples taken from different dairies of Quetta, Baluchistan, Pakistan
445
Pelagia Research Library
Muhammad Javed Khan et al
Euro. J. Exp. Bio., 2013, 3(6):443-447
______________________________________________________________________________
Graph 2:The graphical representation of cattle milk samples taken from organized milk dairy farm Quetta Cantt. Baluchistan, Pakistan
Graph 3: The graphical representation of Buffalo milk samples taken from sariab road, Quetta, Baluchistan, Pakistan
CONCLUSION
According to present study, incidence of bovine tuberculosis is 1.67 % in cattle, while 0% in buffalo. 98.33 % were
free from the M.bovis while in 40 samples of buffalo no positive case came on front.Hence, the current study reveals
that the overall prevalence of Bovine tuberculosis is only 01 % in the selected area.
Acknowledgement
We are thankful to the Center for Advanced Study in Vaccinology and Biotechnology (CASVAB) for providing us
the assistance and material. We are also thankful to Mr.Nazir Ahmed Rodini for providing us the milk samples.
446
Pelagia Research Library
Muhammad Javed Khan et al
Euro. J. Exp. Bio., 2013, 3(6):443-447
______________________________________________________________________________
REFERENCES
[1] Chapman J.S, Speight M., Am Rev Resp Dis., 1968, 98: 1052-1054.
[2] Bryan F.L.. In H Riemann, Food-borne Infections and Intoxications, Academic Press, New York., 1969, p. 224288.
[3] Sweeney R.W, Whitlock R.H, Rosenberger A.E., J ClinMicrobiol., 1992, 30: 166-17[1]
[4] Lunder, T. and Brenne E., Symposium on Bacteriological Quality of raw milk. Wolf Passing, Austria., 1996, 1315.
[5] Kashifa K. M, Ashfaque, Iftikhar H and Masood A., Pak Vet. J., 2001, 21(2).
[6] Griffin, J. M, Dolan L. A., A review. Irish Veterinary Journal., 1995, 228- 234.
[7] O’Reilly L. M, Daborn C. J., Tubercle and Lung Disease., 1995, v. 76, n. 1, p. 46.
[8] Moda G, Daborn C. J, Grange J. M, Cosivi O., Tubercle and Lung Disease.,1996, v. 77, n. 1, p. 103-108.
[9] Pollock J.M, Rodgers J.D, Welsh M.D, and McNair J., . Vet. Microbiol., 2006.
[10] Aranaz A, Cousins D, Mateos A. and Dominiguez L., Int. J. Syst. Evol. Microbiol., 2003, 1999, 5: 1785-1789.
[11] Dr. Hamid J, Dr. Puran D and Dr.Asif S., Threat to the public health., 2003.
[12] OIE, “Manual of Diagnostic Tests and Vaccines for Terrestrial Animals,” OIE Terrestrial Manual, Paris,
France, 2008.
[13] Ayele W. Y, Neill S. D, Zinsstag J, Weiss, M. G, and Pavlik I., “Bovine tuberculosis: an old disease but a new
threat to Africa,” International Journal of Tuberculosis and Lung Disease, vol. 8, no. 8, pp. 2004, 924–937.
[14] Corbett, E. L, Marston B, Churchyard G. J, and De Cock K. M., “Tuberculosis in sub-Saharan Africa:
opportunities, challenges, and change in the era of antiretroviral treatment,” Lancet, vol. 367, 2006, 926–937.
[15] Corbett, E. L, Watt C. J, Walker N. et al.,Archives of Internal Medicine, vol. 163, no. 9, pp. 2003, 1009–
102[1]
[16] O. Cosivi, Grange J. M, Daborn C. J et al., Emerging Infectious Diseases, vol. 4, no. 1, pp. 1998, 59–70.
[17] Kiboss J. K, and Kibitok N. K., Journal of Social Development in Africa, vol. 18, no. 2, pp. 2003, 121–132.
[18] Tan D. H. S, Upshur R. E. G, and Ford N., BMC International Health and Human Rights, vol. 3, article 1, pp.
2003, 1–9.
[19] WHO, Global Tuberculosis Control: Epidemiology, Strategy, Financing, World Health Organization, Geneva,
Switzerland,WHO report 2009.
[20] Greenwald R, Lyashchenko O, Esfandiari ., Miller M, Mikota S, Olsen J.H, Ball R, Dumonceaux G, Schmitt
D., Moller T, Payeur J.B, Harris B, Sofranko D , Water W.R. and Lyashchenko K.P., Clin. Vaccine Immunol., 2009,
16 (5), 605-615.\
[21] Cousins D.V, Bastida R, Cataldi A, Quse V, Redrobe S, Dow S, Duignan P, Murray A, Dupont C, Ahmed A.
Collins D.M, Butler W.R, Dawson D, Rodriguez D, Loureiro J, Romano M.I, Alto A, Zumarraga M. and Bernardelli
A., Int. J. Syst. Evol. Microbiol., 2003, 53: 1305-1314.
[22] Prescott−Harley−Klein: Microbiology., The Study of Microbial Structure: Microscopy and Specimen
Preparation. 5th Edition chapter., 2002, 2: page 28. 112:141-50.
447
Pelagia Research Library