Bovine tuberculosis in Dairy Animals at Lahore,
Threat to the Public Health.

AUTHORS

1. Dr. Hamid Jalil
DVM, M.Sc (Hons) Ph.D (Veterinary Microbiology)
Director Veterinary Services, Metropolitan Corporation Lahore, Pakistan
Email: Dr_hamidjalil@yahoo.com & rafaqatpk@yahoo.co.in

2. Dr. Puran Das
DVM, M.Sc (Hons) (Veterinary Microbiology)
Veterinary Officer (Research) Veterinary Research Institute, Ghazi Road Lahore, Pakistan. E-mail: Pdr_71@yahoo.com

3. Dr. Asif Suleman
DVM, M.Sc (Hons) (Veterinary Microbiology)
Veterinary Officer, Metropolitan Corporation Lahore, Pakistan

CORRESPONDENCE

Dr. Hamid Jalil
DVM, M.Sc (Hons) Ph.D (Veterinary Microbiology)
Director Veterinary Services, Metropolitan Corporation Lahore, Pakistan
Email: Dr_hamidjalil@yahoo.com & rafaqatpk@yahoo.co.in

ABSTRACT

Objectives:- Investigating the incidence of bovine tuberculosis and ruling out Mycobacterium bovis from secretion and excretion of infected milch animals.
Methods:- Single comparative intradermal tuberculin test of thousand dairy animals out of a population of ten thousand. Microscopic examination and culture of milk, faces and nasal secretion of infected animals for demonstration and isolation of M.bovis.
Results:- 6.91% of buffaloes and 8.64% cows were found positive to tuberculin test. Mycobacterium bovis and isolated from milk of 28.07% and nasal secretion of 12.18% tuberculin positive buffaloes and from milk of 25% and nasal secretion of 12.5% tuberculin positive cows.
Conclusion:- Mycobacterium bovis the cause of zoonotic tuberculosis was isolated from lactating animals which makes it most important for public health.

INTRODUCTION:

Bovine tuberculosis is a chronic contagious respiratory disease of cattle spreading horizontally, within and between species, by the aerosol and ingestion 18. 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 program, is also increasing worldwide particularly in the Asian, African and Latin American countries 3.
In Indo Pak the incidence of Bovine tuberculosis in cattle and buffalo is nearly 2.25% of the population, in 1969 13 and in 1989 19 the incidence of disease was increased up to 7.3% in cattle and buffaloes being slaughtered at Lahore abattoirs. In 1975 it was found that 10% of the buffalo population was suffering from Bovine Tuberculosis 1. Bovine tuberculosis is a public health problem. Mycobacterium bovis the cause of bovine tuberculosis and M. tuberculosis, the cause of classical human tuberculosis, are genetically and antigenically very similar and cause identical clinical disease in humans 9. There is considerable and continuing public health significance of M. bovis infection in humans and animals and the disease has emerged as a major zoonotic problem in many African countries 21. In humans the M. bovis is the major cause of extra-pulmonary tuberculosis like tuberculosis of gastrointestinal tract and tuberculosis of cervical and mesenteric lymph nodes, the peritoneum, and the genito-urinary tract 3,9. In countries where bovine milk is not pasteurized before use, bovine tuberculosis has emerged as the single major cause of extra-pulmonary human tuberculosis.
The mammalian, human and avian varieties of mycobacteria possess remarkable adaptive variability and have been found to acquire properties that are species-specific to the animal through whose body they had been consequently passaged for 4 to 8 times 16. Many methods are available for diagnosis of tuberculosis in infected animals but the single comparative intra dermal tuberculin test (SCITT) is most widely used for diagnosis and eradication of Bovine tuberculosis 17.
Unfortunately, bovine TB has never been seriously attended in Pakistan. Sparse information regarding its incidence in cattle and buffaloes and its economic impact on livestock industry is available. No serious efforts are underway to estimate the gravity of risk and its threat to the public health 15. The existing situation calls for a comprehensive program to address this problem in order to improve the lot of livestock and to safeguard the human population from this menace.
The present study was designed to estimate the prevalence of bovine tuberculosis in cattle and buffalo maintained at the Cattle Colonies in Lahore Metropolis using single comparative intra dermal tuberculin test (SCITT) and detection / isolation of M.bovis from secretion and excretion of SCITT positive animals which might be a plausible source of infection to humans.

MATERIALS AND METHODS:

One thousand buffalos and cows, out of a population of ten thousand dairy animals were randomly selected from cattle colonies of Lahore, Metropolitan, Pakistan showing low milk yield, emaciation, and anorexia, intermittent diarrhea, not responding to anthelmintic treatment, irregular febrile episodes, and stubborn recurring mastitis.

Tuberculin test: Single comparative intra dermal tuberculin test (SCITT), 17 was used for screening animals. Both mammalian and avian PPD-Tuberculin (purified protein derivative of tubercle bacilli) were obtained from Veterinary Research Institute, Ghazi Road, Lahore.
The following technique was used.

On the left side, in the center of neck of test animal, an area roughly 6? square was shaved, cleansed with a swab of 70% alcohol and dried.
The thickness of the skin fold was measured with Vernier's caliper and recorded.
A fold of skin was pinched with tips of fingers, its thickness was measured and 0.1 ml (0.5mg / ml) avian tuberculin was injected intra-dermally in the upper site.
A fold of skin was pinched with finger tips, its thickness measured and 0.1 ml (0.5mg / ml) mammalian tuberculin was injected intradermally in the lower site (12cm apart).
Both the injection sites were encircled with indelible ink of different colors.
The results were recorded after 72 hours post-inoculation by measuring the thickness of skin fold.
Any gross/visible changes at the site of the injection were also recorded.
An increase in thickness of skin up to 2.9 mm------------------Negative
An increase in thickness of skin up to 3 to 3.9mm -------------Doubtful
An increase in thickness of skin up to 4mm or more-----------Positive
Inflammation and edema regardless of measurement………..Positive

The animals in which positive reaction was seen at the site of mammalian tuberculin were confirmed as suffering from bovine tuberculosis. All animals with a positive reaction at the site of avian tuberculin were confirmed as Johne's disease cases.

Collection of Samples
From all tuberculin positive animals the following specimens were collected for bacteriological examination.
Nasal secretions: Secretions were collected with sterile cotton swabs that were immediately placed in screw capped test tubes, already containing 35 ml sterile Physiological saline solution. The test tubes containing sample were cooled and stored to 4 ºC.
Fecal samples: Feces, about 4 grams, were collected in sterilized McCartney bottles and mixed well in about 50 ml of sterilized physiological saline (0.85% NaCl aqueous solution) by stirring and stored at 4 ºC for 18 hours.
Milk samples: Milk from all tuberculin positive animals was obtained. The last few streams of milk (usually 10 ml), at the end of milking were collected in sterile pre-cooled McCartney bottles and stored at 4ºC.
Processing of samples:
Nasal secretions: To the sampling bottles equal quantity of N-acetyl and sodium hydro-oxide solutions were added and the mixture was incubated at 37ºC for 30 minutes for decontamination. The mixture was centrifuged at 3000rpm for 15 minutes, the supernatant was discarded and the sediment was used for microscopic and cultural examination.
Fecal samples: About 10 ml of fluid, just above the liquid-solid inter- phase was pipetted out and mixed with an equal quantity of 10% oxalic acid solution for decontamination. The contents were centrifuged at 3000 rpm for 10 minutes, and the sediment was used for microscopic and cultural examinations.

Milk samples: Milk samples were centrifuged at 3000 rpm for 15 minutes and the supernatant was discarded. The sediments were 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 37ºC. Finally the samples were neutralized with sterilized 4-N hydrochloric acid solution and were centrifuged at 3000rpm for 15 minutes, and sediment was used for microscopic and cultural examination 14.
Microscopic examination: From sediments 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.

Cultural examination: All the samples positive for the presence of AFB were cultured on slopes of Lowenstein and Jensen medium, with and without the addition 1% sodium Pyruvate, with for the isolation of Mycobacteria. Thick inoculums of sediments were smeared on the surface of medium slopes and the cultured tubes were incubated at 37 ºC for six to eight weeks 4. The growth appearing after incubation was subjected to nitrate reduction and niacin strip test for further identification and Characterization 14.
Pathogenicity and species identification: All isolated cultures of mycobacterium were inoculated in various animals for the study of pathogenicity. For this purpose eight to 12 weeks-old cockerels, 6 week-old guinea pigs and 6 to 8 week-old rabbits were used for the study. Each isolate was inoculated in a group of 6 animals of each species using following protocol.

Rabbit: The rabbits were given an intraperitoneal injection providing 1.0 mg of organisms/animal 5.

Guinea pigs: The guinea pigs were given an intraperitoneal injection providing 10 µg of organisms/animal 5.

Fowl: The cockerels were given an intraperitoneal injection providing 1.0 mg of organisms/animal 22.

RESULTS:-

In the present study one thousand milch animals including 815 buffaloes and 185 cows were subjected to SCITT. All the test animals were randomly selected from cattle colonies of Lahore, Metropolitan, Pakistan with the complaints of low milk yield, emaciation, anorexia, intermittent diarrhoea, not responding to anthelmintic treatment, and recurring mastitis. In positive reactors the average (mean) increase in thickness of skin at the site of inoculation 72 hours post inoculation was recorded 4.8+ 0.09mm in buffaloes and 4.9 + 0.06mm in cows. Diffused odema at the site of injection was recorded in ten buffaloes and three cows. One adult buffalo characteristically reacted to avian PPD-tuberculin indicating a possible exposure to M.avium or M. paratuberculosis.

Out of 815 buffaloes 57 (6.91%) gave a positive reaction to mammalian tuberculin. Out of 185 cows 16 (8.64%) gave positive reaction to mammalian tuberculin, the respective incidence of Bovine tuberculosis in buffaloes and cattle was 6.91% and 8.64% respectively. A very small percentage 0.12% of buffaloes were suffering from Johnes disease or Avian tuberculosis the overall incidence of Bovine tuberculosis in milch animals of cattle colonies Lahore, Metropolis was therefore 7.3% (Table-1).

During this study acid fast bacilli (AFB) were detected microscopically and isolated from the milk of 16 (28.07%) and nasal secretions of 7 (12.28%) buffaloes positive to SCITT. Two buffaloes from positive reactors (3.5%) were secreting AFB in both their milk and nasal secretions. Acid fast bacilli were present in the milk of 4 (25%) and nasal secretion of 2 (12.5%) cows, positive to SCITT. Mycobacterium bovis was isolated from milk of 20 (27.4%) and nasal secretions of 9 (12.33%) SCITT positive AFB secretors (Table-2).
The growth of M.bovis was very slow and visible growth appeared after six weeks of incubation at 37ºC. The growth of M.bovis was triggered with the addition of sodium Pyruvate 1% to lowenstein and Jensen medium which resulted in the appearance of visible growth in three weeks. All the isolates produced white, moist, slightly rough and friable colonies with no pigmentation composed of acid fast rods. All these culture were negative to nitrate reduction test and niacin strip test (difco).

On animal inoculation all of the isolates produced fatal disease in rabbits and guinea pigs in about 10 weeks period. The lesions resembled the miliary tuberculosis and were present in almost all organs of the body. The isolates produced a small localized lesions at the site of inoculation in cockerels with no evidences of spreading to regional lymphnodes or generalization. This confirmed that all isolates are M.bovis on the basis of negative reaction to nitrate reduction test and niacin strip test and pathogenicity for rabbits and guinea pigs and not for
cockerels.

DISCUSSION:

Of the 815 buffaloes, 57 (6.91%) gave a positive reaction to mammalian tuberculin and 1(0.12%) reacted positively to avian tuberculin. Out of 185 cows 16 (8.64%) gave a positive reaction to mammalian tuberculin (Table-1). Mycobacterium tuberculosis and M. bovis are genetically and antigenically similar. The avian strain M. avium and John's bacillus (M. paratuberculosis) are antigenically similar. The purified protein derivative of Mycobacterium (PPD-tuberculin) is used for diagnosis of tuberculosis in animals and man. The standard single intradermal comparative tuberculin test (SCITT) using purified protein derivative (PPD) of M. bovis and M. avium was used to detect cattle infected with M. bovis 3.
The overall incidence of 7.3% was recorded in this study. In 1959 the prevalence of tuberculosis in Czech Republic in cattle was on average 21.03% and it reached 32.26% in milking cows, i.e. every third cow was suffering from tuberculosis that was also confirmed in slaughterhouses 12. In a survey conducted in 1975, 10% of the of the buffalo population was found to be suffering from Bovine tuberculosis 1. The status of bovine tuberculosis had been different in various periods. The incidence of bovine tuberculosis recorded in 1969, 1989 and 2001 was 2.2%, 7.3% and 13.8% respectively 13,3,19. The findings of this study are in congruent with the results recorded in 1975 1. The difference of present results with that of findings recorded in 1969, 1989 and 2001 may be attributed to a number of epidemiological factors control and eradication measures adopted during the course of period 2. These animals were housed in open sheds and have frequent mixing / association with the other animals of the area at least once a day (grazing in common country side). Moreover all the animals of cattle colonies have access to single pond for drinking water (stagnant water) where all the excreta and wastes of human houses were drained. The role of such handicaps is pivotal in transmission of infections in third world. These observations are supported by the findings that Mycobacteria can survive and retain infectivity in moist conditions up to 18 days 2.
Bovine tuberculosis occurs all over the world but the problem is more acute in the developing Asian, African and Latin American countries where there is no national control and eradication program. It has been found that now a days in Africa some of epidemiological conditions for the spread of M. bovis infection between animals and humans are very similar to those in Europe in the 1930 6. The same may be true about Pakistan, and most of the other developing countries.

One buffalo gave positive reaction to avian-tuberculin. The animal may have been exposed to M. paratuberculosis or M. avium. The village life man and his animals and birds either live in the same yard, or under the same roof or in close proximity to each other increasing the chances of exposure.
Mycobacterium bovis was isolated from all AFB positive milk and nasal secretion samples (Table: 2). In the present work M. bovis was isolated from the milk of 20 (27.4%) and nasal secretion of 9 (12.33%) of tuberculin positive AFB secretors.
The growth of M. bovis was very slow and it appeared after six weeks of incubation at 37 ºC. The growth of M. bovis was promoted with the addition of 1% sodium pyruvate to Lowenstein and Jensen medium. All of the isolates produced white, moist, slightly rough and friable colonies with no pigmentation. All of the cultures were negative to nitrate reduction and niacin strip test 10. The organisms were classified as M. bovis on the basis of findings that colonies were composed of acid fast rods, no pigmentation and being negative for niacin and nitrate reduction tests 17.
Of the milch animals included in the study 27.4% were passing M. bovis in milk. This points out to a very serious situation with regard to the public health. The milk from these colonies, without pasteurization, is directly supplied to the public. Transmission from cattle to humans is not uncommon. Milk is one of the most important links between Bovine tuberculosis and human beings especially children. Mycobacterium bovis is a highly pathogenic organism and in terms of virulence, transmissibility, stability and antigenicity it possesses great potentials for infecting man 14,20. In its natural host i.e. cattle and buffalo tuberculosis is primarily a respiratory disease and transmission of infection within and between species is mainly by the airborne route 18. The tuberculous animals pass out M. bovis, constantly or intermittently, in their environment 22. Humans associated with infected animals can acquire M. bovis infection.

On animal inoculation all of the isolated strains produced fatal disease in rabbits and guinea pigs, in about 10 weeks time 5. This confirmed that all the isolates were M. bovis in type and possessing of pathgenicity for rabbits and guinea pigs and not for the cockerels 22.
The disease is of paramount importance on account of its zoonotic spread. It has been recorded that M. bovis infection accounted for 1.6% cases of tuberculosis in HIV positive human patients, more over the isolated strains of the M. bovis were showing a uniform pattern of drug-resistant pointing out to some common source of infection 11. Scanty epidemiological information about the incidence of Bovine

tuberculosis in man in the developing world requires immediate attention. It has been observed that no effective vaccine or centralized global surveillance or eradication programs currently exist 7,10. It is an established fact that by elimination of tuberculin positive animals and by pasteurization of milk, the animal to human chain of Bovine tuberculosis is cut off at two points and the incidence of disease in humans is reduced to minimal levels 8.

CONCLUSION:-

In Pakistan tuberculosis is prevalent at epidemic proportions, in animals, humans and maybe in the wildlife. According to the present study incidence of bovine tuberculosis is 6.91% in buffaloes and 8.64% in cows. Mycobacterium bovis the principal cause of bovine Tuberculosis is secreted by the infected animals in milk and nasal secretion. The disease has significant importance on account of its transmission to humans. Mycobacterium bovis infection is acquired by humans mostly through consumption of un-pasteurized milk contaminated with tuberculous bacilli. According to a rough estimation, 20 % patients of tuberculosis visiting various hospitals of Lahore are suffering from extra pulmonary tuberculosis. The present situation is worse as the treatment, even in human beings, is very costly and it is almost impossible in animals. 'Test and slaughter' is not feasible under the existing socio-economic conditions of the country. Eradication of tuberculosis from wild reservoirs is almost impossible. The possible way of bringing down the incidence in animals and humans is by adopting some strict and uniform control measures for the animal and human population of the country. The measure may include:

• Public education for their awareness about tuberculosis.
• Mandatory mass scale vaccination of livestock with BCG-bovine strain vaccine in the country.
• Mandatory tuberculin testing of dairy animals, twice a year, and destruction of all tuberculin-positive animals secreting AFB, with full compensation to the owner by the Government.
• Segregation of all tuberculin positive animals.
• Immediate weaning of all calves of tuberculin positive mothers and vaccination.
• Mandatory pasteurization of milk before marketing,
• Mandatory vaccination of human population with BCG vaccine.
• Enactment of proper laws.

REFERENCES:

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bovis infections in animals and man: a review. Tuber Lung Dis. Jun. 77 (3): 291.
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Table. 1: Result of tuberculin test:

Table.2: Results of AFB presence and isolation:

AFB: Acid fast bacterium
SCITT: Single comparative intra dermal tuberculin test
NS: Nasal secretion.

ACKNOWLEDGEMENTS:

I am very thankful to Dr. Mateen Izhar, Director Indus Laborites Lahore,
Dr. Tayyaba Rasool Chairman Diagnostic Laborites Mayo Hospital Lahore and
Dr. M. Zulfiqar Director VRI Lahore for their cooperation and guidance through out the period of this study.

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