Diagnosis of Bovine Tuberculosis: An Overview

Abstract

India is the largest milk producer in the world tonnes and stands first in terms of milch cattle. Yet one of the major challenges faced by the livestock sector is the increased incidence of tuberculosis in cattle. Bovine tuberculosis can be transmitted to humans by consumption of raw/unpasteurized milk or vice versa where human and animal association co-exists. Presumptive and early diagnosis  are  essential to have a control over the disease. Preventive and control measures involves frequent monitoring and screening in endemic areas where there is occurrence of human animal interaction.

Introduction

Bovine Tuberculosis (bTB) is caused by M. bovis belongs to the member of Mycobacterium tuberculosis complex (MTBC) which also includes M. tuberculosis, M. bovis BCG, M. africanum, M. canetti, M. microti, M. caprae and M. pinnipedii (1). M. tuberculosis causes TB in humans and animals where human and association is common. M. bovis causes bovine tuberculosis that affect cattle, human, sheep, goats, pigs and wild animals. Drug resistant tuberculosis in humans is day by day increasing. Incidence on drug resistance in animals are rare due to the fact that animals are not treated for tuberculosis. But this scenario is changing due to the possibility of reverse zoonosis in endemic regions, drug resistant M. tuberculosis from humans may end up as spill – over infection to cattle due to the close human animal interaction (2).

Transmission

Transmission varies depending upon the localization of infection in animal tissue or organs. In open pulmonary lesion, tubercle bacilli are discharged and disseminated by the aerosol route but if it is swallowed it is excreted in faeces. But 90-95% of infected cattle transmit the infection by aerosol route. Menzies and Neill (2000) suggested that probability of single bacillus within a droplet nucleus was sufficient to establish infection within the bovine lung (3).

The route of transmission of  M. bovis from cattle to human is mainly to ingestion of raw milk and rarely through direct contact (via aerosols) (4,5,6). In India, farmers, dairy workers and livestock keepers are in close contact with the animals in their routine life while feeding and milking (7). Even a single cow can excrete sufficient viable mycobacteria in milk to make even pooled milk infective (8).

Recently, transmission at the interface between humans, domestic animals and wild life has increased. Once a disease became endemic in a wildlife population, disease control becomes a much more challenging issue.

Clinical Presentation

The disease caused by M. bovis was indistinguishable to that caused by M. tuberculosis (9). Tuberculosis is a chronic debilitating disease, early infections are often asymptomatic but in later stages signs will be noticed when many organs are involved. Pulmonary involvement usually produces moist cough that is worse in the morning, cold weather or exercise, low-grade fluctuating fever, weakness and inappetence. In terminal stages, animal will be extremely emaciated and have acute respiratory distress. In some animals, lymphnode may enlarge and rupture and drain. If digestive tract is involved intermittent diarrhoea and constipation may be seen (10).

Diagnosis

1. Samples to be collected: In a study, screening of blood, nasal mucus and milk samples revealed that all 3 sample types from a single infected animal were never positive. Nevertheless, nasal mucus was the sample with highest number of Mycobacterium organism. This was probably because tuberculosis infection occurs mainly by the respiratory route and thoracic lesions were much more frequent than those located in the mesenteric region or mammary gland (11). Other sample could be the blood, fine needle aspirates of prescapular lymph gland (PSLG), milk, pharyngeal swab and faecal samples. Among that PSLG were found to be the most suitable specimen for isolation of tuberculosis complex from cattle and were thus of diagnostic importance (12). It was recommended that, a minimum pooled lymphnode samples from head and thorax could be cultured when no visible lesions are detected at post-mortem examination (10).
2. Direct Smear examination: Acid-Fast Bacillus (AFB) staining provided rapid evidence for the presumptive diagnosis of TB in clinical specimens. AFB staining was used by most health care facilities to determine patients with suspected TB. (13). The only limitation with direct smear examination was it’s lack of sensitivity because large number of organisms (more than 10⁴ mycobacteria/ml) must be present to make detection reliable (14).
3. Isolation: Specimens which are used for isolation of mycobacteria may contain other contaminants. The most commonly used method of decontamination is modified Petroff’s method (4% NaOH), a simple, inexpensive technique which will cause liquefaction of the specimen. All the decontamination methods also reduce the mycobacterial population. Decontamination should be followed by neutralization with acid or sterile water (15).

Egg based media with glycerol (Lowenstein Jensen or agar based medium supplemented with bovine albumin- Middlebrook 7H10 or 7H11) allowed abundant growth of M. tuberculosis. M. tuberculosis colonies were buff coloured, rough having the appearance of bread crumbs or cauliflower. In primary culture tubercle bacilli did not grow in less than one week and usually required two to four weeks to give visible growth (16).

The solid media took long time to grow and to see the visible growth but by the use of automated or semi-automated liquid culture system which can detect the growth much earlier than the naked eye. BACTEC 460TB was able to detect the radiolabelled CO_2, produced by the metabolising organism produced by adding radiolabelled C-palmitate. Newer systems that rely on non-radiometric detection of growth such as BACTEC 9000 and MGIT (Mycobacterial Growth Indicator Tube) are also available (17).

4. Molecular Characterization of MTBC

IS6110 PCR: The IS6110 element was usually present in multiple copies (usually 10-20 copies) in M. tuberculosis and in some isolates only single copy was present. M. bovis and M. bovis BCG carried one or two copies of IS6110 element (18).

5. Molecular differentiation of bovis and M. tuberculosis

A single copy of species specific mtp40 genomic fragment present   in  M. tuberculosis was utilized for the species specific identification of  M. tuberculosis and it was a possible alternative for specific direct detection of    M. tuberculosis (19).

Regions of difference (RD1 to RD16) were deleted in the members of MTBC other than M. tuberculosis. This could accurately differentiate the members of MTBC from M. tuberculosis and also reported that RD1, RD9 and RD 10 were sufficient for initial screening followed by the use of RD3, RD5 and RD11 if the results of first three regions were found to be negative (20).

In 12.7kb multiplex PCR,  M. bovis lacks a 12.7-kb fragment present in the genome of M. tuberculosis (21).

6. Drug Resistance In Bovine Tuberculosis

Drug Susceptibility Testing (DST)

Proportion method Due the emergence of MDR-TB there was an urgent need for drug susceptibility testing (DST) for early diagnosis for starting the regimen, patientmanagement and also preventing the transmission in the population especially in humans. Since therapy was not feasible in animals, DST was not attempted for animal tuberculosis. DST could be used with less resource, with the use of LJ medium or Middlebrook agar and it required 6-8 weeks to yield the result (22). The demand for DST increased with the need for an appropriate treatment of MDR-TB.

Minimum Inhibitory Concentration (MIC)

Broth microdilution method (BMM): Wallace et al. (1986) devised microdilution method for slow growing mycobacteria using 7H9 broth and also with regard to M. tuberculosis BMM was the most useful method for drug susceptibility testing and also they reported that this method was not likely to allow direct susceptibility testing because of the problem of contamination but it is easy for evaluation of all first and second line drugs as well as level of resistance (23).

Prevention and Control

Bovine tuberculosis continued to be major problem in countries especially in India where test and slaughter principles are not affordable and these programs are partially effective because wild life act as reservoir host and it maintains the infection. Control programs have to concentrated more in areas where human and animal habitat co-exist because of emerging threat of drug resistant tuberculosis.

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