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Institute of Molecular Pathogenesis (IMP)

National Reference Laboratory for Bovine Tuberculosis

(Mycobacterium bovis und Mycobacterium caprae)

Tuberculosis is an infectious disease caused by mycobacteria of the Mycobacterium tuberculosis complex. It is transmissible between humans and animals and is therefore considered a zoonotic disease.

Mycobacteria

Bacteria of the genus Mycobacterium are non-motile, in most cases short bacilli, which often form aggregates. In principle, they can be stained according to Gram. However, due to their strong stability against acids (acid fastness) they can be differentiated from other bacterial species by specific staining methods, e.g. according to Ziehl-Neelsen.

Mycobacteria grow under aerobic conditions and are characterized by long generation times (2-20h). So-called fast-growing mycobacteria take three to five days, slow-growing four (eugonic) to six (dysgonic) weeks and Mycobacterium avium ssp. paratuberculosis even three months or more to form colonies.

Currently, the genus Mycobacterium comprises more than 150 species, some of which have human and animal pathogenic properties.

Significance of mycobacteria as pathogenic agents

Due to their close relationship, the causative agents of tuberculosis are grouped in the „Mycobacterium tuberculosis complex (MTC)“. They do not show any differences in their 16S rRNA gene and therefore taxonomically belong to one species, Mycobacterium (M.) tuberculosis. However, the members of the MTC show differences in other chromosomal regions, in their adaptation to different hosts and in their pathogenicity for individual mammal species or humans. Therefore, they are classified into different species, which are designated based on their primary hosts or first description.
M. tuberculosis (humans)
M. africanum (humans; Africa)
M. bovis (bovines)
M. caprae /M. bovis ssp. caprae (goats)
M. microti (mice)
M. pinnipedii (seals)
These species designations do not mean to imply, however, that infections with the Mycobycterium sp. in question are restricted to the eponymous host species. Mycobacterium sp. may be pathogenic for different hosts.

Being the causative agent of human tuberculosis, M. tuberculosis still is an important pathogen today, as it was when it was discovered by Robert Koch more than 100 years ago; however it is also pathogenic for many animal species (e.g. elephants, primates, tapirs, dogs, psittacine birds), which may become infected if they are kept in close contact with infected humans. Diseases of farm animals caused by this pathogen are rare cases; however, infections leading to immuno-conversion (positive tuberculin test) may occur.

M. africanum is a pathogen that causes human tuberculosis and mainly occurs in African countries.

Bovine tuberculosis (M. bovis) is distributed with a high prevalence in the cattle populations of many countries worldwide.

The significance of bovine tuberculosis for public health was described by a WHO expert committee on tuberculosis in 1950 as follows: The committee recognizes the seriousness of human infection with bovine tuberculosis in countries where the disease in cattle is prevalent. There is danger of transmission of infection by direct contact between diseased cattle and farm workers and their families, as well as from infected food products.

The World Organisation for Animal Health (OIE) has included bovine tuberculosis into its list of notifiable diseases. These are “transmissible diseases that are considered to be of socio-economic and/or public health importance within countries and that are significant in the international trade of animals and animal products“.

In Germany, bovine tuberculosis is a notifiable animal disease. Due to intensive disease control programs that were initiated in West Germany in the early 1950s and in East Germany in the late 1970s it is considered practically eradicated today. From July 1st, 1996, Germany was officially declared free from bovine tuberculosis pursuant to EU Decision 97/76/EC, replaced by EU Decision 99/467/EC. To maintain this status at least 99.9% of cattle holdings must be officially free from tuberculosis in the respective year. Based on this disease-free status, routine nationwide tuberculin testing of cattle for the presence of tuberculosis was stopped. Control is now based on the “Regulation for Protection from Bovine Tuberculosis” (1997), partly modified by the “Regulation on the Modification of the Regulation on Tuberculosis” (2009 and 2013). The regulation is currently still under modification.

Pursuant to these regulations, control is mainly based on the diagnosis of clinical symptoms, official meat inspection and post mortem examination of dead animals by veterinary service laboratories and tuberculin testing in certain cases. In case of clinical suspicion or pathological tissue lesions, diagnostic investigations (bacteriology, molecular biology, epidemiology, tuberculin testing of contact animals, gamma interferon test) are initiated for clarification. To prevent transmission of tuberculosis by milk, pasteurization still is the most efficient protective measure.

M. bovis also plays a role as causative agent of tuberculosis in sheep, goats, horses, dogs, and cats, various zoo animal species and wildlife. In these species, the diagnosis „M. bovis infection“ usually is based on post mortem examination, as immunological intra vitam detection is not well established yet and usually does not generate reliable results. In distinct cases, pathogen detection in feces (secretions, lavages) may be successful depending on the stage and severity of the disease. Human infections with M. bovis (mainly extrapulmonary manifestations) are rather rare cases in Germany today - approx. 2% of tuberculosis cases -, however, the course of these infections may be of similar severity as infections with M. tuberculosis. The same applies to M. caprae. Re-transmission from humans to cattle has been reported as well.

M. microti is mainly detected in small wild animal species (mice, badgers, foxes, martens), but may also cause tuberculois in wild boar, cats, swine and zoo animals as well as in humans, particularly in immunocompromised patients.

The Mycobacterium species of the so-called MOTT complex (Mycobacteria other than tuberculosis), also called non-tuberculous mycobacteria, are widely distributed in the environment and in civilization-related installations, e.g. in soil, water, and aqueducts. In contrast to tuberculosis, the diseases caused by these pathogens are called mycobacterioses. The most important species are those of the M. avium-intracellulare complex (MAIC).

M. avium ssp. avium is the causative agent of avian tuberculosis.

In pigs, increased rates of mycobacteria-induced lymph node lesions occur in animals kept on wood shavings, peat and peat products. In most cases, members of the M. avium-intracellulare complex (in Germany usually M. avium ssp. hominissuis) are isolated as causative agents. Macroscopically and histologically, the lesions cannot be differentiated from M. bovis-induced lesions. M. avium infections belong to the most frequent mycobacterioses in children and immunocompromised patients (HIV, transplantations, tumor diseases).

The probably most important bovine disease caused by a non-tuberculous mycobacterial species (M. avium ssp. paratuberculosis) is paratuberculosis.

In many other animal species (homoiothermal und poikilothermal), non-tuberculous mycobacteria are isolated from a large number of organs and disease processes, in cattle e.g. from cases of chronic, therapy-resistant mastitis. A bacteriological investigation including species determination is required to exclude the presence of tuberculosis.

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Council Directive 92/117/EEC of 17 December 1992 concerning measures for protection against specified zoonoses and specified zoonotic agents in animals and products of animal origin in order to prevent outbreaks of food-borne infections and intoxications stipulates the establishment and designation of reference laboratories in the EU member states, among others also for bovine tuberculosis. With the notification of the national reference laboratories for zoonoses on 13 June 1996 by the Federal Ministry for Health, the National Veterinary Reference Laboratory for Tuberculosis was established in the Department for Bacterial Animal Diseases and Zoonosis Control of the Federal Institute for Consumer Protection and Veterinary Medicine (BgVV), since 2002 part of the Friedrich-Loeffler-Institut.

The tasks of the reference laboratory focus on the following major issues:

  • Support of veterinary service laboratories to clarify suspect cases
  • Establishment and updating of recommended methods for laboratory diagnosis (isolation and differentiation) of the causative agents of tuberculosis
  • Identification of sources of infection
  • Identification of infection chains
  • Organization of ring trials
  • Scientific consulting of policy makers
  • Licensing of diagnostic test kits
  • Differentiation of mycobacterial isolates 
  • Cultivation of mycobacteria from sample materials – species identification by means of polymerase chain reaction (PCR)
  • Direct detection of pathogen-specific DNA from infected tissues by means of a MTC-specific real-time PCR developed at the FLI
  • Genotyping by means of molecular methods:
    • Spoligotyping (differentiation of members of the MTC)
    • VNTR-typing (analysis of repetitive DNA sequences)
    • DNA sequence analysis
    • MTC-specific PCR restriction analysis
  • Identification of sources of infection and investigations in infectious chains
  • Establishment and update of recommended methods for isolation and differentiation of mycobacteria and for direct detection of DNA from tissue in cooperation with the Working Group for Veterinary Infection Diagnostics (AVID) of the German Veterinary Medical Society (DVG)
  • Development of a genus-specific real time PCR for mycobacteria
  • Optimization of DNA extraction from tissue
  • Performance of diagnostic tests for tuberculosis in rural sub-sahara Africa
  • Regulation on the protection against bovine tuberculosis; Notification of 13 March 1997 (Federal Law Gazette I p. 462)
  • Regulation on the modification of the regulation on tuberculosis and other animal disease regulations of 17 June 2009 and 14 March 2013
  • Regulation on notifiable animal diseases of 18 April 2000 (Federal Law Gazette I p. 531)
  • Council Directive 97/12/EC of 17 March 1997 amending and updating Directive 64/432/EEC on health problems affecting intra-Community trade in bovine animals and swine (Official Journal EC No. L 109 p. 1)
  • Commission Decision of 15 July 1999 establishing the officially tuberculosis-free status of bovine herds of certain Member States or regions of Member States and repealing Decision 97/76/EC (Official Journal EC No. L 181 p. 36)