Mycobacterium tuberculosis complex
Tuberculosis (TB) is the most common fatal infectious disease in humans worldwide. There are different tuberculosis pathogens, the most common pathogen of human tuberculosis is Mycobacterium (M.) tuberculosis. The bacterium can be inactivated by pasteurisation (heating to 72 °C for a short time); however, it is insensitive to dehydration or cold.
Tuberculosis is widespread worldwide, especially in Africa, Asia and Latin America. According to estimates by the World Health Organization(WHO), more than one third of the world's population is infected with tuberculosis(M. tuberculosis). Every year, 1.6 million people die from the infection and around 9 million become newly ill.
In Europe, the pathogen causing bovine tuberculosis(M. bovis) was greatly reduced after World War 2, as a result of which many countries received the officially recognized status of "free of bovine tuberculosis". Austria's cattle population received the status "officially free of bovine tuberculosis" from the EU in 1999, and since then this tuberculosis pathogen has not been detected in any Austrian cattle population. However, since 2008, transmission of M. caprae between red deer and cattle has occurred in individual areas of the provinces of Tyrol and Vorarlberg.
Whether an infection occurs depends on the frequency and intensity of contact, the amount of inhaled or orally ingested pathogens and the physical condition of the affected person. Infection usually occurs through inhalation of fine droplets with the air breathed, which are released during coughing and sneezing by persons suffering from overt tuberculosis. Open pulmonary tuberculosis refers to diseases in which pathogens can be detected in the sputum. Transmission through raw (unpasteurised) milk from infected cattle is possible in principle.
Infection from animal to animal occurs preferentially by the aerogenic route through inhalation of fine air droplets containing the pathogen, which are coughed up by diseased animals. However, it can also occur through contact or orally, e.g. via contaminated feed in feed mangers and salt licks.
Humans: The disease most frequently occurs as pulmonary tuberculosis with the leading symptom cough. If mycobacteria are also coughed up, the disease is referred to as open pulmonary tuberculosis. Other symptoms are nonspecific, and may include fatigue, weakness, loss of appetite and weight loss, swollen lymph nodes, low-grade fever, especially in the afternoon hours, and night sweats, with worsening general condition.
If the infection is fought off by the immune system and the pathogens are closed off, e.g. in nodules (tubercles), it can still happen that the pathogens reactivate under certain circumstances after years and the disease breaks out again as secondary tuberculosis.
As a result of infection with pathogens of bovine tuberculosis, manifestations mainly occurred outside the lungs (extra-pulmonary forms) before the implementation of control programs and pasteurization of milk.
Animal: Chronic pulmonary tuberculosis in cattle is manifested mainly by progressive cough and slowly deteriorating general condition. However, disease processes may also occur in other organs. In cattle, tuberculosis may be latent or subclinical for years. Infected red deer often show no specific symptoms in the early stages of the disease. In advanced cases, red deer may be in poor condition, emaciated, and debilitated.
Since the pathogens are difficult to reach with the drugs, the therapy takes several months and the risk of mycobacteria developing resistance is particularly high. In cases of confirmed tuberculosis, patients must therefore be treated with a combination therapy consisting of several special antibiotics, so-called antituberculotics. The duration of treatment is correspondingly long (over months) in order to avoid possible relapses.
Tuberculosis is a notifiable animal disease. Control focuses on the culling of infected animals.
In 2021, 396 human cases of tuberculosis were reported to the Epidemiological Reporting System (EMS) (as of 05/17/2022), which corresponds to 4.4 cases per 100,000 population. Of these, 296 cases belonging to MTC (NRZ tuberculosis as of 17.05.2022) were confirmed microbiologically. Two cases each caused by M. caprae and M. bovis were detected.
In Austria, bovine tuberculosis is a notifiable animal disease. Since 1999, Austria has been recognized as free of bovine tuberculosis. As of May 2000, nationwide testing of ruminants by means of an intradermal test was discontinued; monitoring of the disease is carried out in the course of ante- and post-mortem inspection. Since 2008, transmission of M. caprae infection between red deer and cattle has occurred in individual areas of the provinces of Tyrol and Vorarlberg during the grazing and alpine pasture period due to the use of the same grazing areas by cattle and red deer. Therefore, to determine the situation in the cattle population, special examination and special surveillance areas (according to the Bovine Tuberculosis Ordinance) are officially designated each year in these regions. In these areas, cattle are examined for tuberculosis by means of a tuberculin test (simultaneous test) before and after the alpine grazing period. These examinations are adapted to the epidemiological situation identified and, if necessary, appropriate area adjustments are made.
In 2022, the bovine tuberculosis pathogen M. caprae (genotype Lechtal) was detected in the NRL for bovine tuberculosis in three cattle from two farms in Vorarlberg (district Feldkirch) after diagnostic killing and in one cattle from one farm in Tyrol (district Reutte) after diagnostic special slaughter. Furthermore, M. microti was detected in one bovine from a farm in Vorarlberg after diagnostic slaughter.
Persons who have close contact with patients with open (i.e., infectious) tuberculosis are particularly at risk. In recent years, there has been a worrying increase in tuberculosis with multidrug-resistant (insensitive at least to the two antituberculotics isoniazid and rifampicin) strains of the pathogen.
After droplet infection, small foci of inflammation usually form in the lungs within the following three to six weeks and encapsulate into nodules (tubercles) (primary infection).
An active infection, usually a reactivation, begins with general symptoms, especially night sweats, increased temperature, fatigue, weight loss, lack of appetite, general feeling of illness. In pulmonary tuberculosis, tissue loss can lead to so-called cavern formation in the lungs. Symptomatic of the disease for this is massive, often bloody sputum, these patient:inside are highly contagious. In rare cases, tuberculous meningitis (meningitis) can occur. One speaks of a miliary tuberculosis when there is a spread via the bloodstream with diffuse infestation of several organ systems, usually also with lung involvement.
The aim of every tuberculosis diagnosis is the cultural detection of the pathogen. This is the only way to identify the resistance pattern and to clarify the outbreak. Due to the danger potential of the Mycobacterium tuberculosis complex, a laboratory with safety level 3 (BSL-3) is required for this purpose. Samples are incubated in the laboratory for up to eight weeks due to the slow growth of the pathogens. Molecular biology methods such as nucleic acid amplification techniques (NAT) shorten the diagnosis time and provide information on the resistance behavior of the pathogen.
To detect infection without disease, the tuberculin skin test can be performed using the Mendel-Mantoux method. Here, the immunological reaction to injected pathogen components is tested. The test becomes positive as early as six weeks after infection. Increasingly, this skin test is being replaced by the so-called interferon-ɣ-release assay (IGRA), a blood test.
Interferon-ɣ-Release Assay (IGRA):
The IGRA test is a diagnostic tool to detect an infection with tubercle bacilli that has occurred. Especially to diagnose latent tuberculosis, the IGRA test is the diagnostic tool of choice. The test becomes positive 2 to 6 weeks after infection. It should be noted that the test cannot distinguish between active and latent tuberculosis. Basically, the IGRA test is used to detect infection with tubercle bacilli in individuals who have had contact with a tuberculosis case (environmental testing), who come from a high-endemic area (geographic region with increased incidence of disease), or before planned immunosuppressive therapy.
The diagnosis of active tuberculosis is usually made by pathogen detection by microscopy, molecular biology methods and culturing (culture), and clinical and radiological assessment. The IGRA test is not designed to prove treatment success; it may remain positive even after successful therapy.
In an IGRA test, the subject's blood is incubated in a blood collection tube coated with Mycobacterium tuberculosis specific antigen (ESAT6 and CFP10). If the subject's immune system has been sensitized to tubercle bacilli (i.e., the subject has had previous contact with tuberculosis pathogens), stimulation of the responsible T lymphocytes results in the production of interferon-gamma, which is measured. The Quantiferon TB Gold Plus test requires 4 ml of blood. It is not necessary to be fasting for the blood draw. Special blood collection tubes are used and filled with 1 ml each. Alternatively, lithium heparin tubes may be used for blood collection and the blood subsequently transferred (within 16 hours) to the Quantiferon Plus tubes. After blood collection, the tubes must be swirled 10 times. Transport to the laboratory must occur within 16 hours of blood collection at room temperature (22 °C ± 5 °C). The samples are then incubated at 37 °C for 16 to 24 hours and can then be stored at 4 - 27 °C for a maximum of 3 days. After incubation, the tubes are centrifuged and can be further processed or stored at 4 °C for a maximum of 4 weeks.
A positive test result indicates that contact with Mycobacterium tuberculosis has occurred. However, the positive IGRA result does not allow any statement about the degree of activity of a disease or even the necessity of a therapy. It is not possible to distinguish whether the person tested has only a latent infection or whether an active disease has developed. Therefore, a positive IGRA test result always requires further diagnostic clarification. A negative result argues against TB infection having occurred and thus against the presence of latent tuberculosis.
Radiographic diagnosis can identify characteristic images of pulmonary infestation, but differential diagnosis cannot exclude some other pulmonary diseases. Therefore, the diagnosis is usually confirmed by combining several investigative procedures.
Detection of mycobacterial nucleic acid provides an initial finding within hours. The time-consuming cultural detection of MTC bacteria confirms the diagnosis of tuberculosis. The advantage of cultural detection is the possibility of testing the mycobacteria for their sensitivity to specific antimicrobial drugs (resistance testing). Obtained isolates are typed by molecular biology.
Molecular biology diagnostics:
In accordance with the latest standards, samples are analyzed by whole genome sequencing (WGS). This allows identification of matching strains and epidemiological clarification of infection chains. In addition, WGS allows the detection of resistance genes and facilitates species assignment within the Mycobacterium tuberculosis complex.
The causative agents of tuberculosis in humans and animals are closely related mycobacterial species grouped as the Mycobacterium tuberculosis complex. This complex includes the described species Mycobacterium (M.) tuberculosis, M. africanum, M. canettii, M. bovis, M. caprae, M. pinnipedii (seals), M. mungi (mongoose), M. orygis (antelope), M. suricattae (meerkat), Dassie Bacillus (klipsheep), and M. microti (mice, secondary hosts e.g., cats, deer, wild boar, camelids, cattle).
In deer, tuberculosis is a chronic disease. Clinical signs, when present, are often nonspecific. The route of infection is usually oral or aerogenic. When the disease generalizes to the regions of the head, thorax, or abdomen, pathogens may be shed in large quantities and, with the potential for transmission to other species, contaminate the environment. Winter feedings of wildlife are problematic: pathogen transmission and lower natural mortality, as well as accumulations of animals in the area of the feedings, promote transmission. Diagnostics in the animal
Early detection of infected cattle is an important issue in the control of bovine tuberculosis and depends on in vivo tests such as the skin and g-interferon test. In Tyrol and Vorarlberg, cattle in certain risk areas for infection with M. caprae (special investigation and special surveillance areas) must be examined annually by means of a simultaneous test (intradermal test). In addition, a blood test (g-interferon test) may be performed. In case of a non-negative test, a diagnostic killing of the cattle is carried out. Direct examination of tissue samples by MTC PCR allows a rapid diagnosis.
Tissue samples from red deer killed during hunting are also examined for the presence of the bovine tuberculosis pathogen M. caprae using MTC PCR and bacterial culture. Selection of animals to be sampled will be based on a sampling design.
Identification of the MTC species and genotyping of the bacterial strains isolated by culture will be performed using various molecular biology techniques (RD4 PCR, DNA strip technology, MIRU VNTR analysis). Due to the classification as a risk group 3 pathogen, the cultivation of the tuberculosis pathogens may only be carried out in the safety level L3 laboratory, the Center for Biological Safety at the National Reference Laboratory for Bovine Tuberculosis in Mödling.
Last updated: 01.06.2023