Bovine tuberculosis (bTB) is a slow progressive infectious disease caused by the acid-resistant bovine mycobacteria
It is mandatory to take action against tuberculosis in cattle. Currently, this obligation is primarily imposed by the Veterinary Inspection Act, concerning the duties of veterinary practitioners employed in the State Inspectorate. The legislation relating directly to the control of infectious diseases is the Act on the Protection of Animal Health and Combating Animal Infectious Diseases (2). The most important Polish legislation on bovine tuberculosis is the Ordinance of the Polish Minister of Agriculture and Rural Development of November 23, 2004, defining Polish procedure for disease suspicion, confirmation and eradication of outbreaks of bovine tuberculosis (25). In summary, cattle with confirmed bovine tuberculosis, based on positive tuberculin tests or comparative tuberculin tests, are removed from the herd and then slaughtered. Subsequently the lymph nodes mentioned in the instructions of the Polish General Veterinary Inspectorate and organs with visible lesions are sent to the National Reference Laboratory of Bovine Tuberculosis, which is located in the Department of Microbiology of the National Veterinary Research Institute (NVRI) in Puławy, Poland.
In 2009, Poland attained the status of a country free from this zoonosis, but despite this fact 12 to 33 outbreaks of tuberculosis in cattle have been recorded each year of the last five (18). In 2003–2018, the largest number of cattle infected with bovine mycobacteria were eliminated and the largest number of outbreaks of this zoonosis were recorded in the Masovian Province (the central region of Poland) (Fig. 1). The close vicinity of farms in which bTB was found led to the suspicion that tuberculosis could have been transmitted between the affected herds. According to data from the literature, horizontal infection of animals is fostered by the trading of infected asymptomatic carriers of mycobacteria, use of common pastures, and lack of knowledge about infectious diseases in people involved in animal husbandry (29, 6, 11).
The material for microbiological tests came from 119 cattle (
In the presented studies, spoligotyping of strains was carried out using a commercial Isogen kit (Isogen Bioscience, the Netherlands). The obtained hybridisation patterns were compared to the patterns registered in the international databases Spol DB4 (
MIRU-VNTR typing was carried out based on the analysis of selected polymorphic microsatellite sequences dispersed in the genome of mycobacteria. The DNA of each strain was subjected to 15 amplification reactions using 15 pairs of primers. The obtained results are presented in the form of a 15-digit code (MIRU-VNTR), where each digit depicts the number of repetitions of subsequent MIRU-VNTR repetitive sequences.
Tuberculosis was confirmed in 54 (45%) animals and pathological tubercular lesions were observed in 40 of them (74%). Bovine tuberculosis was characterised by the formation of granulomas (tubercles), which were located mainly in the bronchial and mediastinal lymph nodes. In one animal, tuberculosis was detected in the peritoneum and the mediastinal lymph node, which was four times larger than normal. There were no clinical signs of the infection.
Materials from all examined cows were pooled. A culture test on Stonebrink and Löwenstein–Jensen media was carried out on each sample pool and yielded 54 mycobacterial strains on Stonebrink solid medium. Positive culture results in an MGIT test confirmed the existence of the disease in all cases. A positive niacin test result allowed
A two-stage analysis of the genetic relationship of the isolated strains was made. In the spoligotyping method, 52 strains were identified as SB2220 according to the
Nine MIRU-VNTR patterns identified among 54 strains
No | Spoligotype | MIRU-VNTR type | LOCUS | ||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
4 | 10 | 16 | 26 | 31 | 40 | 424 | 577 | 2165 | 2401 | 3690 | 4156 | 2163 | 1955 | 4052 | |||
1 | BOVIS 595 | 424722356422327 | 4 | 2 | 4 | 7 | 2 | 2 | 3 | 5 | 6 | 4 | 2 | 2 | 3 | 2 | 7 |
2 | BOVIS 595 | 424722357422325 | 4 | 2 | 4 | 7 | 2 | 2 | 3 | 5 | 7 | 4 | 2 | 2 | 3 | 2 | 5 |
3 | BOVIS 595 | 425532355422434 | 4 | 2 | 5 | 5 | 3 | 2 | 3 | 5 | 5 | 4 | 2 | 2 | 4 | 3 | 4 |
4 | BOVIS 595 | 424721374422525 | 4 | 2 | 4 | 7 | 2 | 1 | 3 | 7 | 4 | 4 | 2 | 2 | 5 | 2 | 5 |
5 | BOVIS 595 | 424732357422325 | 4 | 2 | 4 | 7 | 3 | 2 | 3 | 5 | 7 | 4 | 2 | 2 | 3 | 2 | 5 |
6 | BOVIS 595 | 424731357432215 | 4 | 2 | 4 | 7 | 3 | 1 | 3 | 5 | 7 | 4 | 3 | 2 | 2 | 1 | 5 |
7 | BOVIS 595 | 424832354422437 | 4 | 2 | 4 | 8 | 3 | 2 | 3 | 5 | 4 | 4 | 2 | 2 | 4 | 3 | 7 |
8 | BOVIS 595 | 424731374452525 | 4 | 2 | 4 | 7 | 3 | 1 | 3 | 7 | 4 | 4 | 5 | 2 | 5 | 2 | 5 |
9 | BOVIS 595 | 424731374422525 | 4 | 2 | 4 | 7 | 3 | 1 | 3 | 7 | 4 | 4 | 2 | 2 | 5 | 2 | 5 |
Molecular clusters identified among 50 strains by spoligotypes and MIRU-VNTR patterns
No | Spoligotype | MIRU-VNTR (number of strains) | Number of strains in the cluster | Number of herds with strains in the cluster |
---|---|---|---|---|
I | BOVIS 5951 SB22202 | 425532355422434 (21) | 21 | 2 |
II | BOVIS 5951 SB22202 | 424722357422325 (18) 424732357422325 (9) |
27 | 2 |
III | Not registered | 424731374452525 (1) 424731374422525 (1) | 2 | 2 |
1Assigned by Spol DB4
2Assigned by
In all cases, most of the observed anatomo-pathological changes confirm the data from the literature (9, 24). However, it is worth mentioning that these lesions must be considered in the differential diagnosis of animal infection by
The strain genotyping results of this study confirmed the transmission of tuberculosis between cattle herds from these different provinces. The transfer of the disease took place among nine examined herds, located in five provinces. In one case, the herds were 240 km away from each other and a common source of infection could not be identified. In the second case, the herds were 100 km apart and the source of the infection could not be determined in this case either. The third transmission of tuberculosis occurred between herds in the same province on farms 8 km apart. In each described infection event, animal owners did not trade cattle between themselves as confirmed by veterinary inspection. If no animal trade caused the transmission of the disease, it happened as a result of the transfer of the mycobacteria mechanically or by wild animals.
The MIRU-VNTR molecular analysis method also indicated that tuberculosis in the herd may have more than one source of infection, as additionally demonstrated by García de Vidma
Studies to ascertain if there were more than one source of infection in the same herd were also carried out in Poland and the results confirmed that there can be (19). Researchers involved in the molecular diagnosis of tuberculosis in humans also indicated various sources of
Most European countries and some others have their own collections of MIRU-VNTR patterns and on this basis it is possible to follow the transmission of bovine tuberculosis even at the international level. A study of 3,398 isolates from New Zealand, archived in 1982–2008, indicated that
The published epidemiological studies show that
Along with the establishment of the status of Poland as a country free from bovine tuberculosis in 2009, the principles of testing animal populations changed. The period between successive tuberculin tests was extended from three to five years. This longer interval creates greater opportunities for disease transmission in the herd before it is diagnosed. The revised instruction of the Polish Chief Veterinary Officer for the eradication of bovine tuberculosis prevents such a scenario. It grants the possibility to increase the frequency of testing in an area at risk. This allows a flexible response to any prevailing epidemiological situation (14).
As can be seen in Fig. 1, the largest number of outbreaks of tuberculosis in cattle (134) in the last 15 years were found in the Masovian Province, from where the isolated bovine mycobacteria originated. From 10 to 50 outbreaks were found in seven more provinces and up to 10 outbreaks occurred in seven provinces. During the period, there was no province free from bovine tuberculosis in Poland.
Unpublished data testify that the number of animals eliminated as a result of a single outbreak of the disease has significantly increased. In 2018, there were nine outbreaks of tuberculosis in Poland, and the largest number of animals eliminated from one herd was 127.
In conclusion, molecular tests show variety in spoligotype prevalence of