Definitions of drug resistance on MTBC.
Although Poland is officially tuberculosis (TB) free, meaning that less than 0.1% of her cattle herd is TB-positive, the problem of bovine TB in Poland may be re-emerging: its presence has recently been confirmed in domestic and companion animals, wildlife such as the European bison, and even humans. The aim of this chapter was to review all reports of bovine TB in Poland described to date, with particular emphasis on molecular studies, and determine further research directions. These studies include a range of molecular methods for diagnosis, including genotyping, spoligotyping and MIRU- VNTR; such methods successfully identifies a tuberculosis-positive European bison as the source of wild boar infection in the Bieszczady Mountains based on its spoligotype. This chapter argues that identified trains should be better archived, as such records would allow detailed epidemiological investigations and shed greater light on the activity of Mycobacterium spp. The current epidemiological situation in Poland highlights the need for further studies to determine epidemiological links and confirm possible routes of transmission based on whole genome sequencing; this need is accentuated by the zoonotic potential of such infections and the endangered species at risk.
- epidemiological investigation
- European bison
- molecular methods
- Mycobacterium caprae
- Mycobacterium bovis
Bovine tuberculosis is a highly-contagious bacterial disease whose etiological agents are the acid-fast bovine mycobacteria species
2. Tuberculosis in Poland as a zoonosis
Tuberculosis (TB) remains a leading cause of death worldwide. Its treatment requires supervision, efficient and reliable diagnostics, contact tracing and effective therapy. In 2019, 10 million people with tuberculosis were registered worldwide. The incidence of tuberculosis in Poland is slightly higher than the European average, being 13.9/100.000 in 2019 [1, 2].
Although most cases of human TB are caused by the bacterial species
While transmission can take place directly, through the aerogenic route, bovine tuberculosis (bTB) is most commonly transmitted to humans though an indirect route, possibly through unpasteurized milk or dairy products and raw meat. Those at the highest risk of indirect exposure are people exposed to the source of infection at work, such as farmers and veterinarians, and those working with meat, such as slaughterhouse workers and hunters in contact with contaminated animals .
According to estimates by the World Health Organization (WHO), in 2016, 147,000 new cases and 12,500 deaths were associated with zoonotic tuberculosis worldwide. However, such figures are often underestimated due to financial constraints and the consequent lack of adequate routine control in countries where bovine tuberculosis is endemic. Zoonotic tuberculosis tends to be of low prevalence where its presence is correctly monitored in animals and appropriate safe food production procedures are followed [6, 7]. While over two thirds of human TB cases, i.e. those resulting from
Like other bacterial species, the resistance of
In addition to mutations, mycobacteria can develop phenotypic resistance through a change in cell wall permeability, which can impair penetration of the drug into the cell, or by employing efflux pumps, which allow the active removal of the drug from the cell. Metabolic pathways that bypass “drug-sensitive” sites in the cell may also be altered. Regardless of its mechanisms, mycobacterial drug resistance always occurs as the result of a selection process, i.e. a change in the ratio of drug-sensitive to drug-resistant cells [13, 14].
With the growth of research of the
From the epidemiological and therapeutic point of view, the identification of MDR (Multi Drug Resistant), XDR (eXtremely Drug Resistant) and TDR (Totally Drug Resistant) MTBC strains is of key importance [15, 16].
Drug-resistant tuberculosis is more difficult to treat than drug-resistant tuberculosis. Patients do not recover from the standard six-month treatment regimen, but undergo long-term therapy requiring the use of less effective, more toxic and more expensive drugs (Table 1) .
|Drug||Type of resistance|
|Isoniazid + rifampicin|
|Isoniazid + rifampicin + streptomycin|
|Isoniazid + rifampicin + ethambutol|
|Isoniazid + rifampicin + streptomycin + ethambutol|
|MDR + fluoroquinolone + one of the injectable drugs (amikacin or kanamycin or capreomycin)|
|INH + RMP + SM + EMB + fluoroquinolone + aminoglycoside + polypeptide + thioamide + cycloserine + para-aminosalicylic acid|
In Poland and most other developed countries, the threat of bTB in humans decreased significantly in the middle of the 20th Century following the introduction of tuberculosis management strategies . Thanks to the combined implementation of appropriate eradication and surveillance programs, in 2009, Poland was awarded the status of a tuberculosis-free country.
However, in 2020, the first Polish case of bTB in humans was recorded in a retrospective study by Kozińska and Augustynowicz-Kopeć , which described the case of a 46-year-old male detected in 2012 with bacteriologically-confirmed pulmonary infection with
|Results of microbiological and molecular testing|
|Culture||Growth after four weeks on LJ medium|
|Phenotype||Sensitive to SM, INH, RMP, EMB, PZA|
As tuberculosis is an infectious disease with a complex epidemiology and pathogenesis, it is essential to employ molecular typing (genotyping) methods when testing for
Various genotyping methods are used in human and bovine TB research, such as IS
The spoligotyping method takes advantage of a polymorphism within the chromosomal region DR (
Being a PCR-based method, spoligotyping requires very little DNA and thus, can be used to detect and identify
Another advantage of spoligotyping is the ease with which typing results can be recorded, i.e. in binary and octagonal formats, cataloged, and compared in central databases  (SpolDB4, SITVIT WEB, Mbovis. Org databases). It is therefore commonly employed as a screening method in molecular epidemiological investigations. It can be used to identify species within the
However, the detection of tuberculosis transmission foci in closed populations of humans and animals, as well as their interspecific transmission, requires the use of methods with a higher genome differentiation potential. Therefore, spoligotyping studies are commonly complemented by the use of MIRU-VNTR analysis and WGS .
The largest group of VNTR sequences in the
In the MIRU-VNTR method, individual sequences are amplified, and the size of the resulting products depends on the number of repeats of the core unit. For each locus, the number of repeats of the MIRU or VNTR motif is calculated, which allows the results to be cataloged using a 15- or 24-digit MIRU-VNTR code. The MIRU-VNTR method is characterized by high sensitivity and repeatability. It allows the analyzed strains to be differentiated to a large extent, is relatively easy and is distinguished by a short analysis time .
Although spoligotyping, MIRU-VNTR and RFLP have a very high diagnostic value, they are not suitable for accurately determining the dynamics of TB transmission. The spread of tuberculosis may also occur through short contacts, or in a high-risk population where epidemiological links between patients are difficult to establish. In addition, as they screen less than 1% of the genome, standard genotyping techniques therefore have limited discriminatory power and cannot optimally detect potential transmission chains.
These limitations can be circumvented by the use of whole genome sequencing (WGS). WGS provides comprehensive genetic data as well as information on drug resistance, virulence factors, and genome evolution. However, such sequencing analysis requires high expenditure, the possession of specialized equipment and complex bioinformatic analysis of the results .
An accurate confirmation of the molecular relationship of the studied strains, supplemented with epidemiological data, can form the basis for identifying the transmission of infection between closely-related patients, such as family members, as well as among homeless people and immigrant populations, between wild animals and livestock, and between humans and animals. Unfortunately, not all diagnostic laboratories have the appropriate equipment to perform specialist testing based on the analysis of the mycobacterial genome. As a result, current data on the transmission of tuberculosis as zoonosis may well be underestimated.
Preventing the development of zoonotic TB in humans requires reducing the risk of exposure and transmission at the human-animal interface. However, while the principal routes of transmission are known, more information is needed about their underlying sociocultural and economic bases, and how to promote safer alternatives.
3. Epizootic situation of bovine tuberculosis in cattle and other animal species in Poland, and the molecular characteristics of isolated strains
Bovine tuberculosis is an infectious disease that mainly affects cattle. In 2020, seven outbreaks in cattle were recorded in Poland; in the rest of Europe, only France (n = 105) and Germany (n = 10) reported higher numbers of outbreaks, while seven outbreaks were noted in Italy and Belgium . Bovine bacilli can cause tuberculosis in other farm species (Figure 1). They show high virulence in natural conditions in goats, pigs, sheep and cats ; however, the disease is less common in horses and dogs [30, 31] Cattle are not very susceptible to human bacilli, but infections with
In Poland, the largest reservoir of bovine bacilli is believed to be sick cattle. The spread of infection between herds is usually due to the movement of asymptomatic vector animals. Introducing infected animals into a tuberculosis-free herd may cause infection of other animals and disease development in immunocompromised animals. However, following the eradication program carried out in Poland in 1959–1975, its prevalence has significantly fallen, especially in the eastern part of the country. Further progress in the control of the disease in cattle herds has been made possible by the application of strict rules and their consistent enforcement. As in other European countries, Poland operates a special bovine tuberculosis control program, described in detail in the Regulation of the Minister of Agriculture and Rural Development and in the amended Instruction of the General Veterinary Inspector. These documents require the testing of 1/5 of the total cattle population in each county based on bovine and avian purified protein derivative (PPD) tuberculin using both single and comparative tuberculin tests. All positively reactive animals are eliminated, and all samples from these animals are tested in the National Reference Laboratory of Bovine Tuberculosis, located in the Department of Microbiology of the National Veterinary Research Institute (NVRI) in Puławy, Poland.
All tissue samples are collected
The numbers of outbreaks and sick animals in cattle breeding were found to fall during the course of the program, and Poland was recognized as free from bovine tuberculosis in 2009. However, a total of 372 outbreaks were recorded in cattle herds during the following 10-year period, i.e. in 2009–2019. Almost 1/3 of these outbreaks were found in the Masovian Voivodeship, the central region of Poland, especially in its northern part . A significant number of outbreaks were also recorded in the Greater Poland (Wielkopolskie) (n = 68), Lodzkie (n = 28) and Lesser Poland (Malopolskie) voivodeships (n = 24). The smallest number of disease outbreaks concerned the Lubusz (Lubuskie) (n = 2) and Opole voivodeships (n = 1). Molecular studies to date indicate that 70% of cattle suffering from bovine tuberculosis were infected with
However, the incidence of bTB is not limited to cattle. In the period of 2009–2010, cases of bTB were recorded in three zoos in Poland. Of the 12 strains isolated from 12 captive animals,
In 2014, a bovine tuberculosis outbreak was also identified among American bison (
In contrast, sporadic cases of transmission to other species of livestock and domestic animals have been reported. For example, one case was found in pigs (
Animal strains of MTBC have been analyzed for drug resistance to five basic anti-tuberculosis drugs: streptomycin (SM), isoniazid (INH), rifampicin (RMP) and ethambutol (EMB), known as SIRE, and PZA. fortunately, the findings indicate that Polish strains of bTB obtained from animals do not show environmental resistance [38, 40, 46].
A gap exists in Polish veterinary legislation regarding bovine tuberculosis: so far, it makes no explicit mention of
Poland was declared OTF in 2009 , and the fact that the country has remained this way for the subsequent 10 years indicates that the procedures used to control the disease are effective. Only minor incidents have been reported, and they usually occur as a result of incidental errors in anti-epizootic management and the carelessness of animal owners. More importantly, such errors do not seem to have a decisive impact on the overall bovine tuberculosis situation. Poland currently has a consistent policy of eradicating
4. Bovine tuberculosis in European bison in Poland and the use of molecular methods
Even though bTB-positive cattle are considered to constitute the primary reservoir of the bovine mycobacterium in Poland, tuberculosis has also been found in wildlife such as badgers (
In recent years, of all species diagnosed with bTB in Poland, the European bison is the most common . A total of 45 cases of tuberculosis were confirmed in European bison in the Bieszczady Mountains during the years 1996–2013 . An autopsy identified generalized tuberculosis in a three-year-old female from a free-living herd in the Brzegi Dolne Forest District. Around the same time, in the years 1997–2001, 13 out of 18 culled European bison from the same
Other scattered cases have been found in the region. Tuberculosis was confirmed in two European bison in the Bieszczady Mountains in 2005–2008 . In addition, a positive result in the
Cases of bTB have been recorded in captive European bison in Poland: in Warsaw Zoo, Wolisko and the Smardzewice Bison Breeding Centre. Spoligotyping and MIRU-VNTR analysis of the European bison from Smardzewice identified the presence of as
A number of studies have been undertaken recently to address the problems associated with the
With the current situation of bTB in European bison in Poland in mind, it would clearly be advisable to include molecular methods in routine diagnostics, thus facilitating more accurate epidemiological investigations and more effective disease control.
5. Tuberculosis in wildlife in Poland, other than European bison, including molecular diagnostic methods
Currently, no wildlife tuberculosis monitoring program exists in Poland, except when visible lesions suggestive of TB are found in the animal. Despite this, it seems that tuberculosis cases are rarely found in wildlife in Poland and are limited to the area of the Bieszczady Mountains in Southeast Poland: a region bordered by Slovakia and Ukraine, with the highest peak being Tarnica (1346 m a.s.l.). This area is characterized by high forest coverage, low human population and low livestock abundance , unpublished data of the County Veterinary Inspectorate, Ustrzyki Dolne, Sanok]. Between 1996 and 2020, most TB cases in this area were found in European bison and in wild boar [49, 51, 53, 60, 61, 62, 63], and no cases have been reported in domestic animals or livestock since 2005. Outside this region, only two single cases of TB have been described in wildlife in Poland: the first in a roe deer (
In the Bieszczady Mountains, the first TB case in wildlife was described in 1996 in a European bison from the Brzegi Dolne Forest District . Between 1997 and 2013, TB was recorded in a total of 40 European bison in the region, resulting in the culling of two bison herds (
The first case of TB in a wild boar was reported in 2012 in a four-year-old female from Nasiczne in the Bieszczady, which was found dead due to
Since then, a number of cases of TB have been found in the Bieszczady wild boar population each year. Between 2012 and 2017,
To describe the occurrence of TB in wildlife other than European bison and wild boar, both within the Bieszczady Mountain region and elsewhere, lymph node samples were collected for analysis from red foxes, wolves, badgers, red deer, roe deer and brown bear between 2011 and 2017.
All molecular research of
In conclusion, bovine tuberculosis remains a real threat in Poland, as indicated by the increasing number of cases observed in wildlife and the recent report of the first confirmed case of
We recommend that in Poland, bovine tuberculosis should not only be monitored in cattle but also in wildlife. This is especially true in the European bison population, which seems to be highly sensitive to infection. This is highly important for protecting public health, maintaining the OTF status of Poland and of course, protecting the European bison themselves. In which case, particular attention should be paid to the free-living animal population in the Bieszczady Mountains.
There is also a particular need to monitor alpacas, as TB-positive animals pose a particular risk to children and disabled people due to increased contact during animal therapy.
We recommend the more intensive use of molecular tests in monitoring and the proper archiving of the identified DNA. Such molecular methods play an essential role in epidemiological investigations, as these can accurately identify the source of infection and effectively control the disease. Their findings also allow steps to be taken to reduce the spread of infection. Further studies would be of particular value in this regard, particularly those based on whole genome sequencing of archived strains of
Conflict of interest
The authors declare no conflict of interest.
Acronyms and abbreviations
|DRs||direct repeat spacers|
|LiPA||Line probe assays|
|MIRU-VNTR||mycobacterial interspersed repetitive units-variable number tandem repeats|
|MTBC||Mycobacterium tuberculosis complex|
|OTF||officially tuberculosis free|
|PPD||purified protein derivative|
|RFLP||restriction fragment length polymorphism|
|SIRE||streptomycin, isoniazid, rifampicin, ethambutol|
|TDR||totally drug resistant|
|WGS||whole genome sequencing|
|WHO||World Health Organization|
|XDR||extremely drug resistant|