**1. Introduction**

Bovine tuberculosis is a highly-contagious bacterial disease whose etiological agents are the acid-fast bovine mycobacteria species *Mycobacterium bovis* and *Mycobacterium caprae*. These two species can also cause tuberculosis in humans, although infection with *Mycobacterium tuberculosis* is more common. Although Poland has been an officially tuberculosis free (OTF) country since 2009, cases are still noted in cattle. In addition, *M. bovis* infection has been observed in emergency cases in alpacas and *M. caprae* has been found in endangered European bison (*Bison bonasus*). Tuberculosis infection has been observed in various other wildlife. The aim of this chapter is to present the epizootic situation of bovine tuberculosis in

Poland, including its molecular diagnostics, and to determine which molecular diagnostic methods would be useful in the future.

#### **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 *M. tuberculosis*, this only represents part of a complex that includes various zoonotic forms. According to the latest nomenclature, some of the most prominent members of this complex known to cause disease in humans or/and animals are *M. tuberculosis* (human), *Mycobacterium africanum* (human)*, Mycobacterium canetti* (human), *M. bovis* (cattle and other animals*), M. caprae* (goats, cattle and other animals), *Mycobacterium pinnipedii* (seal), *Mycobacterium microti* (voles and other small rodents) and *M. bovis* BCG (vaccine strain) [3, 4].

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 [5].

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 *M. tuberculosis* infection, primarily affect the lungs [8], zoonotic TB often affects extrapulmonary sites, including lymph nodes and other organs [9]. Since bovine mycobacteria causes clinical, radiological and pathological symptoms that are similar to *M. tuberculosis*, these strains can be distinguished only by bacterial culture, by biochemical and morphological analysis, and by genotyping.

*M. bovis* used to be differentiated from other complex members based on its resistance to pyrazinamide (PZA); however, following the discovery of PZAsusceptible strains of *M. bovis*, the species was split into two subspecies: the PZAresistant *M. bovis* subsp. *bovis*, and the PZA-sensitive *M. bovis* subsp. *caprae* [10, 11]. PZA is one of the four essential drugs used in the current standard first-line anti-TB treatment regimen. However, as most healthcare providers initiate treatment without performing any drug susceptibility testing, patients with zoonotic TB caused by *M. bovis* may demonstrate poorer treatment outcomes and may develop further resistance to other anti-TB drugs; for example, additional resistance to rifampicin and isoniazid has been detected in some *M. bovis* isolates [12].

Like other bacterial species, the resistance of *Mycobacterium tuberculosis* complex members to antimycobacterial drugs arises from the selection of naturally-resistant mutants that are constantly present in every bacterial population. Wild strains of mycobacteria belonging to the *M. tuberculosis* complex that have never been

**83**

**Table 1.**

*Molecular Characterization of* Mycobacterium *spp. Isolated from Cattle and Wildlife in Poland*

exposed to drugs are naturally sensitive to tuberculostats, with one exception; the

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

With the growth of research of the *Mycobacterium* genome and its drug resistance pathways, the main mechanisms and genes by which mutations cause drug resistance have been recognized. Currently, commercial tests based on PCR reactions are used to detect the most common mutations determining the resistance of mycobacteria to antibiotics that are crucial in treatment. One such example is the line probe assay (LiPA); these employ targeted amplification of specific regions of the MTB genome using biotinylated primers followed by reverse hybridization of the amplicons to oligo probes immobilized on nitrocellulose strips. Hybridization is then detected by a colorimetric reaction. Currently, the most widely used tests are those developed by Hain Lifescience (Nehren, Germany): Genotype MTBDRplus and Genotype MTBDRsl, detecting resistance to the most important antituberculo-

From the epidemiological and therapeutic point of view, the identification of MDR (Multi Drug Resistant), XDR (eXtremely Drug Resistant) and TDR (Totally

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

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 [18]. Thanks to the combined implementation of appropriate eradication and surveillance programs, in 2009, Poland

However, in 2020, the first Polish case of bTB in humans was recorded in a retrospective study by Kozińska and Augustynowicz-Kopeć [19], which described the case of a 46-year-old male detected in 2012 with bacteriologically-confirmed pulmonary infection with *M. caprae*. Changes typical for pulmonary TB were

**Drug Type of** 

Isoniazid + rifampicin **MDR**

MDR + fluoroquinolone + one of the injectable drugs (amikacin or kanamycin or

INH + RMP + SM + EMB + fluoroquinolone + aminoglycoside + polypeptide + thioamide +

**resistance**

**XDR**

**TDR**

Drug Resistant) MTBC strains is of key importance [15, 16].

was awarded the status of a tuberculosis-free country.

*DOI: http://dx.doi.org/10.5772/intechopen.96695*

of drug-sensitive to drug-resistant cells [13, 14].

sis drugs of the I- and II- lines [13].

more expensive drugs (**Table 1**) [17].

Isoniazid + rifampicin + streptomycin Isoniazid + rifampicin + ethambutol

cycloserine + para-aminosalicylic acid

*Definitions of drug resistance on MTBC.*

capreomycin)

Isoniazid + rifampicin + streptomycin + ethambutol

PZA-resistant *M. bovis*.

*Molecular Characterization of* Mycobacterium *spp. Isolated from Cattle and Wildlife in Poland DOI: http://dx.doi.org/10.5772/intechopen.96695*

exposed to drugs are naturally sensitive to tuberculostats, with one exception; the PZA-resistant *M. bovis*.

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 *Mycobacterium* genome and its drug resistance pathways, the main mechanisms and genes by which mutations cause drug resistance have been recognized. Currently, commercial tests based on PCR reactions are used to detect the most common mutations determining the resistance of mycobacteria to antibiotics that are crucial in treatment. One such example is the line probe assay (LiPA); these employ targeted amplification of specific regions of the MTB genome using biotinylated primers followed by reverse hybridization of the amplicons to oligo probes immobilized on nitrocellulose strips. Hybridization is then detected by a colorimetric reaction. Currently, the most widely used tests are those developed by Hain Lifescience (Nehren, Germany): Genotype MTBDRplus and Genotype MTBDRsl, detecting resistance to the most important antituberculosis drugs of the I- and II- lines [13].

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**) [17].

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 [18]. 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 [19], which described the case of a 46-year-old male detected in 2012 with bacteriologically-confirmed pulmonary infection with *M. caprae*. Changes typical for pulmonary TB were


#### **Table 1.**

*Definitions of drug resistance on MTBC.*

*Molecular Epidemiology Study of Mycobacterium Tuberculosis Complex*

diagnostic methods would be useful in the future.

rodents) and *M. bovis* BCG (vaccine strain) [3, 4].

**2. Tuberculosis in Poland as a zoonosis**

age, being 13.9/100.000 in 2019 [1, 2].

contaminated animals [5].

Poland, including its molecular diagnostics, and to determine which molecular

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 aver-

Although most cases of human TB are caused by the bacterial species *M. tuberculosis*, this only represents part of a complex that includes various zoonotic forms. According to the latest nomenclature, some of the most prominent members of this complex known to cause disease in humans or/and animals are *M. tuberculosis* (human), *Mycobacterium africanum* (human)*, Mycobacterium canetti* (human), *M. bovis* (cattle and other animals*), M. caprae* (goats, cattle and other animals), *Mycobacterium pinnipedii* (seal), *Mycobacterium microti* (voles and other small

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

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 *M. tuberculosis* infection, primarily affect the lungs [8], zoonotic TB often affects extrapulmonary sites, including lymph nodes and other organs [9]. Since bovine mycobacteria causes clinical, radiological and pathological symptoms that are similar to *M. tuberculosis*, these strains can be distinguished only by bacterial culture, by biochemical and morphological analysis, and by

*M. bovis* used to be differentiated from other complex members based on its resistance to pyrazinamide (PZA); however, following the discovery of PZAsusceptible strains of *M. bovis*, the species was split into two subspecies: the PZAresistant *M. bovis* subsp. *bovis*, and the PZA-sensitive *M. bovis* subsp. *caprae* [10, 11]. PZA is one of the four essential drugs used in the current standard first-line anti-TB treatment regimen. However, as most healthcare providers initiate treatment without performing any drug susceptibility testing, patients with zoonotic TB caused by *M. bovis* may demonstrate poorer treatment outcomes and may develop further resistance to other anti-TB drugs; for example, additional resistance to rifampicin and isoniazid has been detected in some *M. bovis* isolates [12].

Like other bacterial species, the resistance of *Mycobacterium tuberculosis* complex members to antimycobacterial drugs arises from the selection of naturally-resistant mutants that are constantly present in every bacterial population. Wild strains of mycobacteria belonging to the *M. tuberculosis* complex that have never been

**82**

genotyping.

identified on chest X-ray, and a tuberculin test result of 18 mm was obtained. In addition, direct staining of sputum revealed the presence of acid-fast mycobacteria (AFB ++), and *Mycobacterium* colonies were identified after four weeks of culture on Löwenstein-Jensen (LJ) medium. Initial identification in the hospital laboratory confirmed that the isolated strain belonged to the *M. tuberculosis* complex. Phenotypic and molecular methods revealed drug susceptibility, and the strain was thus classified to the species *M. caprae*. Further genotyping identified the unique spoligotype 200003757377600; although this strain was not registered in the international spoligotype databases SpolDB4 and SITVIT WEB, it was found to match SB1690 in Mbovis. Org, this being a Spanish isolate from 2009 (**Table 2**) [20]. The source of infection remained unknown: the patient's history revealed that he had not had recent contact with any person with tuberculosis, nor had he been close to farm animals which had not been tested for tuberculosis. Until now, this has been the only documented case of zoonotic TB in Poland.

As tuberculosis is an infectious disease with a complex epidemiology and pathogenesis, it is essential to employ molecular typing (genotyping) methods when testing for *M. tuberculosis*: such tools are fundamental for guiding effective epidemiological research, defining the dynamics of transmission, and enabling global surveillance of the disease. In addition, genotyping provides an insight into the biodiversity and evolution of the pathogen.

Various genotyping methods are used in human and bovine TB research, such as IS*6110*-RFLP (*Insertion Sequence 6110-Restriction Fragment Length Polymorphism*), spoligotyping, MIRU-VNTR (*Mycobacterial Interspersed Repetitive Units-Variable Number Tandem Repeats*), and WGS (*Whole Genome Sequencing*) [21].

The spoligotyping method takes advantage of a polymorphism within the chromosomal region DR (*Direct Repeat*) found in mycobacteria belonging to the *M. tuberculosis* complex. This region, first described by Hermans in the *M. bovis* BCG P3 strain, is formed by a variable number of direct repeat (DR) sequences, 36 bp long, with short (35–41 bp) unique spacer sequences between them [22]. The spacer sequences are detected by synthetic oligonucleotide probes complementary to the 43 known sequenced spacer sequences identified in *M. tuberculosis* H37Rv and *M. bovis* BCG strains.

Being a PCR-based method, spoligotyping requires very little DNA and thus, can be used to detect and identify *M. tuberculosis* complex bacteria directly in clinical specimens, bypassing the culture step.

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


**85**

alternatives.

*Molecular Characterization of* Mycobacterium *spp. Isolated from Cattle and Wildlife in Poland*

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

The largest group of VNTR sequences in the *Mycobacterium* genome are the 46–100-nucleotide MIRU fragments. Of these 15 to 24 known *loci* with the highest

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

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

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

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

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

**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

transmission of tuberculosis as zoonosis may well be underestimated.

variability were selected for the genetic typing of mycobacteria.

databases [23] (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 *M. tuberculosis* complex, provide information regarding the lineage of various strains, determine their placement in major genetic families and indicate the directions of the global spread of molecular

*DOI: http://dx.doi.org/10.5772/intechopen.96695*

distinguished by a short analysis time [26].

detect potential transmission chains.

complex bioinformatic analysis of the results [27].

families of mycobacteria [24].

WGS [25].

#### **Table 2.**

*Characteristics of* Mycobacterium caprae *– The first human isolate in Poland.*

#### *Molecular Characterization of* Mycobacterium *spp. Isolated from Cattle and Wildlife in Poland DOI: http://dx.doi.org/10.5772/intechopen.96695*

databases [23] (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 *M. tuberculosis* complex, provide information regarding the lineage of various strains, determine their placement in major genetic families and indicate the directions of the global spread of molecular families of mycobacteria [24].

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 [25].

The largest group of VNTR sequences in the *Mycobacterium* genome are the 46–100-nucleotide MIRU fragments. Of these 15 to 24 known *loci* with the highest variability were selected for the genetic typing of mycobacteria.

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 [26].

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 [27].

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.
