**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

*Molecular Epidemiology Study of Mycobacterium Tuberculosis Complex*

the only documented case of zoonotic TB in Poland.

the biodiversity and evolution of the pathogen.

cal specimens, bypassing the culture step.

**Results of microbiological and molecular testing**

Culture Growth after four weeks on LJ medium Phenotype Sensitive to SM, INH, RMP, EMB, PZA

*Mycobacterium caprae*

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

Sputum

Spoligotyping Hybridization pattern

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

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

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* 

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.* 

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 clini-

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

□■□□□□□□□□□□□□□□■■■■■■□■■■■□■■■■■■■■■■□□□□□

*Number Tandem Repeats*), and WGS (*Whole Genome Sequencing*) [21].

**84**

**Table 2.**

*bovis* BCG strains.

Clinical material

Strain identification

Bacterioscopy ++

(n = 105) and Germany (n = 10) reported higher numbers of outbreaks, while seven outbreaks were noted in Italy and Belgium [28]. Bovine bacilli can cause tuberculosis in other farm species (**Figure 1**). They show high virulence in natural conditions in goats, pigs, sheep and cats [29]; however, the disease is less common in horses and dogs [30, 31] Cattle are not very susceptible to human bacilli, but infections with *M. tuberculosis* are known in this species: one case of bovine tuberculosis due to *M. tuberculosis* has been reported in Poland so far [32]. Wild animals living in the close vicinity of farms can also be a mycobacterial reservoir. The largest reservoir of bovine bacilli in Great Britain is the badger population [33]. However, in Spain, wild boar populations represent the largest reservoir of tuberculosis [34]. The transmission of tuberculosis bacilli occurs in shared pastures, less often as a result of fighting or biting.

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

#### **Figure 1.**

*Diagram illustrating transmission of potential tuberculosis cases caused by mycobacteria from the MTBC complex.*

**87**

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

Department of Microbiology of the National Veterinary Research Institute (NVRI)

All tissue samples are collected *post mortem*, prepared and cultured on Stonebrinck and Petragnani media, as stipulated by the State Veterinary

Inspectorate's Instructions for Laboratory Diagnosis of Bovine Tuberculosis [35]. The culture is also supplemented by a biological analysis performed using two guinea pigs, and the strain extraction procedure is complemented by GenoType

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 [36]. 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 *M. bovis* and 30% with *M. caprae*. In 2010, the first case of tuberculosis in a calf caused by *M. tuberculosis* was confirmed [33]. It should be noted, however, that the status of Poland as being officially free of bovine tuberculosis was never threatened; on the contrary, compared to other European countries, Poland has very favorable data on disease control, particularly consider-

Among the *M. bovis* strains isolated from cattle in Poland, the most common individual spoligotype was SB0856, being present in 44% of the tested strains. In addition, SB0127 and SB0119 were also frequently observed [37]. Among *M. caprae* strains isolated from cattle, the most common spoligotypes were SB0418, SB2390

In 2014, a bovine tuberculosis outbreak was also identified among American bison (*Bison bison*) farmed in Poland [42]. In total, three cases of *M. caprae* strains were isolated, all of which were characterized by the spoligo pattern SB1912. Most cases of TB among free-living animals and exotic animals kept in zoos in Poland are caused by *M. caprae* strains, with spoligotypes SB1912, SB2391 and SB2392

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 (*Sus scrofa* f. *domestica*) kept in the vicinity of a herd in which advanced disease was diagnosed [43]. In 2018, bovine tuberculosis was reported among alpacas of British origin in Poland [44]. In both cases, the strain was identified as *M. bovis* spoligotype

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, *viz.* six antelopes, three giraffes, two tapirs and one alpaca, those from ten animals were identified as *M. bovis* and two were identified as *M. caprae* [37, 39, 40]. Transmission was only confirmed in the antelope herd and between tapirs. Unfortunately, it has been suggested that zoos may withhold epidemiological data, making it very difficult to conduct epidemiological investigations and trace the source of infection among rare and valuable animals threatened with extinction. During a tuberculosis outbreak in the Slaski Ogród Zooologiczny (*Silesia Zoo*), the decision was made to treat a giraffe with active tuberculosis, with unfortunately negative results [41]; this raises the question of whether the research team should have undertaken the treatment of an animal with active tuberculosis, particularly when considering the potential consequences for public health.

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

MTBC (HAIN Lifescience, Germany) typing kits.

ing that almost six million cattle are farmed there.

in Puławy, Poland.

and SB2393 [38].

predominating [38].

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

Department of Microbiology of the National Veterinary Research Institute (NVRI) in Puławy, Poland.

All tissue samples are collected *post mortem*, prepared and cultured on Stonebrinck and Petragnani media, as stipulated by the State Veterinary Inspectorate's Instructions for Laboratory Diagnosis of Bovine Tuberculosis [35]. The culture is also supplemented by a biological analysis performed using two guinea pigs, and the strain extraction procedure is complemented by GenoType MTBC (HAIN Lifescience, Germany) typing kits.

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 [36]. 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 *M. bovis* and 30% with *M. caprae*. In 2010, the first case of tuberculosis in a calf caused by *M. tuberculosis* was confirmed [33]. It should be noted, however, that the status of Poland as being officially free of bovine tuberculosis was never threatened; on the contrary, compared to other European countries, Poland has very favorable data on disease control, particularly considering that almost six million cattle are farmed there.

Among the *M. bovis* strains isolated from cattle in Poland, the most common individual spoligotype was SB0856, being present in 44% of the tested strains. In addition, SB0127 and SB0119 were also frequently observed [37]. Among *M. caprae* strains isolated from cattle, the most common spoligotypes were SB0418, SB2390 and SB2393 [38].

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, *viz.* six antelopes, three giraffes, two tapirs and one alpaca, those from ten animals were identified as *M. bovis* and two were identified as *M. caprae* [37, 39, 40]. Transmission was only confirmed in the antelope herd and between tapirs. Unfortunately, it has been suggested that zoos may withhold epidemiological data, making it very difficult to conduct epidemiological investigations and trace the source of infection among rare and valuable animals threatened with extinction. During a tuberculosis outbreak in the Slaski Ogród Zooologiczny (*Silesia Zoo*), the decision was made to treat a giraffe with active tuberculosis, with unfortunately negative results [41]; this raises the question of whether the research team should have undertaken the treatment of an animal with active tuberculosis, particularly when considering the potential consequences for public health.

In 2014, a bovine tuberculosis outbreak was also identified among American bison (*Bison bison*) farmed in Poland [42]. In total, three cases of *M. caprae* strains were isolated, all of which were characterized by the spoligo pattern SB1912. Most cases of TB among free-living animals and exotic animals kept in zoos in Poland are caused by *M. caprae* strains, with spoligotypes SB1912, SB2391 and SB2392 predominating [38].

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 (*Sus scrofa* f. *domestica*) kept in the vicinity of a herd in which advanced disease was diagnosed [43]. In 2018, bovine tuberculosis was reported among alpacas of British origin in Poland [44]. In both cases, the strain was identified as *M. bovis* spoligotype

*Molecular Epidemiology Study of Mycobacterium Tuberculosis Complex*

(n = 105) and Germany (n = 10) reported higher numbers of outbreaks, while seven outbreaks were noted in Italy and Belgium [28]. Bovine bacilli can cause tuberculosis in other farm species (**Figure 1**). They show high virulence in natural conditions in goats, pigs, sheep and cats [29]; however, the disease is less common in horses and dogs [30, 31] Cattle are not very susceptible to human bacilli, but infections with *M. tuberculosis* are known in this species: one case of bovine tuberculosis due to *M. tuberculosis* has been reported in Poland so far [32]. Wild animals living in the close vicinity of farms can also be a mycobacterial reservoir. The largest reservoir of bovine bacilli in Great Britain is the badger population [33]. However, in Spain, wild boar populations represent the largest reservoir of tuberculosis [34]. The transmission of tuberculosis bacilli occurs in shared pastures, less often as a result of fighting

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

*Diagram illustrating transmission of potential tuberculosis cases caused by mycobacteria from the MTBC* 

**86**

**Figure 1.**

*complex.*

or biting.

SB0666, according to the international spoligotype database (www.Mbovis.org); this type was first isolated in Great Britain in 2003. bTB has also been confirmed in both free-living wild animals and those in breeding centers [45].

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 *M. caprae* causing tuberculosis in animals. In the Act of 11 March, 2004 on the Protection of Animal Health and Control of Infectious Diseases of Animals, Annex 2, bovine tuberculosis is listed as a notifiable disease without a disease-causing pathogen. While the disease is mentioned in the Regulation of the Minister of Agriculture and Rural Development of 23 November 2004 on eradication of bovine tuberculosis, it does not indicate an etiological agent. Despite the Amendment of the Instruction of the Chief Veterinary Officer No. GIWpr-02010/2016 of 8 February 2016, the only pathogenic species listed as causing bovine tuberculosis is *M. bovis*.

Poland was declared OTF in 2009 [47], 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 *M. bovis*/*M. caprae* infections in cattle herds, and the country still meets the formal requirements for a TB-free status.

### **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 (*Meles meles*), wild boar (*Sus scrofa*), wolves (*Canis lupus*) and European bison (*Bison bonasus*) [45, 48, 49].

In recent years, of all species diagnosed with bTB in Poland, the European bison is the most common [50]. A total of 45 cases of tuberculosis were confirmed in European bison in the Bieszczady Mountains during the years 1996–2013 [51]. An autopsy identified generalized tuberculosis in a three-year-old female from a freeliving 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 *Brzegi Dolne* herd were microbiologically confirmed to have tuberculosis and the decision was made to liquidate the entire herd [49]; however, not all animals were culled, and several bison from the herd have still not been found [51].

Other scattered cases have been found in the region. Tuberculosis was confirmed in two European bison in the Bieszczady Mountains in 2005–2008 [52]. In addition, a positive result in the *Górny San* herd from Bieszczady in 2009 resulted in the entire herd of 24 European bison being culled. Tuberculosis-like lesions were found in all individuals, and tuberculosis was microbiologically confirmed in 23 [51]. It is possible that the source of infection for the European bison from the *Brzegi Dolne* herd was locally grazed cattle, while the source of infection in the *Górny San* herd may have been individuals that separated from the *Brzegi Dolne* herd. Unfortunately, as no strains from the *Brzegi Dolne* herd were archived, it

**89**

Forest [64, 65].

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

was not possible to compare the mycobacteria strains between the two herds; this underlines the importance of using molecular methods when studying epidemiology among wildlife. Interestingly, a strain isolated from the *Górny San* was found to have the same spoligotype as one isolated from wild boar from the same area, i.e.

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 *M. caprae*–spoligotype *M. bovis* \_4\_ CA 1600 (octagonal pattern: 200003770003600) (SpolDB4 database) [53]. The source of infection remains unknown due to a lack of archived *Mycobacteria* strains, but there are suspicions

A number of studies have been undertaken recently to address the problems associated with the *ante mortem* diagnosis of tuberculosis in wildlife [54–56]. Such studies have also been conducted in European bison [57]. Although a range of serology methods have been tried [58], the material for direct detection is collected from tracheobronchial lavage, and from swabs and biopsy from retropharyngeal lymph nodes. A more recent approach is to combine microbiological testing with molecular tests, allowing accurate results to be obtained in a much shorter time. In one case, MTBC genetic material was confirmed in laryngeal swab and tracheobronchial lavage using the BD ProbeTec *Mycobacterium tuberculosis* Complex (DTB) Direct Detection Reagent Pack (Becton Dikinson, US) which allows direct detection of mycobacterial genetic material in a clinical specimen [57]. The test acts by amplifying and identifying the target DNA simultaneously. However, the method is characterized by *inter alia* intermittent mycobacterial shedding, which can lead to

With the current situation of bTB in European bison in Poland in mind, it would

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 [59], 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, 60–63], 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 (*Capreolus capreolus*) near Gdańsk and the second in a European bison in Borecka

In the Bieszczady Mountains, the first TB case in wildlife was described in 1996 in a European bison from the Brzegi Dolne Forest District [52]. 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 (*Bison bonasus caucasicus*) (see section 4) [37, 39, 49, 52, 60, 66–68]. Since then, no new TB cases have been detected within this species

clearly be advisable to include molecular methods in routine diagnostics, thus facilitating more accurate epidemiological investigations and more effective disease

**5. Tuberculosis in wildlife in Poland, other than European bison,** 

**including molecular diagnostic methods**

that it may have been acquired from an individual from *Silesia Zoo*.

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

the Bieszczady Mountains [49].

false positive results.

control.

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

was not possible to compare the mycobacteria strains between the two herds; this underlines the importance of using molecular methods when studying epidemiology among wildlife. Interestingly, a strain isolated from the *Górny San* was found to have the same spoligotype as one isolated from wild boar from the same area, i.e. the Bieszczady Mountains [49].

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 *M. caprae*–spoligotype *M. bovis* \_4\_ CA 1600 (octagonal pattern: 200003770003600) (SpolDB4 database) [53]. The source of infection remains unknown due to a lack of archived *Mycobacteria* strains, but there are suspicions that it may have been acquired from an individual from *Silesia Zoo*.

A number of studies have been undertaken recently to address the problems associated with the *ante mortem* diagnosis of tuberculosis in wildlife [54–56]. Such studies have also been conducted in European bison [57]. Although a range of serology methods have been tried [58], the material for direct detection is collected from tracheobronchial lavage, and from swabs and biopsy from retropharyngeal lymph nodes. A more recent approach is to combine microbiological testing with molecular tests, allowing accurate results to be obtained in a much shorter time. In one case, MTBC genetic material was confirmed in laryngeal swab and tracheobronchial lavage using the BD ProbeTec *Mycobacterium tuberculosis* Complex (DTB) Direct Detection Reagent Pack (Becton Dikinson, US) which allows direct detection of mycobacterial genetic material in a clinical specimen [57]. The test acts by amplifying and identifying the target DNA simultaneously. However, the method is characterized by *inter alia* intermittent mycobacterial shedding, which can lead to false positive results.

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 [59], 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, 60–63], 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 (*Capreolus capreolus*) near Gdańsk and the second in a European bison in Borecka Forest [64, 65].

In the Bieszczady Mountains, the first TB case in wildlife was described in 1996 in a European bison from the Brzegi Dolne Forest District [52]. 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 (*Bison bonasus caucasicus*) (see section 4) [37, 39, 49, 52, 60, 66–68]. Since then, no new TB cases have been detected within this species

*Molecular Epidemiology Study of Mycobacterium Tuberculosis Complex*

both free-living wild animals and those in breeding centers [45].

resistance [38, 40, 46].

ing bovine tuberculosis is *M. bovis*.

**of molecular methods**

and European bison (*Bison bonasus*) [45, 48, 49].

bison from the herd have still not been found [51].

SB0666, according to the international spoligotype database (www.Mbovis.org); this type was first isolated in Great Britain in 2003. bTB has also been confirmed in

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

A gap exists in Polish veterinary legislation regarding bovine tuberculosis: so far, it makes no explicit mention of *M. caprae* causing tuberculosis in animals. In the Act of 11 March, 2004 on the Protection of Animal Health and Control of Infectious Diseases of Animals, Annex 2, bovine tuberculosis is listed as a notifiable disease without a disease-causing pathogen. While the disease is mentioned in the Regulation of the Minister of Agriculture and Rural Development of 23 November 2004 on eradication of bovine tuberculosis, it does not indicate an etiological agent. Despite the Amendment of the Instruction of the Chief Veterinary Officer No. GIWpr-02010/2016 of 8 February 2016, the only pathogenic species listed as caus-

Poland was declared OTF in 2009 [47], 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 *M. bovis*/*M. caprae* infections in cattle herds, and

the country still meets the formal requirements for a TB-free status.

**4. Bovine tuberculosis in European bison in Poland and the use** 

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 (*Meles meles*), wild boar (*Sus scrofa*), wolves (*Canis lupus*)

is the most common [50]. A total of 45 cases of tuberculosis were confirmed in European bison in the Bieszczady Mountains during the years 1996–2013 [51]. An autopsy identified generalized tuberculosis in a three-year-old female from a freeliving 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 *Brzegi Dolne* herd were microbiologically confirmed to have tuberculosis and the decision was made to liquidate the entire herd [49]; however, not all animals were culled, and several

Other scattered cases have been found in the region. Tuberculosis was confirmed in two European bison in the Bieszczady Mountains in 2005–2008 [52]. In addition, a positive result in the *Górny San* herd from Bieszczady in 2009 resulted in the entire herd of 24 European bison being culled. Tuberculosis-like lesions were found in all individuals, and tuberculosis was microbiologically confirmed in 23 [51]. It is possible that the source of infection for the European bison from the *Brzegi Dolne* herd was locally grazed cattle, while the source of infection in the *Górny San* herd may have been individuals that separated from the *Brzegi Dolne* herd. Unfortunately, as no strains from the *Brzegi Dolne* herd were archived, it

In recent years, of all species diagnosed with bTB in Poland, the European bison

**88**

in the Bieszczady region [63]. Even so, over the past 20 years, TB has been found in other species of wild animals in the Bieszczady Mountains, mostly in wild boars.

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 *Metastrongylus* spp. invasion. Postmortem examination showed small caseous, yellowish tubercules in submandibular lymph nodes, from which *M.caprae* was isolated (at that time *M. bovis* ssp. *caprae*). The strain was found to have the same spoligo pattern as those strains isolated in 2011 from European bison from the Bieszczady area [45], this being 200003777377400, or SB2391 as assigned by www.Mbovis.org [69].

Since then, a number of cases of TB have been found in the Bieszczady wild boar population each year. Between 2012 and 2017, *M. caprae* was isolated from the lymph nodes of 21 out of 55 investigated wild boar [63]. These strains were subjected to molecular analysis based on spoligotyping according to Kamerbeek et al. [70], and MIRU-VNTR typing, as given in the public protocol [71]. A total of 15 loci were investigated: MIRU 4, MIRU 10, MIRU 16, MIRU 26, MIRU 31, MIRU 40, VNTR 424, VNTR 577, VNTR 2165, VNTR 2401, VNTR 3690, VNTR 4156, VNTR 2163b, VNTR 1955 and VNTR 4052. All 21 isolated strains shared an identical spoligotype 200003777377400 – SB2391. From this group, 19 strains shared a single MIRU-VNTR pattern (464652364413423), while the other two had patterns that differed with regard to a single locus (464552364413423 and 463652364413423) [63].

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. *M. caprae* was isolated from the lymph nodes of one roe deer and three wolves. Those animals had no visible TB-like lesions [48, 63].

All molecular research of *M. caprae* strains isolated from wildlife in the Bieszczady Mountains has been performed based on hsp65 sequence analysis, the GenoType®MTBC (Hain Lifescience, Germany) test, spoligotyping and MIRU-VNTR analysis. Further studies to determine the epidemiological link and the possible route of transmission of the source of infection are needed based on whole genome sequencing.

#### **6. Conclusions**

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 *M. caprae* infection in human. *M. caprae* is the main etiological agent of bovine tuberculosis in wildlife, and *M. bovis* in cattle.

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

**91**

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

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

MIRU-VNTR mycobacterial interspersed repetitive units-variable number

strains of *M. bovis* and *M. caprae* from different species in Poland.

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

The authors declare no conflict of interest.

tandem repeats MTBC *Mycobacterium tuberculosis* complex

RFLP restriction fragment length polymorphism

SIRE streptomycin, isoniazid, rifampicin, ethambutol

OTF officially tuberculosis free PPD purified protein derivative

**Conflict of interest**

**Acronyms and abbreviations**

EMB ethambutol INH isoniazid

LiPA Line probe assays LJ Löwenstein-Jensen MDR multidrug resistant

PZA pyrazinamide

RR rifampicin resistant

TDR totally drug resistant

WGS whole genome sequencing WHO World Health Organization XDR extremely drug resistant

RMP rifampicin

SM streptomycin

TB tuberculosis

AFB acid-fast mycobacteria bTB bovine tuberculosis DRs direct repeat spacers

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

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 *M. bovis* and *M. caprae* from different species in Poland.
