Mycobacterial Infections in Invasive Turtle Species in Poland.

Over the last 30 years, the number of invasive turtle species living in the wild has significantly increased in Poland. This proliferation carries many threats, which mainly include the displacement of native species of animals from their natural habitats. Turtles can also be reservoirs for pathogens, including bacteria from the Mycobacterium genus. In order to confirm or rule out the presence of acid-fast mycobacteria in the population of invasive turtle species, samples from carapace, plastron, internal organs and mouth cavity swabs from 125 animals were tested. Twenty-eight mycobacterial strains were isolated in culture, which were classified as atypical following multiplex-PCR reactions. The GenoType Mycobacterium Common Mycobacteria (CM) test, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, PCR-restriction fragment length polymorphism (PRA)-hsp65 and DNA sequencing were used to identify the species of isolates. Of the 28 strains, 11 were identified as M. fortuitum, 10 as M. chelonae, 3 as M. avium ssp. avium, 2 as M. nonchromogenicum and 1 each of M. neoaurum and M. scrofulaceum. The results of the research will also strengthen the understanding that these animals can be vectors for pathogens when living in the wild.

Pulmonary mycobacteriosis of sitatunga antelope caused by M. avium ssp. hominissuis.

INTRODUCTION AND OBJECTIVE: Mycobacteriosis are diseases caused by acid-fast mycobacteria other than M. leprae and tuberculous mycobacteria. Animal mycobacteriosis is often caused by M. avium ssp. hominissuis. Many species of animals are susceptible to infection with this bacterium, even those kept in Zoological Gardens. The aim of the study was to determine the species of bacterium responsible for causing the disease in the tested animals. MATERIAL AND METHODS: Tissue samples of two male sitatunga antelopes (Tragelaphus spekii) were analyzed. Lymph node and lung samples were subjected to anatomical examination and Ziehl-Neelsen staining. Real-time PCR was performed to confirm or rule out tuberculosis mycobacteria infection. In order to isolate the bacterial strain, tissue samples were inoculated on both solid and liquid media. HainLifescience CM tests, mass spectrometry and New Generation Sequencing were used to determine the mycobacterial species. RESULTS: Results showed that atypical mycobacteria are responsible for the antelope disease. The results of the HainLifescience CM test and mass spectrometry indicated that the mycobacterium responsible for causing mycobacteriosis was M. avium. New Generation Sequencing helped to identified a subspecies that was M. avium ssp. hominissuis. CONCLUSIONS: The sitatunga antelope is an animal susceptible to infection by M. avium ssp. hominissuis. Considering the wide range of hosts and the easiness of interspecies transmission of the pathogen, as well as its zoonotic nature, the mycobacteriosis induced by this microorganism should not be underestimated.

The Potential Risk of International Spread of Mycobacterium Bovis Associated with Movement of Alpacas.

Introduction: The study highlights the transboundary nature of tuberculosis (TB) in alpacas and the failure of current ante-mortem testing protocols (the tuberculin skin and Enferplex Camelid TB tests) to identify TB-free alpaca herds and individuals for export. Our research and the available literature indicate that the alpaca (Vicugna pacos) is extremely susceptible to Mycobacterium bovis infection, and that testing periodicity fails to take into account that animals do not manifest disease symptoms for a long time. The skin test failed to identify Mycobacterium bovis infection in two alpacas prior to their movement from the UK to Poland. The animals were purchased by a breeding centre in Poland, and were then shown at an international animal exhibition. The last owner of the alpacas before their deaths from TB bought the infected animals unwittingly in order to run rehabilitation activities with disabled children on his farm. Material and Methods: Thoracic lymph node, lung and liver tissue samples obtained at necropsy were examined histopathologically after Ziehl-Neelsen staining. Tissue samples were homogenised and mycobacteria present there were cultured on Stonebrink's medium during a 6-week incubation. A commercial test using polymorphism of the chromosomal direct repeat region provided species identification and additional identification was by spacer oligonucleotide typing and mycobacteria interspersed repetitive unit-variable number tandem repeat analysis with a gel electrophoresis protocol. Results: The microbiological examination confirmed multiorgan TB caused by the SB0666 spoligotype of Mycobacterium bovis. Conclusion: Due to the suboptimal performance of current diagnostic tests for TB in alpacas, there is a risk that infected animals may be moved unwittingly. A risk of TB spread associated with the international movement of alpacas is implied by this study.

Infection of a Free-Living Wild Boar (Sus scrofa) with a Bacterium from the Mycobacterium kansasii Complex.

The most numerous group of bacteria in the genus Mycobacterium is the nontuberculous mycobacteria. Currently, over 200 species of bacteria have been classified as belonging to this group, of which approximately 30 are pathogenic to humans and animals. Mycobacterium kansasii complex numbers among these pathogenic species. The submandibular lymph nodes of a wild boar shot by a hunter were examined in order to confirm or exclude infection with bacteria of the genus Mycobacterium. In culture, a bacterial isolate was obtained after 12 days of incubation on Petragnani and Stonebrink media. A multiplex PCR clearly indicated that the isolate was a nontuberculous mycobacterium. The results of species identification attempts via both molecular biology methods and mass spectrometry confirmed that the isolated strain belonged to MKC. The described case of a wild boar infection with MKC is the first documented case in Poland and only the second in Europe, and in confirming the presence of this pathogen among free-living animals, this report implies that MKC is of great concern. Our research elucidates some specifics of wild boar mycobacteriosis and may be used to instill awareness in the public of the dangers of dressing hunt prey or consuming its meat in ignorance of safe procedures, which can contribute to the transmission of the pathogen to humans.

Molecular Characterisation of the Mycobacterium Bovis Causing Bovine Tuberculosis Outbreaks in Poland.

INTRODUCTION: Since 2009, Poland has been recognised as a country officially free of bovine tuberculosis (bTB), although in each year of the last five there were from 8 to 18 outbreaks of the disease. In 2008-2016, the largest number of cattle infected with bovine mycobacteria were eliminated in the Masovian Province (the central region of Poland) and the largest number of outbreaks of this zoonosis were recorded in this area. The close proximity of farms where bTB was found led to the suspicion that tuberculosis could have been transmitted between the affected herds. The aim of the study was the molecular characterisation of the pertinent M. bovis/caprae strains and determination of the epidemiological relationship of various bTB outbreaks. MATERIAL AND METHODS: The material for microbiological tests came from 119 cattle (Bos taurus) from nine herds located in five provinces, neighbouring the Masovian Province. RESULTS: Laboratory tests of tissue material gave results confirming tuberculosis in 54 (45%) animals. All strains belonged to the Mycobacterium bovis species. A two-step analysis of genetic affinity allowed 50 strains to be identified as phylogenetically closely related and separated between three genetic clusters consisting of 2 to 27 strains. CONCLUSION: Based on the results of genotyping, bTB outbreaks were found in three herds, and three transmission chains were identified among these herds.

Transboundary tuberculosis: Importation of alpacas infected with Mycobacterium bovis from the United Kingdom to Poland and potential for serodiagnostic assays in detecting tuberculin skin test false-negative animals.

The present study highlights the transboundary nature of tuberculosis (TB) in alpacas and the failure of current antemortem testing protocols to identify TB-free alpaca herds and individuals for exportation. The tuberculin skin test (TST) failed to identify Mycobacterium bovis-infected animals prior to movement from the United Kingdom (UK) to Poland. This study describes the use of four serological assays [Enferplex Camelid TB, dual-path platform (DPP) VetTB and BovidTB assays, and multi-antigen print immunoassays (MAPIAs)] to detect TB in an alpaca herd with negative TST results. The breeding in Poland purchased alpacas for several years from the UK with the last group arriving in May 2018. In July 2018, two sick alpacas from the centre were hospitalized in a veterinary clinic and both died of TB a few weeks later. In November 2018, 20 alpacas remaining in this M. bovis-affected herd were euthanized and samples were collected. The study population included 20 M. bovis-infected and 20 uninfected alpacas, but only 15 infected animals were tested by all serology tests. The DPP VetTB and DPP BovidTB assays detected antibodies in 14 of the 20 infected alpacas, with results confirmed by MAPIA, and in none (MAPIA and DPP BovidTB) or one (DPP VetTB) of the 20 uninfected animals. None of the infected alpacas tested positive using the Enferplex assay. In addition, the group included three orphans and two cria-dam pairs, which provided an opportunity to analyse immune aspects of cria-mother relationships in this herd. The results suggest high susceptibility of this host species to M. bovis infection and rapid progression to disease. The serological tests used in this study offer useful tools for the detection of M. bovis infection in TST and Enferplex test non-reactive alpacas. These tests should be further evaluated for implementation into TB management and control strategies for camelid species.

Human as a potential vector of bovine tuberculosis in cattle.

INTRODUCTION AND OBJECTIVE: Bovine tuberculosis (bTB, bovine TB) is caused by mycobacteria which are grouped within the MTBC. TB in animals is a highly infectious and progressive disease which can be transmitted to humans. Since 2009, Poland has gained official bTB-free status. Despite the official fact of bTB-free status, a dozen bTB outbreaks are still noted each year. Since 2000 in Poland, every year 1/5 of the national herd is subject to intradermal skin TB testing to control the bTB outbreaks in the cattle population. Application, with 5-year intervals between each government-funded skin test, undoubtedly resulted in financial savings. However it also seems to have caused several adverse and worrying events, e.g. an increase in the number of reactors detected and removed from a single tested herd. The objective of this study was the examination of 898 cattle imputed with bTB infection in Poland between 2008-2012. MATERIAL AND METHODS: The study concerned a potential epidemic outbreak with suspected bTB transmission. 20 cows came from 3 herds in the same county located in the same province in southern Poland. RESULTS: 134 MTBC strains were identified. In MIRU-VNTR, all isolates showed the same genetic pattern 322532243421232. Based on molecular investigation, the characteristics of M. bovis strains isolated from cattle from 3 different herds confirmed the common source of this zoonotic disease. CONCLUSIONS: Although not bacteriologically proven, everything points to the fact that humans were the vector of bovine tuberculosis transmission between herds. This finding confirms transmission between 3 cattle herds in the Malopolskie Province in southern Poland (Podhale). The outbreak of tuberculosis in animals finally compromised public health.

A case of bovine tuberculosis in pigs in Poland - a country free from the disease.

INTRODUCTION: Bovine tuberculosis is a chronic contagious disease caused by Mycobacterium bovis or Mycobacterium caprae. Before widespread action conducted in Poland between 1959-1975 to combat bovine tuberculosis (BTB), about 40% of all tuberculosis cases in pigs was caused by the bovine bacillus. At the present time, correctly carried out, long-term control of cattle has resulted in cases of bovine tuberculosis in pigs and humans being extremely rare and sporadic. In pigs, tuberculosis is most often caught in a slaughterhouse during slaughter. MATERIAL AND METHODS: Samples came from pigs kept on the farm. Traditional bacteriological methods on solid media (Stonebrink, LJ with pyruvate) supported by the semi-automatic, liquid indicative culture method (MGIT) and PCR test were applied in targeted studies. The GenoType Mycobacterium MTBC and CM tests (Hain Lifescience, Germany) were used to additionally confirm that isolated strains classification was used. RESULTS: Strains of mycobacteria were isolated from all examined pigs. Mycobacterium bovis was determined by real time PCR and Hain Genotype methods. CONCLUSIONS: In order to effectively fight against BTB, all animals on farms should be tested, regardless of species, while the milk of suspected cows should be utilized without being used for feed. It is important to adapt the current legal regulations to the current epidemiological situation.

Badgers as a potential source of bovine tuberculosis - first studies in Poland.

Since 2009, Poland has been recognized as a country officially free of bovine tuberculosis (BTB). However, new outbreaks are each year quoted. In many countries it has been shown that badgers (Meles meles) are a vector of Mycobacterium bovis/caprae (M.bovis/caprae) and a source of bovine tuberculosis for many domestical species, mainly for cattle. The aim of the presented study was to determine, for the first time in Poland, the occurrence of tuberculosis in badgers in areas where the disease occurs in cattle. Tissue samples were examined by classical microbiology methods, mycobacteria growth indicator tube (MGIT), and real time PCR. A total of 155 samples from 31 badgers were examined. In any case Mycobacterium bovis/caprae infection has not been diagnosed. This indicates that badgers probably are not a vector of bovine tuberculosis in Poland.

Tuberculosis in Antelopes in a Zoo in Poland--Problem of Public Health.

Bovine tuberculosis is an infectious disease that occurs in many species of both domestic and wild animals, as well as those held in captivity. The etiological factor is the acid resistant bacillus (Mycobacterium bovis or Mycobacterium caprae), which is characterized by the major pathogenicity among mycobacteria belonging to the Mycobacterium tuberculosis complex. The material from 8 antelopes from the zoo, suspected for tuberculosis were examined, and M. bovis strains were isolated from 6 of them. The spoligotyping method showing spoligo pattern 676763777777600. In Poland, this spoligotype has not been observed so far.

Bovine tuberculosis in a wild boar (Sus scrofa) in Poland.

Poland is officially tuberculosis free and bovine tuberculosis (BTB) cases are rarely found except in bovids. We found BTB in a wild boar (Sus scrofa) in the Bieszczady Mountains, southeastern Poland. Studies suggest possible transmission of infection between free-living European bison (Bison bonasus caucasicus) and wild boar in this area.