Epidemiological characterization of Mycobacterium caprae strains isolated from wildlife in the Bieszczady Mountains, on the border of Southeast Poland.

BACKGROUND: The majority of animal tuberculosis (TB) cases reported in wildlife in Poland over the past 20 years have concerned the European bison inhabiting the Bieszczady Mountains in Southeast Poland: an area running along the border of Southeast Poland. As no TB cases have been reported in domestic animals in this region since 2005, any occurrence of TB in the free-living animals inhabiting this area might pose a real threat to local livestock and result in the loss of disease-free status. The aim of the study was to describe the occurrence of tuberculosis in the wildlife of the Bieszczady Mountains and determine the microbiological and molecular characteristics of any cultured strains. Lymph node samples were collected for analysis from 274 free-living animals, including European bison, red foxes, badgers, red deer, wild boar and roe deer between 2011 and 2017. Löwenstein-Jensen and Stonebrink media were used for culture. Molecular identification of strains was performed based on hsp65 sequence analysis, the GenoType®MTBC (Hain Lifescience, Germany) test, spoligotyping and MIRU-VNTR analysis. RESULTS: Mycobacterium caprae was isolated from the lymph nodes of 21 out of 55 wild boar (38.2%; CI 95%: 26.5%, 51.4%) and one roe deer. Since 2014, no new TB cases have been reported in the Bieszczady European bison population. CONCLUSIONS: The identification of TB in wild boar in the Bieszczady is an alarming phenomenon, which requires further investigation. The Bieszczady mountains are a precious, unique area, home to many protected species. However, it is also the only area in Poland where TB cases have been reported in free-living animals. The occurrence of TB in wild boar inhabiting this area might pose a real threat to local livestock and many of the protected species (for example European bison that can share feeding places with wild boar). Given this situation, ongoing monitoring of the prevalence of TB should be conducted, and protective measures should be considered.

Molecular methods in diagnostics of post-BCG vaccine adverse events.

INTRODUCTION: Bacillus Calmette-Guérin (BCG) is the only tuberculosis vaccine available and although it has been routinely used for more than 80 years, its protective effect varies depending on the age and the form of tuberculosis. Due to the close analogy between the vaccine strain and other species of the Mycobacterium tuberculosis complex, molecular methods are recommended for differential diagnosis of post-BCG complications. The aim of the study was to assess usefulness of molecular methods in diagnosis of post-BCG vaccine adverse events (VAEs). MATERIAL AND METHODS: M. tuberculosis complex strains obtained in 2011-2017 from 68 ill children were subjected to molecular analysis. RESULTS: Molecular analysis of 68 strains showed 100% agreement between the results in the GenoType MTBC method and the multiplex PCR method. For the strains isolated from 45 patients with suspected VAE, M. bovis BCG was obtained, whereas the strains isolated from the remaining 23 children were identified as M. tuberculosis. The analysis confirmed the close relationship between the result of identification and the type of material as well as the patient's age. CONCLUSIONS: The use of genetic methods enables quick and detailed diagnostics of infections caused by M. bovis BCG, which allows for the confirmation or exclusion of VAE.

From Latent Tuberculosis Infection to Tuberculosis. News in Diagnostics (QuantiFERON-Plus).

It is estimated that one third of the world's population have latent tuberculosis infection and that this is a significant reservoir for future tuberculosis cases. Most cases occur within two years following initial infection. The identification of individuals with latent tuberculosis infection is difficult due to the lack of an ideal diagnostic assay and incomplete understanding of latent infection. Currently, there are three tests: the oldest tuberculin skin test, T-SPOT.TB and the latest QuantiFERON-Plus for the detection of Mycobacterium tuberculosis infection. The interpretation of the test results must be used in the conjunction with a patient's epidemiological history, risk assessment, current clinical status, radiography and microbiological methods to ensure accurate diagnosis.

PCR-based genomic deletion analysis of RD-regions in the identification of mycobacteria isolated from adverse events following BCG vaccination or TB suspected cases.

Early identification of mycobacterial species is crucial for early diagnosis. PCR-multiplex method performed on randomly chosen 54 mycobacteria isolates originating from clinical samples was found to be an inexpensive, quick and reliable alternative for commercially available diagnostics tests. Although the results of gene probes identification performed by NTLDR were generally consistent with multiplex PCR, two mixed Mycobacterium bovis BCG/Mycobacterium tuberculosis infections and a single misdiagnosis of M. tuberculosis with M. bovis were found. The routine application of multiplex-PCR has the potential to make diagnostics surveillance studies feasible.

Mycobacterium avium and Klebsiella pneumoniae co-infection in the domestic ferret (Mustela putorius furo) - Case Report.

Introduction and Objective. Pets infected with zoonotic pathogens might become a source of infections for their owners, especially those who are immuno-compromised. The aim of this report is to describe a case of chronic, untreatable pneumonia in a domestic ferret. Materials and method. The subject was a 5-year-old female ferret suffering from recurrent pneumonia. Ante-mortally, swabs from the nasal cavity, alveolus and throat were collected from the animal. Post-mortally, lesioned organ fragments were collected. Standard microbiological testing was performed. Additionally, mycobacterial diagnosis including culture and molecular tests was performed. Results. The co-infection of Mycobacterium avium and Klebsiella pneumoniae was microbiologically confirmed. Conclusions. This case demonstrates the need to pay attention to the possibility of zoonotic pathogens in ferrets. Veterinarians diagnosing ferrets are potentially exposed to Mycobacteria spp. infections and other pathogens.

Mycobacterium caprae tuberculosis in a captive lion in Ukraine - Case Report.

Introduction and Objective. Because of the armed conflict in Ukraine, companion, farm and captive wild animals have been moved in a simplified procedure across the Polish-Ukrainian border. For that reason, in 2022, Poznan Zoo provided support for almost 200 wild animals before movement to facilities in other countries. The aim of the study is to identify the under-recognized risk of moving animals that may be infected with zoonotic pathogens, such as Mycobacterium caprae, between countries. Materials and Method. Sections of the heart, lungs, and mesentery of 4-year-old lioness from Ukraine were taken for histopathological and bacteriological examination. Results. Microbiological examinations confirmed the presence of Mycobacterium caprae, SB0418 spoligotype. Conclusion. TB is a zoonotic disease present globally. Movement of captive wild animals from regions with MTBC cases, or lack of MTBC surveillance, such as UA may pose a potential threat to public health.

Multiplex-PCR as an identity assay for Mycobacterium bovis BCG Moreau descendants.

In the study, we assessed the identity of locally produced BCG vaccine via screening for the presence of genetic markers specific for particular Mycobacterium bovis BCG substrains - RD8, RD2, senX3-regX3, RD14, RD16, ΔRD1, DU2, a second copy of IS6110, mutation D322G in phoR, and deletions in fadD26-ppsA and Rv3887c regions. In order to increase the specificity of the multiplex-PCR test for locally produced BCG vaccine, we have modified previously developed primer sets by the introduction of a primer pair specific for deletion in Rv3887c. The modified multiplex-PCR specifically and reproducibly distinguished both BCG Moreau sublineages, and allowed, with no decrease in power, differentiation of BCG substrains of different origin. The growing knowledge of genetic differences among BCG vaccine strains enables improvements in the specificity of identity tests that will be useful both for routine release of vaccines and potential applications in clinical practice. Modified multiplex-PCR accompanied by PFGE analysis can serve as specific tools to monitor consistency in BCG manufacture.

Mycobacterium avium Subspecies paratuberculosis in Asymptomatic Zoo Herbivores in Poland.

Mycobacterial infections are significant issues in zoo animals, influencing animal welfare, conservation efforts, and the zoonotic potential of pathogens. Although tuberculosis is recognised to be highly dangerous, paratuberculosis can also lead to animal losses and is potentially dangerous for humans. The aim of the current study was to confirm whether Mycobacterium avium spp. paratuberculosis (MAP) infections are currently present in zoos in Poland. Faeces samples (n = 131) were collected from different animal species from eight zoos in Poland. The faeces were decontaminated and inoculated into Herrold's Egg Yolk Media. The species was determined using commercial DNA testing. The IS900 was checked using RT-PCR. The culture was positive in seven samples: five with M. avium, one with Mycobacterium fortiatum, and one without any identified Mycobacterium species. RT-PCR confirmed MAP genetic material in nine animals. Our findings represent the first confirmation of MAP in bongo (Tragelaphus eurycerus), indicating that it is present in Polish zoological gardens. Fortunately, the disease can be monitored more easily due to recent legislation (the Animal Health Law).

Molecular Identification of Extrapulmonary Vaccine Adverse Events after BCG in Paraffin-Embedded Specimens.

According to the World Health Organization (WHO), around 1 million children worldwide are diagnosed with tuberculosis each year. The Bacillus Calmette-Guérin (BCG) vaccine has been used around the world for over 100 years. The complications of the BCG vaccination can occur in about 0,06% of children and include local or systemic adverse reactions. Due to the close analogy between the vaccine strain and other species of the Mycobacterium tuberculosis complex (MTBC), molecular methods are recommended for differential diagnosis of Vaccine adverse events (VAE) after BCG. The ability to quickly and specifically identify BCG is important in view of different treatment regimens. The aim of the study was to assess the usefulness of genetic testing for Mycobacterium bovis BCG in the paraffin-embedded specimens' methods. We describe two cases of VAE in immune-compromised children presenting with osteoarticular changes that had been clinically suspected of tuberculosis and led to molecular identification through GeneXpert, GenoType MTBC, and Spoligotyping. Results: Mycobacterium bovis BCG was detected in osteoarticular changes embedded in paraffin block of two patients. Conclusion: Genetic tests using paraffin-embedded materials allow for quick identification and differential diagnosis of patients with Tuberculosis and VAE after BCG. This is an important issue, especially in cases where the tissue has only been submitted for histopathological examination without microbiological diagnostics for tuberculosis.

Detection of Mycobacterium tuberculosis Complex Genetic Material in a Free-Living Brown Bear (Ursus arctos).

Mycobacterium tuberculosis complex (MTBC) has rarely been detected in bears (Ursidae). We describe detection of MTBC genetic material using a single-tube, high-multiplex PCR and fluorescence-based detection system in a throat swab collected from a free-living, problem individual during immobilization and telemetry collar deployment. Mycobacterial culture was negative in all samples.

The capacity of Mycobacterium tuberculosis complex species and M. bovis BCG substrains specific identification - implications for optimized PCR-based diagnostics in adverse events following vaccination suspected cases.

The capacities of differentiation of Mycobacterium bovis BCG from other members of M. tuberculosis complex species using PCR-RFLP, multiplex PCR, and PCR-based genomic deletion analysis approaches were compared. In the study, mycobacteria isolated from patients suspected of adverse events following vaccination with BCG, primarily classified according presence of RD1 marker as virulent and avirulent mycobacteria, were used. The PCR-based genomic deletion analysis was found the best option for mycobacteria diagnostics improvement, as it was capable precisely differentiate virulent and avirulent mycobacteria or virulent species of M. tuberculosis complex. The routine confirmation of mycobacteria species in the cases of adverse events following BCG vaccination is highly expected, especially in clinical practice of patients with primary immunodeficiency.

[Molecular analysis of strains from tuberculosis patients in Polish prisons in 2004-2008. Initial analysis of the project]. / Molekularne dochodzenia epidemiologiczne wsród polskich wiezniów chorych na gruzlice w latach 2004­2008. Badania wstepne.

INTRODUCTION: Correctional facilities are recognised breeding ground for infectious diseases. As The World Health Organization reported the incidence of infectious diseases in prison's population is 10-100 times higher than in general population. The incidence of tuberculosis among correctional inmates in Poland in 2008 was 270/100000, that is around 10 times higher than among non-prisoners. MATERIALS AND METHODS: The study included 57 M. tuberculosis isolates from patients in Polish prisons in 2004-2008 (5% of all diagnosed TB patient in Polish prisons 2004-2008). Primary isolation was performed with Löwenstein-Jensen (L-J) medium, species identification was done with the niacin test and gene probes test. Bacterial DNA was extracted from the L-J medium slants with the cetyltrimethylammonium bromide (CTAB) method. Mycobacterium tuberculosis strains were analyzed with two methods: screening for epidemiological discrimination of M. tuberculosis - spoligotyping and high-throughput - MIRU/VNTR. RESULTS: Isolates that are grouped in clusters (33 isolates) were analyzed by means of MIRU/VNTRs. In MIRU/VNTRs all strains showed different genetic patterns. Most isolates of the prisoners were grouped into two clusters: T1 53 and H3 50. CONCLUSIONS: 1. MIRU/VNTR is a high-throughput method. 2. MIRU/VNTR is a promising method to diagnose TB transmission in Polish jails. 3. To identify the probable source of transmission, molecular analysis of strains from patients of the general population is needed.

Could the type and severity of gross lesions in pig lymph nodes play a role in the detection of Mycobacterium avium?

The Mycobacterium avium complex (MAC) comprises a widespread group of slowly-growing bacteria from the Mycobacteriaceae. These bacteria are responsible for opportunistic infections in humans and animals, including farm animals. The aim of the study was to determine whether it is possible to predict the presence of M. avium in pig lymph nodes based on the size and type of lesions found during post-mortem examination at a slaughterhouse. Lymph nodes were collected from 10,600 pigs subjected to such post-mortem examination. The nodes were classified with regard to their quality, and the number of tuberculosis-like lesions; following this, 86 mandibular lymph nodes with lesions and 113 without visible macroscopic lesions were selected for further study. Cultures were established on Löwenstein-Jensen and Stonebrink media, and a commercial GenoType Mycobacterium CM test was used to identify and differentiate M. avium species. The prevalence of M. avium was 56.98% in the lymph nodes with lesions and 19.47% in the unchanged ones. Statistical analysis indicated that visual assessment of lesions in the mandibular lymph nodes, in particular the number of tuberculous lesions, is a highly-efficient diagnostic tool. Similar results were obtained for estimated percentage area affected by the lesion, i.e. the ratio of the changed area of the lymph node in cross-section to the total cross-sectional area of the lymph node; however, this method is more laborious and its usefulness in slaughterhouse conditions is limited. By incising the lymph nodes and assessing the number of tuberculosis-like lesions, it is possible to limit the inclusion of meat from pigs infected with M. avium into the human food chain.

Pregnancy in European bison (Bison bonaus) with generalized tuberculosis - no evidence of vertical transmission.

INTRODUCTION AND OBJECTIVE: One of the main health threats to the endangered European bison (Bison bonsasus) is bovine tuberculosis, the pathogenesis of which in this species is not fully known. The aim of the study was to confirm a possible case of vertical transmission from a pregnant European bison with generalized tuberculosis to its 12-week-old foetus. MATERIAL AND METHODS: During the autopsy it was found that the bison had become pregnant, despite an advanced stage of tuberculosis. Material collected from the organs and foetus was placed on Lowenstein and Stonebrink media and incubated at 37 °C for 12 weeks. RESULTS: Mycobacteria were isolated from the lungs and lymph nodes; however, the tissue of the foetus and fragments of the reproductive system were negative. CONCLUSIONS: Vertical transmission was excluded, although it cannot be ruled out that infection could occur as pregnancy progresses.

MIRU-VNTR Typing of Atypical Mycobacteria Isolated from the Lymph Nodes of Slaughtered Pigs from Poland.

No regulations currently require the excision of lymph nodes from pig carcasses or the thermal processing of pork before consumption. Therefore, the presence of anatomopathological lesions with signs of coagulation necrosis in lymph nodes from pigs during post-mortem inspection is concerning, as is the increasing incidence of mycobacteriosis in humans. Therefore, the aim of the present study is to verify whether mycobacteria can be isolated from tuberculous-like lesions in mandibular lymph nodes in slaughtered pigs, and whether further molecular analysis based on MIRU-VNRT, used to identify mycobacteria from the Mycobacterium avium complex, can indicate zoonotic potential. Forty of the fifty isolates from the lymph nodes with signs of coagulation necrosis were classified as Mycobacterium avium complex. MIRU-VNTR analysis allowed for the isolation of six strains, one of which was classified as M. avium subsp. paratuberculosis (MAP). Our findings confirm the presence of atypical mycobacteria in the lymph nodes of slaughtered pigs. While the isolated strains (other than MAP) do not pose a significant or direct health risk to consumers, further research and monitoring are necessary. Atypical mycobacteria can cause a wide range of diseases in children and compromised adults, and often show resistance to many classes of antibiotics, including those used to treat tuberculosis.

Mycobacterium bovis Transmission between Cattle and a Farmer in Central Poland.

INTRODUCTION: Zoonoses have recently become an increasing public health problem. Zoonoses are estimated to account for 60% of all emerging infectious diseases. One particularly important zoonosis is human tuberculosis, especially tuberculosis due to Mycobacterium bovis (M. bovis), which is naturally resistant to pyrazinamide (PZA). MATERIAL AND METHODS: The patient had a pulmonary form of tuberculosis accompanied by a cough and fever. At the same time, the disease was also confirmed in 20 out of 25 cattle on the farm. The clinical specimen (sputum) was examined in accordance with the European Union (EU) laboratories' methodology. Tissue materials from cattle were verified in the National Veterinary Research Institute (NVRI), in the Bovine tuberculosis (BTB) Reference Laboratory, Pulawy, Poland and tested in accordance with the guidelines for the laboratory diagnosis of BTB. RESULTS: All M. bovis isolates represented one spoligotype, SB0120. The results of mycobacterial interspersed repetitive unit variable number tandem repeat (MIRU-VNTR) evaluation showed the same genetic pattern. CONCLUSIONS: Findings from this study suggest the first confirmed interspecific transmission of Mycobacterium bovis, between a farmer and his cattle, in Poland. Present findings support the increasing concern regarding zoonotic TB that has been highlighted elsewhere.

[Granuloma of epididymis of patient treated with intravesical BCG therapy--complication of BCG therapy or tuberculosis?]. / Ziarnina stwierdzona w najadrzu chorego leczonego dopecherzowymiwlewkami BCG--powiklanie leczenia BCG czy gruzlica?

We present a case of 72-year old man treated from 2007 for superficial bladder carcinoma. Patient had undergone surgical intervention for transitional cell carcinoma of the bladder, followed by BCG therapy. Two years later enlarged right testis and epididymis was observed. A resection was carried out. Histologic examination revealed in epididymis cauda granulomatous infiltration with eosinophilic necrosis. No bacteriologic tests of resected material were performed. Suspecting BCG infection or TB patient was referred to the Institute of Tuberculosis and Lung Diseases in Warsaw for pulmonary evaluation. Chest X-ray, chest CT scan and bronchoscopy were performed but beside revealing scars in bronchi suggesting a history of TB did not contribute to the diagnosis. Tuberculin skin test was 21 mm. Diagnosis was determinated by spoligotyping which found bacille of Mycobacterium tuberculosis in specimen preserved in paraffin block. Tuberculosis of right epididymis and past pulmonary tuberculosis was diagnosed. Patient was treated with rifampin, isoniazid and pirazynamide.

[Interferon-gamma assays T-SPOT.TB for the diagnosis of latent tuberculosis infection]. / Interferonowy test T-SPOT.TB w diagnostyce latentnego zakazeniapratkiem gruzlicy.

INTRODUCTION: The diagnosis of latent tuberculosis infection (LTBI) is currently based on the century-old tuberculin skin test (TST). However a positive reaction can result from infection by Mycobacterium tuberculosis, BCG vaccination or cross-reaction with nontuberculous mycobacteria. T-SPOT.TB assay is a new test to diagnose tuberculosis infection by measuring in vitro T-cell interferon gamma release in response to two Mycobacterium tuberculosis-specific antigens: ESAT-6 and CFP 10. MATERIAL AND METHODS: T-SPOT.TB assay has been performed on whole blood samples (n = 137) from March to September 2010. A tuberculin skin test result was available for 96 of participants. A positive TST result was considered if the induration was 10 mm or more. RESULTS: Of the 137 patients tested, T-SPOT.TB assay results were positive in 37 (27%), negative in 98 (71.5%) and indeterminate in only 2 (1.5%) persons. We analyzed T-SPOT.TB and TST results in the 96 patients for whom both test were available. Concordance between T-SPOT.TB and TST results (10 mm skin reaction interpreted as positive) was 79%. Fifteen (15.6%) patients had a positive TST result and a negative T-SPOT.TB and 5 (5.2%) patients had a negative TST result and a positive T-SPOT.TB. We observed good correlation between positive T-SPOT.TB results and the size of induration ≥ 15 mm in TST results. CONCLUSIONS: T-SPOT.TB offers a more accurate approach than TST for identification tuberculosis infection. The study shows that the test T-SPOT.TB is a good diagnostic tool in identifying persons with tuberculosis infection. For full confirmation of this assessment, it is necessary to examine more cases.

Mycobacterium chimaera as an Underestimated Cause of NTM Lung Diseases in Patients Hospitalized in Pulmonary Wards.

Mycobacterium chimaera is the newly described species belonging to Mycobacterium avium complex (MAC), with morphology and growth characteristics closely related to Mycobacterium intracellulare. The aim of this retrospective study was to analyze the frequency and clinical significance of M. chimaera identification in the population of patients with previous positive respiratory cultures for M. intracellulare or MAC. 200 strains of M. intracellulare or MAC, isolated from respiratory specimens of patients hospitalized in pulmonary wards, between 2011 and 2020, were retrospectively analyzed with GenoType NTM-DR test. 88 (44%) of strains were re-classified to M. chimaera species. Analysis of clinical data in 30 patients with positive M. chimaera isolates revealed that they were diagnosed with chronic obstructive pulmonary disease (COPD) - 27%, past tuberculosis - 20%, or interstitial lung diseases - 17%, respectively. Non-tuberculous mycobacterial lung disease (NTMLD) caused by M. chimaera has been recognized in 53% of patients, most often in those presenting with post-tuberculous lung lesions. M. chimaera was almost exclusively isolated from respiratory specimens of patients with underlying lung diseases, especially those with COPD and/or past tuberculosis. NTMLD due to M. chimaera was diagnosed predominantly in patients with past tuberculosis.

Antibody responses in European bison (Bison bonasus) naturally infected with Mycobacterium caprae.

Mycobacterium caprae, a member of the Mycobacterium tuberculosis complex, infects humans and animals causing lesions and disease like that of Mycobacterium bovis. The aim of this study was to evaluate antibody responses in European Bison (EB, Bison bonasus; a vulnerable species) naturally infected with M. caprae using dual path platform (DPP) BovidTB test and multi-antigen print immunoassay (MAPIA). Study cohorts consisted of naturally M. caprae-infected EB (n = 4), M. caprae-exposed but uninfected (n = 3), EB infected with non-tuberculous mycobacteria or other respiratory pathogens (n = 3), and negative controls (n = 19). M. caprae-infected EB were seropositive by both DPP and MAPIA; 3/4 were seropositive by DPP; and 4/4 were seropositive by MAPIA. One M. caprae-infected animal that developed generalized disease with most advanced gross lesions in the group produced the most robust antibody response. All 25 EB with no culture-confirmed M. caprae infection, including three animals exposed to M. caprae and three other animals infected with non-tuberculous pathogens, were seronegative on both tests. Antibody responses to M. caprae infection included IgM antibodies against MPB70/MPB83 and IgG antibodies to both MPB70/MPB83 and CFP10/ESAT-6. This study demonstrates the potential for use of serological assays in the ante-mortem diagnosis of M. caprae infection in EB.