Mycobacteriosis: the most common causative agents.

The annual number of diagnosed diseases caused by non-tuberculous mycobacteria in predisposed individuals remains constant in the Czech Republic. Their clinical characteristics vary depending on the properties of the causative species and its presence and quantity in the immediate environment of the patient. The most common clinically relevant species are Mycobacterium avium, M. kansasii, and M. xenopi. The most important source of M. avium is peat and products derived from it. M. avium may colonise warm water systems, posing a high risk of exposure to users (jacuzzi users in particular). M. kansasii is still present in waters of areas affected by industrial and mining activities. Its recently isolated genetic variants are mostly of no clinical significance but may be present as contaminants in medical preparations. M. xenopi permanently colonises most warm water systems, and its practical ubiquity makes difficult the interpretation of ambiguous findings on imaging. The antibiotic treatment, which may not always be successful, should be initiated after a comprehensive assessment of the patient's condition, imaging data, and disease progression. Similarly, the results of laboratory tests may not always be authoritative in decision making.

Epidemiology of selected Mycobacterium tuberculosis complex members in the Czech Republic in 2000-2016.

The paper concerns the epidemiology of Mycobacterium tuberculosis complex (MTBC) members except for M. tuberculosis in the Czech Republic in 2000 to 2016. M. bovis was confirmed in 18 patients. M. caprae was diagnosed in two patients in 2001 and 2016 and M. microti in one patient in 2007. M. africanum was detected in one HIV infected woman from Nigeria in 2011. As regards animals, M. pinnipedii was isolated in 2009 from one Southern sea lion (Otaria flavescens) imported from Germany. In 2002, M. caprae was isolated from two Bactrian camels (Camelus ferus) kept in a zoological garden. M. tuberculosis was isolated from one dog in 2004 and from two domestic pigs in 2007. In both cases, the source of M. tuberculosis was an infected patient. Upon examination of 3 727 environmental samples of water and sediments, none of the MTBC members was detected in the stu-died period. Infected persons coming from M. africanum endemic countries (especially West African countries) and infected animals can be considered as the current risk factors for transmission of MTBC species. If the epidemiological situation remains as it is now, there is no risk of transmission of MTBC species via milk or unpasteurised dairy products. Keywords: mycobacterial ecology - domestic and wild animals - food safety.

Mycobacterium avium subsp. avium and Mycobacterium neoaurum detection in an immunocompromised patient.

Non-tuberculous mycobacteria are increasingly described as infectious agents in immunocompromised patients. A 17-year-old male patient suffering from secondary non-Hodgkin's lymphoma and treated with chemotherapeutic agents was admitted to hospital due to pleuropneumonia. Mycobacterium neoaurum was cultured repeatedly from his sputum and, Mycobacterium avium subsp. avium (M. a. avium) was detected by IS901 qPCR from detached fragments of his intestinal mucosa. We attempted to determine the possible sources of infection by analysing environmental samples from the closed oncology unit and conventional unit in the hospital, and from the patient's home residence and places which he frequented. The environment of the patient harboured mycobacteria (41 isolates in total); however, M. neoaurum was not recovered. M. a. avium was detected by qPCR in the environmental samples from a small flock of hens kept by his neighbour. Although it was not confirmed by DNA fingerprinting methods, the M. a. avium infection could have been acquired through the eating of incompletely cooked eggs.

Spread of Mycobacterium avium subsp. paratuberculosis through soil and grass on a mouflon (Ovis aries) pasture.

The aims of this study were to describe spatial contamination of the environment on a mouflon pasture, as well as to assess the contamination of grass and roots after surface contamination and in depth contamination with feces and buried tissues from animals infected with Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis). Samples of soil, roots, and aerial parts of plants were collected from different locations inside the mouflon pasture, and one control sample site was chosen outside the area where the animals are living. M. a. paratuberculosis DNA was present in all the examined sites and was more often detected in roots than in soil. DNA was detected at up to 80 cm of depth and was spatially more widespread than the initial hypothesis of M. a. paratuberculosis leaching vertically into deeper layers of soil. This study broadens our knowledge of the spread and persistence of M. a. paratuberculosis in an environment with highly infected animals.

Relative prevalence of Mycobacterium marinum in fish collected from aquaria and natural freshwaters in central Europe.

A survey was carried out on occurrence of Mycobacterium marinum in fish kept in aquaria and those living in their natural environment. Species-specific qPCR targeting the erp and IS2404 genes together with the conventional culture method were used. The analysis of 72 ornamental fish (n = 216 samples: gills, muscle and intestine) collected from aquaria revealed the presence of M. marinum in 30 individuals (41.7%) of whom 17 (23.6%) were later culture positive. Culture-independent detection revealed the presence of M. marinum in 16 of 83 environmental samples (19.3%) collected in aquaria. The presence of viable M. marinum cells was later confirmed in 5 samples (6.0%). No qPCR or culture positivity was observed when 123 groundwater fish and their corresponding environmental samples (n = 142) were analysed.

Mycobacterium avium subsp. paratuberculosis detected in the reproductive tract of cows from an infected herd.

Mycobacterium avium subspecies paratuberculosis (MAP), the causal agent of paratuberculosis, was detected by quantitative real-time IS900 PCR in the follicular fluid from the reproductive tracts of cows originating from one infected herd. As well as being detected in follicular fluid of cows shedding bacteria in their faeces, MAP was also detected in the follicular fluid of one apparently healthy, non-shedding individual cow. The finding of MAP in follicular fluid is unexpected and could contribute to the lower viability of embryos and resultant lower pregnancy rate. In addition to finding contaminated follicular fluid, vaginal and uterine flush fluids were determined to be positive for the presence of MAP in 75% and 56.3% of the time of the cattle currently shedding MAP in their faeces, respectively. The presence of MAP in different parts of the reproductive tract was seen in clinically as well as subclinically infected cows. These findings extend our currently scant and contradictory knowledge about the dissemination of MAP in the reproductive tract of female cattle.

Culture- and quantitative IS900 real-time PCR-based analysis of the persistence of Mycobacterium avium subsp. paratuberculosis in a controlled dairy cow farm environment.

The aim of this study was to monitor the persistence of Mycobacterium avium subsp. paratuberculosis in environmental samples taken from a Holstein farm with a long history of clinical paratuberculosis. A herd of 606 head was eradicated, and mechanical cleaning and disinfection with chloramine B with ammonium (4%) was carried out on the farm; in the surrounding areas (on the field and field midden) lime was applied. Environmental samples were collected before and over a period of 24 months after destocking. Only one sample out of 48 (2%) examined on the farm (originating from a waste pit and collected before destocking) was positive for M. avium subsp. paratuberculosis by cultivation on solid medium (Herrold's egg yolk medium). The results using real-time quantitative PCR (qPCR) showed that a total of 81% of environmental samples with an average mean M. avium subsp. paratuberculosis cell number of 3.09 × 10(3) were positive for M. avium subsp. paratuberculosis before destocking compared to 43% with an average mean M. avium subsp. paratuberculosis cell number of 5.86 × 10(2) after 24 months. M. avium subsp. paratuberculosis-positive samples were detected in the cattle barn as well as in the calf barn and surrounding areas. M. avium subsp. paratuberculosis was detected from different matrices: floor and instrument scrapings, sediment, or scraping from watering troughs, waste pits, and cobwebs. M. avium subsp. paratuberculosis DNA was also detected in soil and plants collected on the field midden and the field 24 months after destocking. Although the proportion of positive samples decreased from 64% to 23% over time, the numbers of M. avium subsp. paratuberculosis cells were comparable.

A low prevalence of mycobacteria in freshwater fish from water reservoirs, ponds and farms.

A survey of the occurrence of mycobacteria was conducted from 717 freshwater fish (25 species) in two water reservoirs, five ponds and two farms in the Czech Republic. A total of 2182 tissue samples from these fish were examined using the conventional culture method. Thirteen mycobacterial isolates were obtained from 12 (1.7%) fish belonging to nine species. Isolates were identified using sequence analysis of the 16SrRNA gene as: Mycobacterium algericum, M. fortuitum, M. gordonae, M. insubricum, M. kumamotonense, M. nonchromogenicum, two isolates of M. peregrinum, M. terrae and M. triplex. Mycobacteria were isolated more frequently from fish skin and gills than from internal organs or muscles.

Short communication: Examination of milk filters by real-time PCR as a herd-level indicator of the presence of Mycobacterium avium subspecies paratuberculosis in dairy herds.

The aim of this study was to assess the suitability of real-time quantitative PCR (qPCR) for the detection of Mycobacterium avium ssp. paratuberculosis (MAP) in milk filters as a herd level indicator of paratuberculosis infection. Seventy-nine samples from textile or metal milk filters from 15 herds with defined MAP prevalence (infection status = noninfected, 0-5%, 5-10%, or >10% of animals with clinically confirmed paratuberculosis) were analyzed. The MAP DNA was isolated by a modified commercially available protocol for feces, and detection and quantification of the pathogen was performed by the IS900 qPCR. Mycobacterium avium ssp. paratuberculosis DNA was detected in 63 (79.7%) samples. Determination of MAP infection established by fecal and tissue culture was correctly confirmed by the analysis of milk filters on 11 of 12 infected farms; MAP was not detected in filters from 3 farms where paratuberculosis was never diagnosed. Statistical analysis of the data supports the evidence that milk filters can be used as a template for the direct detection of MAP on the herd level. The probability of successful MAP detection in milk filters in a herd with MAP-infected cows is at least 94.3%. Absolute numbers of MAP detected on the milk filter can be used for a rough estimation of paratuberculosis prevalence >10% in the herd. Analysis of milk filters for the presence of MAP can be a useful tool for the detection of paratuberculosis on the herd level before any individual control strategies.

[Methods used to detect mycobacterioses in humans--assessment of advantages and disadvantages of mycobacterial identification by sequencing analysis]. / Metody pouzívané pro detekci mykobakterióz u lidí--posouzení výhod a nevýhod identifikace mykobakterií pomocí sekvencní analýzy.

The isolation of potentially pathogenic mycobacteria (PPM) from clinical specimens has become very frequent in the last years. Such organisms are typically environmental and occasionally pathogenic for humans. Standard diagnosis of mycobacterial infections relies on direct examination and culture. Nowadays, molecular tools are available, allowing quicker accurate diagnosis. Detection of PPM can be performed directly from clinical samples, although in most cases identification is carried out after isolation. Sequencing of genomic targets (such as 16S rRNA, rpoB or hsp65) allows accurate and quick identifications but has some technical limitations. Problems concerning sequencing analysis used for PPM identification together with description of available algorithms for PPM identification are the major objectives of this review.

Persistence of Mycobacterium avium subsp. paratuberculosis at a farm-scale biogas plant supplied with manure from paratuberculosis-affected dairy cattle.

In this study, products from all steps of anaerobic digestion at a farm-scale biogas plant supplied with manure from paratuberculosis-affected dairy cattle were examined and quantified for the presence of the causal agent of paratuberculosis, Mycobacterium avium subsp. paratuberculosis, using culture and quantitative real-time PCR (qPCR). Viable M. avium subsp. paratuberculosis cells were detected using culture in fermentors for up to 2 months; the presence of M. avium subsp. paratuberculosis DNA (10(1) cells/g) was demonstrated in all anaerobic fermentors and digestate 16 months after initiation of work at a biogas plant, using IS900 qPCR. F57 qPCR was able to detect M. avium subsp. paratuberculosis DNA (10(2) cells/g) at up to 12 months. According to these results, a fermentation process that extended beyond 2 months removed all viable M. avium subsp. paratuberculosis cells and therefore rendered its product M. avium subsp. paratuberculosis free. However, M. avium subsp. paratuberculosis DNA was found during all the examined periods (more than 1 year), which could be explained by either residual DNA being released from dead cells or by the presence of viable cells whose amount was under the limit of cultivability. As the latter hypothesis cannot be excluded, the safety of the final products of digestion used for fertilization or animal bedding cannot be defined, and further investigation is necessary to confirm or refute this risk.

Mycobacterium marinum epididymoorchitis: case report and literature review.

Mycobacterium marinum is the most frequent non-tuberculous Mycobacterium in humans. We report the first ever described case of epididymoorchitis resulting from hematogenous spread of M. marinum from hand oligoarthritis. This was initially mistaken for rheumatoid disease and methylprednisolone-induced immunosuppression led to hematogenous spread of infection to the testis and epididymis.

Morphology and distribution of granulomatous inflammation in freshwater ornamental fish infected with mycobacteria.

Mycobacteriosis in fish is a chronic progressive ubiquitous disease caused by Mycobacterium marinum, M. gordonae and M. fortuitum in most cases. The aim of this study was to describe the morphology and distribution of lesions in 322 freshwater ornamental fish across 36 species. Granulomatous inflammation was diagnosed by gross examination and histopathology testing in 188 fish (58.4%); acid-fast rods (AFR) were determined in only 96 (51.1%) fish from 19 species after Ziehl-Neelsen staining. The most often affected organs with AFR were the kidney (81.2%), digestive tract (54.1%), liver (48.2%), spleen (45.9%) and skin (21.2%); sporadically, AFR were found in the branchiae (9.4%) and gonads (4.7%). In 14 randomly selected fish originating from four different fish tanks, the distribution of mycobacterial infection was studied by culture examination of the skin, gills, muscle tissue, digestive tract, liver, spleen and kidney. In 12 fish, the species M. marinum, M. gordonae, M. fortuitum, M. triviale, and M. avium subsp. hominissuis (serotypes 6 and 8 and genotype IS901- and IS1245+) were detected; mixed infection caused by different mycobacterial species was documented in five of them.

Determinants of the prevalence of Heligmosomum costellatum (Heligmosomidae: Trichostrongyloidea) in a common vole population in southern Moravia, Czech Republic.

A study of nematode (Heligmosomum costellatum) prevalence in the common vole (Microtus arvalis) population was performed in 2002 and 2003 in an agricultural region of southern Moravia, Czech Republic. The influence of the trapping date, body weight, age, gender and reproduction of the common vole on the prevalence of the larvae of H. costellatum encapsulated in the stomach wall was examined. Of 503 common voles examined, 27.6% were H. costellatum positive. The prevalence of H. costellatum in the common vole population was influenced mainly by the weight, trapping date, year and reproductive state, as well as by the interactions between year and gender and between year and reproductive state. The influence of age was weak. The most infected common vole groups were the older, heavier and already reproducing females captured from April to August and all voles captured in 2002. In general, parasite prevalence seems to be dependent on the population parameters of the host species. However, the H. costellatum prevalence study in a single common vole population was not sufficient to fully describe and explain the nematode-host interactions.

[Mycobacteria at extrapulmonary sites]. / Mykobakterie v mimoplicní lokalizaci.

Two cases of mycobacterial infections are presented - one of rare hepatic tuberculosis and second of cutaneous mycobacteriosis caused by Mycobacterium chelonae. The aim of the second report is to point to nontuberculous, atypical, otherwise potentially pathogenic mycobacteria. These mycobacteria may cause diseases of various localization and severity in immunocompromised patients.

Statistical analysis of symptoms, endoscopy and urothelial morphology in 58 female bladder pain syndrome/interstitial cystitis patients.

INTRODUCTION: The goal of the study was to assess the course of painful syndrome in patients with bladder pain syndrome/interstitial cystitis and to assess the changes in endoscopic and histopathological findings in relation to the type of treatment. PATIENTS AND METHODS: We included a total of 58 patients with histologically diagnosed interstitial cystitis. Out of these, 31 patients were treated with oral pharmacotherapy and 27 patients were treated by intravesical application of heparin. The patients were followed from time of diagnosis for 6.9 +/- 2.5 and 6.6 +/- 2.7 years, respectively. RESULTS: Treatment - irrespective of its type - had a clear demonstrable effect on the monitored parameters; intravesical treatment was more effective than oral. Statistically significant (p < 0.05) changes could be observed in both groups (with two exceptions). When evaluating the relationship between subjective symptoms and objective criteria, and patients' age and time to diagnosis, it is clear that the higher the age and the longer the time from symptoms to diagnosis, the more severe the symptoms. CONCLUSIONS: When evaluating the monitored parameters, we found significant correlations (both positive and negative). However, these relationships cannot be used to simplify the evaluation algorithm (according to ESSIC) and the initial criteria cannot predict the course of the disease.

Mycobacterium avium subsp. avium distribution studied in a naturally infected hen flock and in the environment by culture, serotyping and IS901 RFLP methods.

Mycobacterium avium subsp. avium (MAA) of serotype 2 and genotype IS901+ and IS1245+ was cultured from 21 naturally infected hens (Gallus domesticus) from one smallholder aviary. From a total of 330 samples taken from hens, 124 mycobacteria were detected. Out of which MAA was detected in 103 (35.7%) of 288 tissues, in 4 (19.0%) of 21 swabs of cloacae and in 9 (42.9%) of 21 faeces samples, 8 other conditionally pathogenic mycobacterial species were also isolated. Tuberculous (TB) lesions were found in the liver, spleen and intestinal organs of seven hens. The isolates of MAA (n=58) from 16 infected hens (7 with TB lesions and 9 without TB lesions) were found to be of 3 IS901 RFLP types AE (n=48), AD (n=4) and E (n=6), where these MAA isolates are highly virulent to hens. Mixed infections with IS901 RFLP types (AE and AD) and (AE and E) were also evident in seven hens. From a total of 35 examined environmental samples, 23 mycobacterial isolates were detected. Out of which four (17.4%) MAA isolates of IS901 RFLP type AE and 19 (82.6%) other isolates of conditionally pathogenic mycobacteria were detected. The finding of identical IS901 RFLP types from both tissues and faecal isolates confirms that infected domestic hens are the principal source of infection for other susceptible hosts and lead to the contamination of the surrounding environment. The presence of different IS901 RFLP types in tissue isolates may indicate the repeated incidence of MAA infection and the occurrence of polyclonal infection.

Strategy for the detection and differentiation of Mycobacterium avium species in isolates and heavily infected tissues.

The members of Mycobacterium avium species, comprising M. avium subsp. paratuberculosis, M. a. hominissuis, M. a. avium, M. a. silvaticum, are currently the most prevalent opportunistic pathogenic mycobacteria causing mycobacterial infection in animals and humans. The ability to distinguish between these subspecies is of relevance for proper diagnosis and control programmes of the diseases. The aim of this study was to design a fast and specific PCR strategy for the detection and differentiation of M. avium subspecies from the solid plate cultures for use in routine veterinary diagnosis. We have developed a multiplex PCR based on IS900, IS901, IS1245 and the dnaJ gene. This method allows the detection of M. a. paratuberculosis, M. a. hominissuis and M. a. avium/M. a. silvaticum in one PCR reaction and theoretically enables mixed infections of M. a. paratuberculosis and M. a. avium or M. a. paratuberculosis and M. a. hominissuis to be revealed. The sensitivity of this multiplex PCR is 10(3)CFU for each bacterial strain in one PCR reaction, which also enabled the use of this test directly for DNA isolated from the tissue of the heavily infected sheep.

Avian tuberculosis in naturally infected captive water birds of the Ardeideae and Threskiornithidae families studied by serotyping, IS901 RFLP typing, and virulence for poultry.

Avian tuberculosis was detected in one flock of 38 water birds of the families Ardeideae (n = 20) and Threskiornithidae (n = 18). Mycobacterium avium subsp. avium (MAA, serotype 1, genotype IS901+ and IS1245+) was more often (p = 0.01) detected in tissue and/or faecal samples in 18 (90.0%) birds form the Ardeideae family: little egret (Egretta garzetta), buff-backed heron (Bubulcus ibis), great white egret (Egretta alba), and bittern (Botaurus stellaris) in comparison to two (11.1%) birds from the Threskiornithidae family: sacred ibis (Threskiornis aethiopicus). Avian tuberculosis was not diagnosed in spoonbills (Platalea leucorodia). Tuberculous lesions were found in nine birds. MAA isolates of IS901 RFLP type F-C3 were present in all of the 20 infected birds and in all environmental isolates. A mixed infection with the MAA isolates of three RFLP types F-C3 (tissue isolate), G-C3, and T-C3 (faecal isolates) was found in one sacred ibis. All 20 tissue isolates of IS901 RFLP type F-C3 from 20 birds and 8 environmental MAA isolates were fully virulent in pullets, whilst the isolates of RFLP types G-C3 and T-C3 were non-virulent in pullets. All of the tested MAA isolates had the same IS1245 RFLP "bird profile". In 12 of 20 infected birds with MAA M.a. hominissuis isolates of serotypes 4, 8, 9 and genotype IS901- and IS1245+ were detected and in 8 other birds mycobacteria not belonging to the M. avium complex were found. The presence of MAA in the environment may be a source for further spread of the causal agent of avian tuberculosis among other groups of animals in zoological gardens, farm animals, and also among their keepers.

Prevalence of Neospora caninum and Toxoplasma gondii antibodies in wild ruminants from the countryside or captivity in the Czech Republic.

In the Czech Republic, sera from 720 wild ruminants were examined for antibodies to Neospora caninum by screening competitive-inhibition enzyme-linked immunosorbent assay and confirmed by indirect fluorescence antibody test (IFAT); the same sera were also examined for antibodies to Toxoplasma gondii by IFAT. Neospora caninum antibodies were found in 14% (11 positive/79 tested) roe deer (Capreolus capreolus), 14% (2/14) sika deer (Cervus nippon), 6% (24/ 377) red deer (Cervus elaphus), 1% (2/143) fallow deer (Dama dama), 3% (3/105) mouflon (Ovis musimon), and none of 2 reindeer (Rangifer tarandus). Toxoplasma gondii antibodies were found in 50% (7/14) sika deer, 45% (169/377) red deer, 24% (19/79) roe deer, 17% (24/143) fallow deer, 9% (9/105) mouflon, and 1 of 2 reindeer. In 42 samples of wild ruminants that tested positive for N. caninum antibodies, 28 (67% of the positive N. caninum samples) reacted solely to N. caninum. This is the first evidence of N. caninum infection in mouflon, the first N. caninum seroprevalence study in farmed red deer, and the first survey of N. caninum in wild ruminants from the Czech Republic.