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.

Virological Quality of Irrigation Water in Leafy Green Vegetables and Berry Fruits Production Chains.

This study condenses data acquired during investigations of the virological quality of irrigation water used in production of fresh produce. One hundred and eight samples of irrigation water were collected from five berry fruit farms in Finland (1), the Czech Republic (1), Serbia (2), and Poland (1), and sixty-one samples were collected from three leafy green vegetable farms in Poland, Serbia, and Greece. Samples were analyzed for index viruses of human or animal fecal contamination (human and porcine adenoviruses, and bovine polyoma viruses), and human pathogenic viruses (hepatitis A virus, hepatitis E virus, and noroviruses GI/GII). Both index and pathogenic viruses were found in irrigation water samples from the leafy green vegetables production chain. The data on the presence of index viruses indicated that the highest percentage of fecal contamination was of human origin (28.1 %, 18/64), followed by that of porcine (15.4 %, 6/39) and bovine (5.1 %, 2/39) origins. Hepatitis E virus (5 %, 1/20) and noroviruses GII (14.3 %, 4/28) were also detected. Samples from berry fruit production were also positive for both index and pathogenic viruses. The highest percentage of fecal contamination was of human origin (8.3 %, 9/108), followed by that of porcine, 4.5 % (4/89) and bovine, 1.1 % (1/89) origins. Norovirus GII (3.6 %, 2/56) was also detected. These data demonstrate that irrigation water used in primary production is an important vehicle of viral contamination for fresh produce, and thus is a critical control point which should be integrated into food safety management systems for viruses. The recommendations of Codex Alimentarius, as well as regulations on the use of water of appropriate quality for irrigation purposes, should be followed.

Mycobacterium avium subsp. paratuberculosis sheep strains isolated from Cyprus sheep and goats.

Paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis (Map), is a chronic incurable infection of intestinal tract of animals. Molecular characterization of Map isolates classifies them into two major groups, 'Cattle' or Type II and 'Sheep' or Type I/III with a different phenotype, epidemiology, virulence and pathogenesis. The aim of this study was to examine 192 Map ELISA-positive sheep and goats from Cyprus using faecal culture and genotype Map isolates using IS1311 PCR and restriction endonuclease analysis (IS1311 PCR-REA) with HinfI restriction enzyme. Map was isolated from only four (4.6%) faecal samples out of 88 sheep and 15 (14.4%) faecal samples out of 104 goats. Genotyping of the isolates using IS1311 PCR-REA revealed that sheep and goat populations on the island are infected primarily by 'Sheep' strains. Only three Map isolates from goats originated from one farm were characterized as 'Cattle' strains.

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.

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.

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.

First identification of Mycobacterium avium paratuberculosis sheep strain in Argentina

We here identified for the first time the presence of Mycobacterium avium paratuberculosis (MAP) sheep (S) strain in Argentina. IS900 polymerase chain reaction (PCR) was positive. The S strain was compared with MAP cattle (C) strains by using IS1311 PCR-restriction endonuclease analysis (PCR-REA), multiplex PCR and restriction fragment length polymorphism (RFLP) analysis.

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.

First identification of Mycobacterium avium paratuberculosis sheep strain in Argentina.

We here identified for the first time the presence of Mycobacterium avium paratuberculosis (MAP) sheep (S) strain in Argentina. IS900 polymerase chain reaction (PCR) was positive. The S strain was compared with MAP cattle (C) strains by using IS1311 PCR-restriction endonuclease analysis (PCR-REA), multiplex PCR and restriction fragment length polymorphism (RFLP) analysis.

[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.

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.

Mycobacterium avium subsp. paratuberculosis survival during fermentation of soured milk products detected by culture and quantitative real time PCR methods.

Mycobacterium avium paratuberculosis (MAP), etiological agent of paratuberculosis in ruminants, is able to survive extreme conditions like very low pH (stomach), high temperature (pasteurization) or low temperature (refrigerated storage). Cheese, infant powder milk, cream and other milk and dairy products might thus be considered as possible sources of MAP for humans. The aim of this study was to investigate the survival of two MAP field isolates during fermentation of three different types of soured milk products (SMP; yogurt, acidophilus milk and kefir) under laboratory conditions. Pasteurized MAP-free milk was artificially contaminated with 10(6)MAPcells/mL and survival and absolute numbers of MAP were monitored during fermentation (4 or 16 h) and after six weeks of storage at 4°C by culture and quantitative real time PCR (qPCR). Viability of MAP was determined by culture using Herrold's egg yolk medium and Middlebrook 7H10 with antibiotics, supplemented with Mycobactin J and incubated at 37°C for up to 12 weeks. The absolute numbers of MAP were quantified by previously published qPCR assays targeting F57 and IS900 loci in MAP genome. We herein confirm that MAP can survive pH reduction, however, longer exposure to pH below 4 in SMP seems to be critical because it inhibits growth. Therefore, it is suggested that probiotic cultures that can decrease pH below 4 during fermentation could provide better inactivation of MAP in SMP.

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.

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.

Real-time quantitative PCR detection of Mycobacterium avium subspecies in meat products.

The aim of this work was to examine various purchased meat products and to find out if any traces of Mycobacterium avium subsp. avium, M. avium subsp. hominissuis, and M. avium subsp. paratuberculosis could be detected in these samples. Analysis of the meat products (raw, cooked, and fermented) was performed using a real-time quantitative PCR (qPCR) method for the detection of specific insertion sequences: duplex qPCR for the detection of IS900 specific for M. avium subsp. paratuberculosis, and triplex qPCR for the detection of IS901 specific for Mycobacterium avium subsp. avium and IS 1245 specific for M. avium subsp. hominissuis. Of the 77 analyzed meat samples, 17 (22%) were found to contain M. avium subsp. paratuberculosis DNA, 4 (5%) samples contained Mycobacterium avium subsp. avium DNA, and in 12 (16%) samples M. avium subsp. hominissuis DNA was detected. The concentration of M. avium subsp. paratuberculosis and M. avium subsp. hominissuis DNA in some meat products exceeded 10(4) genomes per g. Culture examination of these mycobacterial subspecies was negative. By analyzing a range of meat products, we have provided evidence to support the hypothesis that M. avium is present in everyday commodities sold to the general public.

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.

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.

[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.