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Lepidopterans affect crop production worldwide. The use of transgenes encoding insecticidal proteins from Bacillus thuringiensis ( Bt ) in crop plants is a well-established technology that enhances protection against lepidopteran larvae. Concern about widespread field-evolved resistance to Bt proteins has highlighted an urgent need for new insecticidal proteins with different modes or sites of action. We discovered a new family of insecticidal proteins from ferns. The prototype protein from Pteris species (Order Polypodiales) and variants from two other orders of ferns, Schizaeales and Ophioglossales, were effective against important lepidopteran pests of maize and soybean in diet-based assays. Transgenic maize and soybean plants producing these proteins were more resistant to insect damage than controls. We report here the crystal structure of a variant of the prototype protein to 1.98 Å resolution. Remarkably, despite being derived from plants, the structure resembles the 3-domain Cry proteins from Bt but has only two out of three of their characteristic domains, lacking the C-terminal domain which is typically required for their activities. Two of the fern proteins were effective against strains of fall armyworm that were resistant to Bt 3-domain Cry proteins Cry1Fa or Cry2A.127. This therefore represents a novel family of insecticidal proteins that have the potential to provide future tools for pest control.

Mycobacteria are a unique group of microorganisms. They are characterised by exceptional adaptability and durability. They are capable of colonisation and survival even in very unfavourable conditions. In addition to the well-known obligate human pathogens, Mycobacterium tuberculosis and M. leprae , more than 200 other species have been described. Most of them form a natural part of the microflora of the external environment and thrive in aquatic and soil environments especially. For many of the mycobacterial species associated with human disease, their natural source has not yet been identified. From an ecological point of view, mycobacteria are saprophytes, and their application in human and animal diseases is opportunistic. Most cases of human disease from saprophytic mycobacteria occur in immunocompromised individuals. This adaptability and resilience to environmental pressures makes treatment of mycobacterial diseases (most often sapronoses and less often zoonoses) and permanent eradication of mycobacteria from the environment very difficult. Saprophytic mycobacterial diseases (sapronoses) are chronic and recurrent due to the fact of repeated endogenous or exogenous re-exposure. Therefore, knowledge regarding their occurrence in soil and dust would aid in the prevention of saprophytic mycobacterioses. In conjunction, their presence and ecological significance in the environment can be revealed.

A total of 152 aerosol and spider web samples were collected: 96 spider's webs in karst areas in 4 European countries (Czech Republic, France, Italy, and Slovakia), specifically from the surface environment ( n = 44), photic zones of caves ( n = 26), and inside (aphotic zones) of caves ( n = 26), 56 Particulate Matter (PM) samples from the Sloupsko-Sosuvsky Cave System (speleotherapy facility; n = 21) and from aerosol collected from the nearby city of Brno ( n = 35) in the Czech Republic. Nontuberculous mycobacteria (NTM) were isolated from 13 (13.5%) spider's webs: 5 isolates of saprophytic NTM ( Mycobacterium gordonae , M. kumamotonense , M. terrae , and M. terrae complex) and 6 isolates of potentially pathogenic NTM ( M. avium ssp. hominissuis , M. fortuitum , M. intracellulare , M. peregrinum and M. triplex ). NTM were not isolated from PM collected from cave with the speleotherapy facility although mycobacterial DNA was detected in 8 (14.3%) samples. Temperature (8.2 °C, range 8.0-8.4 °C) and relative humidity (94.7%, range 93.6-96.6%) of air in this cave were relatively constant. The average PM 2.5 and PM 10 mass concentration was 5.49 µg m -3 and 11.1 µg m -3 . Analysed anions (i.e., F - , Cl - , NO 2 - , SO 4 2- , PO 4 3- and NO 3 - ) originating largely from the burning of wood and coal for residential heating in nearby villages in the surrounding area. The air in the caves with speleotherapy facilities should be monitored with respect to NTM, PM and anions to ensure a safe environment.

Mycobacterium fortuitum group (MFG) members are able to cause clinical mycobacteriosis in fish and other animals including humans. M. alvei , M. arceuilense , M. brisbanense , M. conceptionense , M. fortuitum , M. peregrinum , M. porcinum , M. senegalense, M. septicum , and M. setense were isolated from fish with mycobacteriosis. In other animals only three MFG species have been isolated: M. arceuilense from camels' milk, M. farcinogenes from cutaneous infections often described as "farcy", and M. fortuitum from different domestic and wild mammals' species. Out of 17, only 3 MFG species ( M. arceuilense , M. lutetiense and M. montmartrense ) have never been reported in humans. A total of eight MFG members ( M. alvei , M. brisbanense , M. conceptionense , M. fortuitum subsp. acetamidolyticum , M. houstonense , M. peregrinum , M. porcinum , and M. septicum ) have been isolated from both pulmonary and extrathoracic locations. In extrathoracic tissues five MFG species ( M. boenickei , M. farcinogenes , M. neworleansense , M. senegalense , and M. setense ) have been diagnosed and only one MFG member ( M. fortuitum subsp. acetamidolyticum ) has been isolated from pulmonary infection.

A total of 281 guano samples were collected from caves (N = 181) in eight European countries (Bulgaria, Czech Republic, France, Hungary, Italy, Romania, Slovakia and Slovenia) and attics in the Czech R. (N = 100). The correlation of detection of mycobacteria between Ziehl-Neelsen (ZN) microscopy and culture examination and qPCR was strong. ZN microscopy was positive in guano from caves (58.6%) more than double than positivity in guano from attics (21.0%; p < 0.01). From 89 mycobacterial isolates (73 isolates from cave guano and 16 isolates from attics' guano), 68 (76.4%) isolates of 19 sp., ssp. and complex were identified as members of three Groups ( M. fortuitum , M. chelonae , and M. mucogenicum ) and four complexes ( M. avium , M. terrae , M. vaccae , and M. smegmatis ). A total of 20 isolates (22.5%) belonged to risk group 1 (environmental saprophytes), 48 isolates (53.9%) belonged to risk group 2 (potential pathogens), and none of the isolates belonged to risk group 3 (obligatory pathogens). When comparing bat guano collected from caves and attics, differences ( p < 0.01; Mann-Whitney test) were observed for the electrical conductivity, total carbon, total organic, and total inorganic carbon. No difference ( p > 0.05; Mann-Whitney test) was found for pH and oxidation-reduction potential parameters.

For epidemiology studies, a decontamination method using a solution containing 4.0% NaOH and 0.5% tetradecyltrimethylammonium bromide (TDAB) represents a relatively simple and universal procedure for processing heavily microbially contaminated matrices together with increase of mycobacteria yield and elimination of gross contamination. A contamination rate only averaging 7.3% (2.4% in Cluster S; 6.9% in Cluster R and 12.6% in Cluster E) was found in 787 examined environmental samples. Mycobacteria were cultured from 28.5% of 274 soil and water sediments samples (Cluster S), 60.2% of 251 samples of raw and processed peat and other horticultural substrates (Cluster R), and 29.4% of 262 faecal samples along with other samples of animal origin (Cluster E). A total of 38 species of slow and rapidly growing mycobacteria were isolated. M. avium ssp. hominissuis , M. fortuitum and M. malmoense were the species most often isolated. The parameters for the quantitative detection of mycobacteria by PCR can be significantly refined by treating the sample suspension before DNA isolation with PMA (propidium monoazide) solution. This effectively eliminates DNA residue from both dead mycobacterial cells and potentially interfering DNA segments present from other microbial flora. In terms of human exposure risk assessment, the potential exposure to live non-tuberculous mycobacteria can be more accurately determined.

Non-tuberculous mycobacteria (NTM) are ubiquitous environmental bacteria that can induce pulmonary and non-pulmonary diseases in susceptible persons. It is reported that the prevalence of NTM diseases is increasing in developed countries, but this differs by regions and countries. NTM species distribution and the rate of diseases caused by NTM vary widely in the historical territories of Moravia and Silesia (Czech Republic). This epidemiologic study of NTM diseases covers the period 2012-2018, reviews isolates obtained from patients with clinical disease and investigates correlations with related socio-economic and environmental factors. Individual NTM patients were included only once during the studied period and results were presented as incidence rate per year. The most frequently isolated NTM meeting the microbiological and clinical criteria in the study were the Mycobacterium avium-intracellulare complex, followed by Mycobacterium kansasii and M ycobacterium xenopi . A previously described endemic incidence of M. kansasii in the Karviná district and M. xenopi in the Ostrava district was also observed in this study. The incidence of NTM patients in the whole studied territory was 1.10/100,000 inhabitants (1.33/100,000 in men and 0.88/100,000 in women). The annual incidence of lymphadenitis in children (≤5 years of age) was 2.35/100,000 of the population of children during the 7 year period but increased in the year 2018 to 5.95/100,000. The rate of human tuberculosis in the studied area was 1.97/100,000 inhabitants. The incidence of NTM pulmonary diseases correlated with a lower socio-economic status ( r = 0.63) and a higher concentration of benzo[a]pyrene pollution in the air ( r = 0.64)., Competing Interests: The authors declare no conflict of interest.

In the eukaryotic cell, the endoplasmic reticulum (ER) plays an important role as the site of lipid synthesis, protein folding and protein maturation. Stringent regulation of redox and calcium homeostasis is paramount, failure of which leads accumulation of unfolded and aggregating proteins resulting in a condition known as ER stress. Eukaryotic cells deal with ER stress by eliciting the unfolded protein response (UPR). This pathway splits into two streams depending on the severity and longevity of the ER stress, where the cell must make a choice for the good of the organism between survival and programmed cell death. The BCL-2 family of proteins is central to the cell death arm of the UPR pathway. This chapter discusses the recent findings on the involvement of BCL-2 family members in the apoptotic process initiated by ER stress and a related process called autophagy. Understanding the molecular mechanisms involved in ER stress and autophagy could have a profound implications developing new therapies for many ER stress associated diseases and cancer.

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.

We defined the role of the syrphid fly Eristalis tenax in the survival and transmission of mycobacteria in pigs. The conditionally pathogenic mycobacterial (CPM) species Mycobacterium chelonae was isolated from 10 % of liquid dung samples, and both M. chelonae and another CPM species M. fortuitum were isolated from 7 (78 %) of the examined E. tenax larvae collected from the same location. Mycobacteriosis of the lymph nodes of pigs from 3 infected farms was caused by M. avium subsp. avium, M. avium subsp. hominissuis, and M. fortuitum. M. avium subsp. avium and M. avium subsp. hominissuis of identical genotype and serotypes and M. fortuitum were isolated from 7 (1.9 %) larvae, 2 (7.4 %) puparia, and one (1.6 %) imago. The count of colony forming units isolated from larval skin covering (pouch) was higher (p < or = 0.01) than that isolated from the internal organs of larvae. These results showed the potential for E. tenax larvae to spread mycobacteria throughout pig herds and the surrounding environment.

The syrphid Eristalis tenax Linnaeus (Diptera: Syrphidae) may be found in and around dung storage pits at cattle farms at various developmental stages of their life cycle. The purpose of this study was to investigate the occurrence of Mycobacterium avium ssp. paratuberculosis in 1044 E. tenax samples at various developmental stages, as well as fresh and stored dung originating from nine cattle farms. Mycobacterium fortuitum was isolated from one (1.5%) larva from the vicinity of three paratuberculosis-free herds of cattle. Mycobacterium a. paratuberculosis was isolated from 111 (21.4%) of E. tenax larvae collected from two of seven farms known to be infected with the causal agent of paratuberculosis. Mycobacteria were not isolated from any of the 340 pupae, 41 adults of 78 samples of exoskeletal exuviae. Mycobacterium a. paratuberculosis isolates from E. tenax larvae were of the IS900 restriction fragment length polymorphism (RFLP) type B-C1, identical to that detected in faecal samples from cattle herds infected with paratuberculosis. Larvae artificially infected with mycobacteria of IS900 RFLP type B-C9 did not contain statistically more CFU of identical IS900 RFLP type B-C9 in the intestinal tract and internal organs than on the body surface. These results show that M. a. paratuberculosis can survive in the intestinal tract and internal organs of E. tenax.

Mycobacteria were not isolated from any of 229 beetle imagoes of 29 species originating from 14 distinct localities in the Czech and Slovak Republics: 186 imagoes (34 samples) and 43 imagoes (12 samples) from the wild and herds with paratuberculosis infected ruminants, respectively. From 75 environmental samples taken from barns with infected ruminants, Mycobacterium avium subsp. paratuberculosis was isolated from five scrapings of the floors in barns and a feed processing room. From bran and peat taken from pig farms, M. a. hominissuis was diagnosed in 13% of 72 samples and in 69% of 70 samples, respectively. M. a. avium was isolated from 2 (2.9%) and atypical mycobacteria from 12 (17.1%) peat samples. In the respective experiments, larvae of Tenebrio molitor Linnaeus and Zophobas atratus Fabricius were infected in vitro with isolates of M. a. paratuberculosis of IS900 RFLP type B-C1 and M. a. avium of IS901 RFLP type F-C3. T. molitor larvae were also infected with M. a. hominissuis by naturally contaminated bran and peat. M. a. paratuberculosis and M. a. avium were diagnosed in larvae of both species on days 1 to 3 post infection (p.i.). M. a. hominissuis was isolated from T. molitor larvae fed by bran on days 4 to 9 p.i. and from imagoes on day 35 p.i. and from larvae fed by peat on days 4 to 14 p.i. RFLP types of all the isolates identified before infection and after isolation from larvae were identical. Thus, beetles could mechanically transmit mycobacteria, this hazard should be considered for both the implementation of control measures and feeding captive animals with larvae.

Mycobacterium avium subsp. paratuberculosis (Actinomycetales: Mycobacteriaceae) isolates of identical restriction fragment length polymorphism (RFLP) type B-C1 were isolated from: intestinal mucosa of two cows showing clinical signs of paratuberculosis, a specimen of the blowfly Calliphora vicina Robineau-Desvoidy (Diptera: Calliphoridae) captured while perched on these cattle intestines in a waste container at the site of the slaughter, and the blowflies C. vicina and Lucilia caesar Linnaeus captured the next day at the same site when no infected cattle with paratuberculosis were slaughtered. Subsequently, second-stage larvae of the blowflies C. vicina and Lucilia sericata (Meigen) were experimentally infected by feeding them liver from hens with avian tuberculosis caused by M. a. avium (serotype 1, genotype IS901+ and IS1245+) and small cuts of pork meat contaminated with M. a. hominissuis (serotype 8, genotype IS901- and IS1245+). Mycobacterium a. avium of identical serotype, genotype and RFLP type F-C3 was isolated from C. vicina larvae on days 4 and 11 post infection (p.i.) and from L. sericata larvae on day 4 p.i. Identical RFLP type B-C1 of M. a. paratuberculosis was isolated from adult C. vicina fed with artificially contaminated saccharose solution on day 2 p. i. Investigation of M. a. paratuberculosis distribution inside the adult C. vicina showed that the majority of Colony Forming Units (CFU) were isolated from the abdomen and head, fewer from the thorax and wings and none from the legs. Larvae and adults may participate in spreading causal agents of mycobacterial infections and this fact should be considered during sanitation of infected herds and in slaughterhouses when materials from animals affected by mycobacterial infections are processed.

A standardised method for PvuII-PstI-IS901 restriction fragment length polymorphism (RFLP) typing was developed and evaluated against 173 isolates of Mycobacterium avium subsp. avium and M. avium subsp. silvaticum originating from birds (N=46) and their aviaries (N=5), pigs (N=85), cattle (N=18), reference serotype strains (N=9), humans (N=7), a horse (N=1), a nutria (N=1), and strain M. avium subsp. avium ST 18 (formerly M. avium subsp. paratuberculosis ST 18). PvuII-IS1245 RFLP typing was also performed on all isolates. DNA was digested in parallel by restriction endonucleases PvuII or PstI and hybridised to standard probes prepared by PCR. DNA fingerprints were scanned by CCD camera and analysed by the Gel Compar (Applied Maths, Version 4.1, Kortrijk, Belgium) software using a standard isolate control profile. A total of 52 PvuII-PstI RFLP profiles was described including 25 PvuII RFLP profiles designated A to Y and 25 PstI RFLP profiles designated A1-L3. Profiles were found to be stable in vivo and in vitro after multiple subcultures. High IS901 copy number was associated with a "bird" PvuII-IS1245 RFLP profile and low IS901 copy number with M. avium subsp. avium isolates from humans and the nutria. A virulence assay of 100 IS901-positive isolates using intramuscular infections of pullets showed 83 isolates differentiated into 32 RFLP types to be virulent and 17 isolates differentiated into 12 RFLP types as nonvirulent. Attenuation of virulence for pullets could be attributed to either multiple in vitro subculture, polyclonal infection or human passage and was not related to IS901 or IS1245 profiles.

Mycobacteria were isolated from 14 (4.5%) of 314 samples, containing 7791 adult Diptera, which were collected in the Czech Republic and Slovakia in 1997-2000. These flies were collected from three cattle herds with paratuberculosis, two pig herds with mycobacterial infections and one farm that kept both cattle and pigs and that did not have problems of mycobacterial infections. Mycobacterium intracellulare was isolated from Eristalis tenax Linnaeus (Diptera: Syrphidae) captured from a pig herd. Mycobacterium avium ssp. avium (serotype 8) was isolated from flies of the genera Drosophila Fallen (Diptera: Drosophilidae) and Musca Linnaeus (Diptera: Muscidae) originating from a pig herd. Mycobacterium spp. were isolated from Musca spp. and Mycobacterium fortuitum was isolated from dung flies of the genus Scatophaga Meigen (Diptera: Scatophagidae), Musca spp. and Stomoxys calcitrans Linnaeus (Diptera: Muscidae) captured in the same herd. Mycobacterium scrofulaceum was isolated from S. calcitrans from the farm with both cattle and pigs. Mycobacterium avium ssp. paratuberculosis was isolated from Scatophaga spp. collected from pastures grazed by one of the cattle herds and from Calliphora vicina Robineau-Desvoidy (Diptera: Calliphoridae) and Lucilia caesar Linnaeus (Diptera: Calliphoridae) captured in a slaughterhouse, where cattle infected with paratuberculosis were slaughtered. Mycobacterium phlei was isolated from flies of the genus Lucilia captured at a waste bin. These data indicate that mycobacteria may be spread by adult flies that have been in contact with material contaminated with these pathogens.