Timber-colonizing gram-negative bacteria as potential causative agents of respiratory diseases in woodworkers.

OCCURRENCE: Gram-negative bacteria occur commonly in the inner tissues of stored coniferous and deciduous timber, showing a marked variation in numbers. The greatest maximal numbers are found in the sapwood of coniferous timber. The common constituents of the Gram-negative biota are potentially pathogenic species of Enterobacteriaceae family of the genera Rahnella, Pantoea, Enterobacter, and Klebsiella. The air of wood-processing facilities is polluted with the wood-borne Gram-negative bacteria and produced by them endotoxin, as demonstrated worldwide by numerous studies. EFFECTS: There are three potential pathways of the pathogenic impact of wood-borne Gram-negative bacteria on exposed woodworkers: allergic, immunotoxic, and infectious. Allergic impact has been underestimated for a long time with relation to Gram-negative bacteria. Hopefully, the recent demonstration of the first documented case of hypersensitivity pneumonitis (HP) in woodworkers caused by Pantoea agglomerans which developed in extremely large quantities in birch sapwood, would speed up finding of new wood-related cases of HP caused by Gram-negative bacteria. The second pathway is associated with endotoxin, exerting strong immunotoxic (excessively immunostimulative) action. It has been demonstrated that endotoxin is released into wood dust in the form of nano-sized microvesicles, by peeling off the outer membrane of bacteria. Endotoxin microvesicles are easily inhaled by humans together with dust because of small dimensions and aerodynamic shape. Afterwards, they cause a nonspecific activation of lung macrophages, which release numerous inflammatory mediators causing an inflammatory lung reaction, chest tightness, fever, gas exchange disorders, and bronchospasm, without radiographic changes. The resulting disease is known as "Organic Dust Toxic Syndrome" or "toxic pneumonitis." The potential third pathway of pathogenic impact is infection. The suspected species is Klebsiella pneumoniae that may occur commonly in wood dust; however, until now this pathway has not been confirmed. CONCLUSION: Summarizing, Gram-negative bacteria-inhabiting timber should be considered, besides filamentous fungi and actinobacteria, as important risk factors of occupational disease in woodworkers that could be either HP with allergenic background or toxic pneumonitis elicited by endotoxin.

Cathelicidin Treatment Silences Epithelial-Mesenchymal Transition Involved in Pulmonary Fibrosis in a Murine Model of Hypersensitivity Pneumonitis.

Pulmonary fibrosis is becoming an increasingly common pathology worldwide. Unfortunately, this disorder is characterized by a bad prognosis: no treatment is known, and the survival rate is dramatically low. One of the most frequent reasons for pulmonary fibrosis is hypersensitivity pneumonitis (HP). As the main mechanism of pulmonary fibrosis is a pathology of the repair of wounded pulmonary epithelium with a pivotal role in epithelial-mesenchymal transition (EMT), we assumed that EMT silencing could prevent disease development. Because of several biological features including wound healing promotion, an ideal candidate for use in the treatment of pulmonary fibrosis seems to be cathelicidin. The aim of the studies was to understand the influence of cathelicidin on the EMT process occurring during lung fibrosis development in the course of HP. Cathelicidin's impact on EMT was examined in a murine model of HP, wherein lung fibrosis was induced by chronic exposure to extract of Pantoea agglomerans (SE-PA) by real-time PCR and Western blotting. Studies revealed that mouse exposure to cathelicidin did not cause any side changes in the expression of investigated genes/proteins. Simultaneously, cathelicidin administered together or after SE-PA decreased the elevated level of myofibroblast markers (Acta2/α-smooth muscle actin, Cdh2/N-cadherin, Fn1/Fibronectin, Vim/vimentin) and increased the lowered level of epithelial markers (Cdh1/E-cadherin, Ocln/occludin). Cathelicidin provided with SE-PA or after cessation of SE-PA inhalations reduced the expression of EMT-associated factors (Ctnnd1/ß-catenin, Nfkb1/NFκB, Snail1/Snail, Tgfb1/TGFß1 Zeb1/ZEB1, Zeb2/ZEB2) elevated by P. agglomerans. Cathelicidin's beneficial impact on the expression of genes/proteins involved in EMT was observed during and after the HP development; however, cathelicidin was not able to completely neutralize the negative changes. Nevertheless, significant EMT silencing in response to cathelicidin suggested the possibility of its use in the prevention/treatment of pulmonary fibrosis.

A Scoping Analysis of Cathelicidin in Response to Organic Dust Exposure and Related Chronic Lung Illnesses.

Over two billion people worldwide are exposed to organic dust, which can cause respiratory disorders. The discovery of the cathelicidin peptide provides novel insights into the lung's response to organic dust; however, its role in the lung's response to organic dust exposure and chronic lung diseases remains limited. We conducted a scoping review to map the current evidence on the role of cathelicidin LL-37/CRAMP in response to organic dust exposure and related chronic lung diseases: hypersensitivity pneumonitis (HP), chronic obstructive pulmonary disease (COPD) and asthma. We included a total of n = 53 peer-reviewed articles in this review, following the process of (i) a preliminary screening; (ii) a systematic MEDLINE/PubMed database search; (iii) title, abstract and full-text screening; (iv) data extraction and charting. Cathelicidin levels were shown to be altered in all clinical settings investigated; its pleiotropic function was confirmed. It was found that cathelicidin contributes to maintaining homeostasis and participates in lung injury response and repair, in addition to exerting a positive effect against microbial load and infections. In addition, LL-37 was found to sustain continuous inflammation, increase mucus formation and inhibit microorganisms and corticosteroids. In addition, studies investigated cathelicidin as a treatment modality, such as cathelicidin inhalation in experimental HP, which had positive effects. However, the primary focus of the included articles was on LL-37's antibacterial effect, leading to the conclusion that the beneficial LL-37 activity has not been adequately examined and that further research is required.

Pantoea agglomerans chronic exposure induces epithelial-mesenchymal transition in human lung epithelial cells and mice lungs.

Pantoea agglomerans is gram-negative bacteria widely distributed in nature. It predominates in inhalable dust from grain, herbs, and flax, and was identified as the most important cause of hypersensitivity pneumonitis (HP) in eastern Poland. To better understand the molecular mechanism of HP development studies focused on the interactions between P. agglomerans and alveolar epithelial cells as well as lung tissue with particular emphasis on the epithelial-mesenchymal transition (EMT). The studies were conducted on human normal lung epithelial NL20 cells and mice strain C57BL/6J. Cells and mice underwent chronic exposure to saline extract of P. agglomerans (SE-PA). Morphological changes were evaluated under light microscopy, the concentration of fibrosis markers was examined by the ELISA method, while the expression of genes involved in EMT was evaluated by RealTime PCR. During incubation with SE-PA epithelial cells underwent conversion and assumed fibroblast phenotype characterized by a decrease in epithelial cells markers (CDH1, CLDN1, JUP) and increase in mesenchymal cells markers (FN1, VIM, CDH2). Mice lungs collected after 14 days of SE-PA treatment revealed inflammation with marked lymphocytes infiltration. The intensified inflammatory process accompanied by increased proliferation of fibrous connective tissue was noted in mice lungs after 28 days of SE-PA exposure. Histological changes correlated with an increase of fibrosis markers (hydroxyproline, collagens), downregulation of epithelial markers (Cdh1, Cldn1, Jup, Ocln) and upregulation of myofibroblasts markers (Acta2, Cdh2, Fn1, Vim). Obtained results revealed SE-PA ability to induce EMT in human lung epithelial cells and mice lung tissue, with the scale of changes proportional to the time of treatment.

Detection and Molecular Characteristics of Toxoplasma gondii DNA in Retail Raw Meat Products in Poland.

Raw and undercooked meat are regarded as important sources of Toxoplasma gondii infection of people in Europe; however, data concerning this issue in Poland are still insufficient. The aim of this study was to determine the prevalence of T. gondii DNA isolated from raw meat products retailed in Poland. The molecular characteristics of detected DNA were also performed. Samples of cured bacon, raw or smoked sausages, ham, and minced meat were examined for the presence of T. gondii DNA. Samples were digested by pepsin solution, followed by the DNA isolation. Nested and real-time polymerase chain reaction (PCR) was performed based on the amplification of 35-fold-repetitive B1 fragment gene of T. gondii. For selected B1-positive samples, multiplex PCR was performed using SAG1, SAG2 (5'-SAG2 and 3'-SAG2), altSAG2, SAG3, GRA6, BTUB, C29-2, and L358 genetic markers. Amplicons were sequenced and analyzed with NCBI database. Among 3223 examined samples, 175 (5.4%) were PCR positive. The highest percentages of positive results were found for samples originating from south-east regions of Poland-Podkarpackie (17.9%), Malopolskie (12.6%), and Lubelskie (10.8%) (p < 0.001). The percentages of positive results for particular types of meat products-sausages, smoked meat products, ham, and minced meat-ranged from 4.5% to 5.8% and the differences between them were not significant (p > 0.05). Sequence analysis of selected B1-positive samples demonstrated mostly the alleles of clonal type III (49.0%), and less-type II (17.3%), and type I (10.2%) based on nine used genetic markers. The combinations of types I/II or II/III or I/III alleles at different loci were also found in 23.5% of cases. Detection of T. gondii DNA in raw meat products may indicate the potential health threat for consumers in Poland; however, for complete risk assessment of T. gondii infection, the additional studies, including detection of live parasite, are needed.

Middle age enhances expression of innate immunity genes in a female mouse model of pulmonary fibrosis.

The lungs are highly sensitive to tissue fibrosis, with a clear age-related component. Among the possible triggers of pulmonary fibrosis are repeated inhalations of fine organic particles. How age affects this response, is still far from being fully understood. We examined the impact of middle-age on gene expression in pulmonary fibrosis, using the novel "inhalation challenge set" mouse model. Our results demonstrate that the response of female mice to exposure of Pantoea agglomerans extract primarily involves various immune-related pathways and cell-cell/cell-extracellular matrix interactions. We found that middle-age had a strong effect on the response to the P. agglomerans-induced lung fibrosis, featured by a more rapid response and increased magnitude of expression changes. Genes belonging to innate immunity pathways (such as the TLR signaling and the NK-cell mediated cytotoxicity) were particularly up-regulated in middle-aged animals, suggesting that they may be potential targets for the treatment of pulmonary fibrosis caused by inhalations of organic particles. Our analysis also highlights the relevance of the "inhalation challenge set" mouse model to lung aging and related pathology.

Presence of Pathogenic Rickettsiae and Protozoan in Samples of Raw Milk from Cows, Goats, and Sheep.

OBJECTIVE: The aim of the present work was to determine the presence of various rickettsiae and protozoan in raw milk and the assessment the potential, milk-borne route in the spread of selected zoonotic pathogens. MATERIALS: A total of 119 raw milk samples collected randomly from 63 cows, 29 goats, and 27 sheep bred on 34 farms situated on eight communities in eastern Poland were examined by polymerase chain reaction (PCR) method for the presence of pathogenic rickettsiae (Coxiella burnetii, Anaplasma phagocytophilum, and Rickettsia spp.) and protozoan (Toxoplasma gondii). RESULTS: The only prevalent pathogen was T. gondii, which was found in 10 samples of cow milk (15.9%), in one sample of goat milk (3.4%), and in one sample of sheep milk (3.7%). One sample of cow milk was positive for C. burnetii; however, the sequence analysis did not confirm any species of Coxiella or Coxiella-like organisms, but showed 100% homology to Psychrobacter alimentarius. None of the examined samples showed the presence of A. phagocytophilum or Rickettsia spp. CONCLUSIONS: The results of this study suggest a potential hazard of milk-borne Toxoplasma infection, mostly by consumption of raw cow milk. The milk-borne spread seems to be limited or nonsignificant in the case of C. burnetii, A. phagocytophilum, and Rickettsia spp. The false-positive sample for Coxiella spp. suggests that some care should be taken in the interpretation of the results obtained by using the PCR method.

Seroprevalence of Toxoplasma gondii infection in goats from the south-west region of Poland and the detection of T. gondii DNA in goat milk.

Toxoplasma gondii (Nicolle et Manceaux, 1908) is an obligatory intracellular protozoan parasite prevalent in animals and humans worldwide having medical and veterinary importance on account of causing abortion or congenital disease in intermediate hosts, including man. Since T. gondii has already been identified in the milk of goats, Capra aegagrus hircus (Linnaeus), the possibility of acquiring infection by ingesting unpasteurised goat milk should be taken into consideration. Thus, the aim of the present study was to determine the presence of T. gondii DNA in goat milk. First, 73 goats (females) from 36 farms located in Poland were examined serologically by direct agglutination test (DAT) to estimate the T. gondii serological status. Milk samples from 60 selected lactating females were examined for the presence of T. gondii DNA by Real time PCR and nested PCR (B1 gene). To estimate the clonal type of detected T. gondii, multiplex PCR was performed using 6 markers. In DAT, positive results were found in 70% of 73 goats. Among examined 60 milk samples, 65% were positive in Real time PCR and 43% in nested PCR. It is noteworthy that 11 samples positive in PCR were collected from seronegative goats. The multilocus PCR analysis mostly revealed the occurrence of genotype III, which is relatively rare in Europe. The recorded high prevalence of anti-Toxoplasma antibodies in tested goats (70%), associated with a high prevalence of T. gondii DNA in goat milk samples (65%), indicates a potential risk of the parasite transmission through goat milk ingestion.

Infections and mixed infections with the selected species of Borrelia burgdorferi sensu lato complex in Ixodes ricinus ticks collected in eastern Poland: a significant increase in the course of 5 years.

In the years 2008-2009 and 2013-2014, 1620 and 1500 questing Ixodes ricinus ticks, respectively, were examined on the territory of the Lublin province (eastern Poland). The presence of three pathogenic species causing Lyme disease was investigated: Borrelia burgdorferi sensu stricto, B. afzelii and B. garinii. The proportion of I. ricinus ticks infected with B. burgdorferi sensu lato showed a highly significant increase between 2008-2009 and 2013-2014, from 6.0 to 15.3%. A significant increase was noted with regard to all types of infections with individual species: single (4.7-7.8%), dual (1.2-6.6%), and triple (0.1-0.9%). When expressed as the percent of all infections, the frequency of mixed infections increased from 21.4 to 49.2%. Statistical analysis performed with two methods (by calculating of odds ratios and by Fisher's exact test) showed that the frequencies of mixed infections in most cases proved to be significantly greater than expected. The strongest associations were found between B. burgdorferi s. s. and B. afzelii, and between B. burgdorferi s. s. and B. garinii. They appeared to be highly significant (P < 0.0001) when assessed by two methods for 2013-2014, and for the sum of findings for both time periods. The proportions of the individual species detected in the mixed infections in 2008-2009 and 2013-2014 revealed highly significant increases for B. burgdorferi s. s. and B. garinii (from 33.9 to 71.1% and from 18.2 to 82.9%, respectively), and an insignificant decrease for B. afzelii (from 51.4 to 41.6%). The proportions of the species B. burgdorferi s. s., B. afzelii and B. garinii (with combined single and mixed infections) for 2008-2009 and 2013-2014 were: 51.2/44.0 %, 30.6/24.9% and 18.2/31.1%, respectively. In conclusion, our results seem to indicate the detrimental trend of the increasing infection rate of I. ricinus ticks with B. burgdorferi s. l. in eastern Poland, and dramatic enhancement of mixed infections with individual species, which may result in mixed infections of humans and exacerbation of the clinical course of Lyme disease cases on the studied area.

Babesia spp. in questing ticks from eastern Poland: prevalence and species diversity.

A total of 853 questing Ixodes ricinus males, females, and nymphs and of 582 questing Dermacentor reticulatus males and females were collected from vegetation on the territory of the Lublin province (eastern Poland). The ticks were examined for the presence of Babesia by PCR detecting part of 18S ribosomal RNA (rRNA) gene and nuclear small subunit rRNA (SS-rDNA) for determining of Babesia spp. and Babesia microti, respectively. The overall incidence of Babesia strains in I. ricinus ticks was 4.6%. Three species of Babesia were identified. The prevalent species was B. microti which occurred in 2.8% of ticks, while Babesia venatorum, Babesia divergens, and unidentified Babesia species were found at the frequency of 1.2, 0.2, and 0.3%, respectively. Altogether, B. microti constituted 61.5% of the total strains detected in I. ricinus, B. venatorum-25.7%, B. divergens-5.1%, and unidentified Babesia species-7.7%. The prevalence of Babesia species in I. ricinus did not depend significantly on locality (χ(2) = 1.885, P = 0.390) nor on the tick stage (χ(2) = 4.874, P = 0.087). The incidence of Babesia strains in D. reticulatus ticks was 2.7%. Two species of Babesia were identified. Again, the prevalent species was B. microti which occurred in 2.1% of ticks, while B. canis was found in 0.7% of ticks. In one D. reticulatus female, B. canis and B. microti co-infection was found. Altogether, B. microti constituted 75% of the total strains detected in D. reticulatus while B. canis formed 25% of the total strains. The frequency of the occurrence of Babesia species in D. reticulatus did not depend significantly on locality (χ(2) = 0.463, P = 0.793). The difference between the prevalence of Babesia in males and females of D. reticulatus was insignificant (P = 0.0954); nymphs were not found. The dominance of B. microti in the species composition of tick-borne Babesia found in this study was typical for eastern Europe. In conclusion, the results revealed that the population inhabiting the forested area of eastern Poland could be exposed to Babesia parasites, especially to those from the species B. microti, by a bite of I. ricinus, a competent vector of human babesiosis, and probably also by a bite of D. reticulatus whose role in the transmission of human babesiosis needs to be clarified.

Prevalence and molecular typing of Giardia duodenalis in wildlife from eastern Poland.

Faecal samples from 162 wild animals were collected from 32 distinct sites of Leczynsko-Wlodawskie Lakeland (eastern Poland). The presence of Giardia duodenalis (Stiles, 1902) was assessed by a Direct Fluorescence Assay (DFA) and by Polymerase Chain Reaction (PCR) and sequencing of a fragment of the beta-giardin gene. DFA showed the presence of cysts of G. duodenalis in 12 of 162 faecal samples (7%), namely in four wild boars (15%), four foxes (19%), two roe deer (4%), and two wolves (29%). PCR identified 34 of the 162 (21%) samples as positive, including 11 wild boars (41%), five red deer (18%), 11 roe deer (23%), four moose (17%), two wolves (29%) and a single sample from the European badger. Thus, PCR detected a significantly higher number of infection than DFA (P = 0.0005). However, 14 of 34 PCR products could not be sequenced because of their insufficient amount; the low number of cysts, poor conservation of the faeces or presence of PCR inhibitors may have contributed to weak DNA amplification. Sequence analysis of the remaining 20 products showed the presence of assemblage B in wild boars, red deer and roe deer, whereas samples from wolves were identified as assemblage D. This is the first detection of assemblage B in wild boars and deer. As assemblage B has zoonotic potential, wild animals from eastern Poland may act as reservoirs of cysts of G. duodenalis infectious for humans.

Toxoplasma gondii (Nicolle et Manceaux, 1908) detected in Dermacentor reticulatus (Fabricius) (Ixodidae).

The aim of the present work was to determine whether Dermacentor reticulatus (Fabricius), tick species common in eastern Poland could be infected with Toxoplasma gondii (Nicolle et Manceaux, 1908). A total of 664 unfed D. reticulatus ticks were collected from six localities of Lublin province (eastern Poland) within the framework of study for the presence of bacterial, viral and parasitological infections, with use of PCR and confirmed by sequencing analysis. The prevalence of T. gondii DNA of B1 gene in the total examined D. reticulatus ticks was 3.2%. The infection varies greatly depending on the locality of tick collection (0-16.7%). Preliminary identification of clonal type (I or II/III) by Restriction Fragments Length Polymorphism PCR (RFLP-PCR) with use B1 gene showed that all isolates of T. gondii belonged to type I. RFLP analysis using genetic markers SAG1, 5'-SAG2, 3'-SAG2, SAG3, and GRA6 on B1-positive samples showed that only a single isolate proved to be type I with all five markers, another type was classified to type I according to four markers, while another five isolates had only type I alleles at GRA6, which cannot be regarded as type I confirmation. It must be pointed out that the used DNA isolation method by boiling with ammonium hydroxide enables to receive the total DNA from ticks, but may be not quite suitable for genotyping. In conclusion, this study indicates that besides Ixodes ricinus (Linnaeus), also D. reticulatus should be considered as a potential vector of T. gondii. The presumption of tick-borne transmission as an alternative pathway of disease spreading could well explain the high prevalence of toxoplasmosis among the herbivorous mammals and birds. However, this hypothesis needs verification by further experimental and ecological studies.

Prevalence of tick-borne pathogens at various workplaces in forest exploitation environment.

BACKGROUND: The objective of the study was the evaluation of the infection of ticks with pathogenic microorganisms at various workplaces (timber acquisition, forest growing, forest cultivation, forest protection). MATERIAL AND METHODS: Eight hundred sixty one Ixodes ricinus ticks collected from 4 workplaces were examined for the presence of Borrelia burgdorferi, Anaplasma phagocytophilum and Babesia microti by polymerase chain reaction (PCR). Then, a comparative analysis of the relative density and infection of ticks at individual workplaces was done. In the statistical analysis, Chi2 test, and Pearson's test for correlation were applied. RESULTS: The differences in infection (15.9-50%) of ticks with B: burgdorferi between the examined workplaces were highly significant, with the highest percentage observed at forest growing. The percentages of infection of ticks with A. phagocytophilum at individual workplaces ranged from 1.1-3.7%, and differences were statistically insignificant. The percentages of infections of ticks with Babesia microti at individual workplaces fluctuated from 3.6-4.4% and differences were also insignificant. Co-infections of ticks with 2 or 3 pathogens were rare. CONCLUSIONS: Co-infections with B. burgdorferi and B. microti showed a significant relationship with the workplaces, while those with B. burgdorferi and A. phagocytophilum did not show such a dependence. No significant positive correlation was found between the relative density of ticks and the frequencies of infections with B. burgdorferi, A. phagocytophilum and B. microti.

Age influence on hypersensitivity pneumonitis induced in mice by exposure to Pantoea agglomerans.

Hypersensitivity pneumonitis (HP) represents the immunologically mediated lung disease induced by repeated inhalations of a wide variety of certain finely dispersed organic antigens. In susceptible subjects, these inhalations provoke a hypersensitivity reaction characterized by intense inflammation of the terminal bronchioles, the interstitium and the alveolar tree. The inflammation often organizes into granulomas and may progress to pulmonary fibrosis. Our previous work indicated that cell extract of gram-negative bacteria Pantoea agglomerans (SE-PA) causes, in young C57BL/6J mice, pulmonary changes that are very similar to the clinical manifestations of HP in men. The purpose of presented studies was to describe the response of mice immune system while exposed to SE-PA. Particular attention was paid to examine the age influence on SE-PA induced inflammation and fibrosis in lung tissue. We used 3- and 18-month-old C57BL/6J mice. Lung samples were collected from untreated mice and animals exposed to harmful agent for 7 and 28 days. HP development was monitored by histological and biochemical evaluation. Using ELISA tests, we examined concentration of pro- and anti-inflammatory cytokines in lung homogenates. Our study demonstrated again that SE-PA provokes in mice changes typical for the clinical picture of HP, and that successive stages of disease (acute, subacute and chronic) might be obtained by modulation of time exposure. Furthermore, we found that animals' age at the time of sensitization influences the nature of observed changes (cytokine expression pattern) and the final outcome (reaction intensity and scale of fibrosis).

Mycobiota and mycotoxins in various kinds of vegetables and fruits as potential health risk factors for consumers - summary of a multiyear study.

INTRODUCTION: Between 2015-2020, the concentrations and diversity of filamentous fungi as well as the levels of mycotoxins in 4 classes of vegetables and fruits were studied in samples from farms located in eastern Poland. The short communication summarizes the results with the use of statistical analysis and indicates the potential health hazards associated with the consumption of the produce contaminated with fungi and/or mycotoxins. The concentrations of filamentous fungi in all examined samples were moderate (2.813 - 4.146 log10 CFU g -1). The highest values were noted in root vegetables, whereas the lowest values in fruit vegetables. The mycobiota revealed a marked biodiversity (67 species and 33 genera). Penicillium and Fusarium prevailed in the mycobiota of root vegetables, whereas Cladosporium and Alternaria in the remaining classes. Most of identified species (88.1%) were described as potentially pathogenic. The importance of mycotoxins as a potential health risk factor for vegetable consumers, was confirmed by the detection of a high prevalence (>55%) of total aflatoxin (AFT). The level of AFT in berry fruits was significantly higher than in other classes. The high prevalence (58.5%) of deoxynivalenol was noted in raspberries. CONCLUSIONS: Filamentous fungi present in the examined vegetables and fruits may pose a potential health risk for consumers due to a high prevalence of potentially pathogenic species, mostly those producing mycotoxins.

Mycobiota of berry fruits - levels of filamentous fungi and mycotoxins, composition of fungi, and analysis of potential health risk for consumers.

INTRODUCTION AND OBJECTIVE: The aim of the study was to determine the presence, concentration and generic composition of filamentous fungi. Selected mycotoxins were also determined: total aflatoxins and deoxynivalenol. MATERIAL AND METHODS: In 2017-2018, 40 samples of strawberry fruits and 40 samples of red raspberry fruits were collected. In 2019-2020, 37 samples of fresh strawberry fruits and 41 samples of fresh red raspberry fruits were collected on conventional farms located in eastern Poland and were subjected to mycological examination. The concentration and species composition of filamentous fungi were determined by the method of plate dilutions on malt agar. The isolated strains were identified using macroscopic and microscopic methods. Samples were also analysed for the presence of aflatoxin B1, total aflatoxin and deoxynivalenol using ELISA tests. RESULTS: The median concentrations of fungi were moderate or low: 3.079 and 3.491 log10 CFU g-1 for strawberries and raspberries, respectively. Fungi of the genus Cladosporium prevailed in the mycobiota of berry fruits, accounting for 84.3% of total isolates in raspberries and 34.8% in strawberries. The occurrence of total aflatoxins was detected in the majority of tested samples (73.2% in raspberries and 70.3% in strawberries) but never exceeded the level of 4.0 µg kg-1 assumed as safe. Deoxynivalenol has been detected only in raspberries with the prevalence of 58.5%. Its median concentration was 242.0 µg kg-1 and in 7 out of 41 samples (17.0%) exceeded the level of 750.0 µg kg-1, assumed as safe. CONCLUSIONS: Filamentous fungi and mycotoxins occurred in the examined berries at levels that mostly do not represent a health risk for immunocompetent people, but might pose such risk for immuno-compromised and/or atopic consumers.

COVID 19 - Possible interrelations with respiratory comorbidities caused by occupational exposure to various hazardous bioaerosols. Part II. Clinical course, diagnostics, treatment and prevention.

INTRODUCTION AND OBJECTIVE: The course of COVID-19 caused by the SARS-CoV-2 may be aggravated by bioaerosols containing other viruses, bacteria, and fungi, occurring mainly in the occupational environment. Hence, the diagnostics and treatment of COVID-19 should address such a possibility in the anamnesis, treatment and final recommendations for avoiding of adverse exposure. ABBREVIATED DESCRIPTION OF THE STATE OF KNOWLEDGE: As SARS-CoV-2 attacks primarily the respiratory system and the severe manifestation of COVID-19 is interstitial pneumonia, diagnostics should include the following clinical and laboratory examinations: chest X-ray; high resolution computed tomography (HRCT); pulmonary function tests; arterial-blood gas test; genetic tests for the presence of SARS-CoV-2, in the future with the use of highly specific and sensitive nano-based biosensors; tests for the presence of specific immunity against the antigens of microorganisms causing other infectious or allergic pulmonary diseases (in the case of anamnestic indications). Because an universally accepted treatment for COVID-19 does not exist, the hitherto prescribed antiviral and immune-modulating drugs should be used be with caution. In many cases, a better alternative could be a safe supportive therapy, such as supplementation of the diet with probiotics, prebiotics, vitamins and microelements. SUMMARY: The most important preventive measures against COVID-19 should include: vaccination; the use of filter or surgical masks; disinfection and sterilization; maintaining of well-functioning ventilation and air conditioning systems; reduction of the community air pollution which has been identified as an important factor increasing the COVID-19 severity. In the choice of preventive measures, the above should be considered for their potential efficacy against other bioaerosols as potential disease-aggravating agents.

Levels of filamentous fungi and selected mycotoxins in leafy and fruit vegetables and analysis of their potential health risk for consumers.

INTRODUCTION AND OBJECTIVE: The aim of the study was to determine the presence, concentration and generic composition of filamentous fungi. Considering the significant role of mycotoxins in the pathogenicity of fungal contaminants of vegetables, the scope of the study was extended by determination of aflatoxins and deoxynivalenol. MATERIAL AND METHODS: In the years 2019-2020, samples of vegetables (lettuce, spinach, tomato, red pepper) collected on conventional farms located in eastern Poland were subjected to mycological examination. The concentration and species composition of filamentous fungi were determined by the method of plate dilutions on malt agar. The isolated strains were identified with the use of macroscopic and microscopic methods. Samples were also analyzed for the presence of aflatoxin B1 (AFB1), total aflatoxin (AFT) and deoxynivalenol (DON) using the immunoenzymatic ELISA method. RESULTS: The median concentrations of filamentous fungi ranged from 2.778-3.204 log10 CFU g -1. Overall, 40 fungal species were identified in the examined vegetables, of which 38 are classified as potentially pathogenic for humans. The mean prevalence values for AFB1 and AFT were moderate or high (16.0-60.0% and 57.8-75.6%, respectively) and very low for DON (0-2.2%). The median concentrations of filamentous fungi, AFB1 and AFT were distinctly greater in leafy vegetables than on non-leafy tomato and pepper fruits, and the differences were highly significant (P<0.001). CONCLUSIONS: The levels of filamentous fungi and mycotoxins in Polish vegetables could be classified as moderate or low. The abundant presence of species with various pathogenic abilities may pose a risk for some categories of people consuming raw vegetables, mostly for immuno-compromised persons or atopics susceptible to food allergy caused by ingested moulds.

Beneficial impact of cathelicidin on hypersensitivity pneumonitis treatment-In vivo studies.

Cathelicidin (CRAMP) is a defence peptide with a wide range of biological responses including antimicrobial, immunomodulatory and wound healing. Due to its original properties the usefulness of CRAMP in the treatment of pulmonary fibrosis was assessed in a murine model of hypersensitivity pneumonitis (HP). The studies were conducted on mouse strain C57BL/6J exposed to a saline extract of Pantoea agglomerans cells (HP inducer). Cathelicidin was administered in the form of an aerosol during and after HP development. Changes in the composition of immune cell populations (NK cells, macrophages, lymphocytes: Tc, Th, Treg, B), were monitored in lung tissue by flow cytometry. Extracellular matrix deposition (collagens, hydroxyproline), the concentration of cytokines involved in inflammatory and the fibrosis process (IFNγ, TNFα, TGFß1, IL1ß, IL4, IL5, IL10, IL12α, IL13) were examined in lung homogenates by the ELISA method. Alterations in lung tissue morphology were examined in mouse lung sections stained with haematoxylin and eosin as well as Masson trichrome dyes. The performed studies revealed that cathelicidin did not cause any negative changes in lung morphology/structure, immune cell composition or cytokines production. At the same time, CRAMP attenuated the immune reaction induced by mice chronic exposure to P. agglomerans and inhibited hydroxyproline and collagen deposition in the lung tissue of mice treated with bacteria extract. The beneficial effect of CRAMP on HP treatment was associated with restoring the balance in quantity of immune cells, cytokines production and synthesis of extracellular matrix components. The presented study suggests the usefulness of cathelicidin in preventing lung fibrosis; however, cathelicidin was not able to reverse pathological changes completely.