Prevalence of Borrelia, Neoehrlichia mikurensis and Babesia in ticks collected from vegetation in eastern Poland.

In Poland, tick-borne diseases constitute the majority of diseases related to exposure to biological agents with a predominance of Lyme borreliosis; therefore, research on ticks as a reservoir of various pathogens remains crucial in the epidemiology of human diseases after tick bites. This study aimed to identify the occurrence of Borrelia burgdorferi sensu lato, Borrelia miyamotoi, Neoehrlichia mikurensis, and Babesia spp. in ticks collected from vegetation in eastern Poland. Additionally, the prevalence of co-infections in the adult Ixodes ricinus ticks was determined. Among I. ricinus ticks the predominantly detected pathogen was B. burgdorferi s.l. (23%) with B. burgdorferi sensu stricto as the most frequently identified species, followed by B. garinii. In 2013, the double or triple infections of B. burgdorferi s.s., B. afzelii, and B. garinii species did not exceed 9% in adult ticks, whereas in 2016, the prevalence of mixed infections reached 29%. The prevalence of N. mikurensis and B. miyamotoi in I. ricinus was determined at the same level of 2.8%. Four Babesia species were identified in the examined I. ricinus population: B. microti (1.5%), B. venatorum (1.2%), B. divergens (0.2%), and B. capreoli (0.1%). Co-infections were detected in 10.1% of all infected ticks with the highest prevalence of co-infections with B. burgdorferi s.l. and Babesia species. The changes in the prevalence and the distribution of particular pathogens within tick populations indicate the need for monitoring the current situation related to tick-borne pathogens from the aspect of risk to human health.

Chlamydia psittaci in Faecal Samples of Feral Pigeons (Columba livia forma urbana) in Urban Areas of Lublin city, Poland.

Pigeons are a typical host and natural reservoir of Chlamydia psittaci, the etiological agent of avian chlamydiosis, considered as a neglected zoonotic diseases. The aim of the study was to determine the prevalence of C. psittaci in faecal samples of feral pigeons (Columba livia forma urbana) as a potential source of infection related to the presence of synanthropic birds in urban areas. A total of 143 samples of dry and fresh faeces of feral pigeons, were collected in the city of Lublin (Poland), from April to September 2021. Molecular detection of C. psittaci was performed by nested-PCR and real-time PCR, confirmed by sequencing. Among the collected samples, 5 positive results were obtained in nested-PCR (3.5%), while in real-time PCR, the number of positive samples increased to 11 (7.7%). The positive samples showed 100% identity to the C. psittaci strain AMK (CP047319.1). C. psittaci was found in 7 out of 111 (6.3%) faecal samples collected in public places, and in 4 out of 32 (12.5%) samples from the nesting site (4.9% and 2.8% among a total of 143 samples, respectively). The infection was detected in both dry and fresh faeces (9.1% and 4.5%, respectively). The highest number of positive results was obtained in June-5 (3.5%). Feral pigeons occurring in urban areas are a natural reservoir of C. psittaci posing a potential risk of zoonotic infections. However, further studies on exposure to contaminated pigeon faeces in terms of occupational and non-occupational risk of chlamydiosis are needed.

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.

Diversity and Drug Resistance of Filamentous Fungi Isolated from the Fresh Raspberries.

Fungi are one of the most widely distributed microorganisms in the environment, including food such as fruits, vegetables and other crops, posing a potential threat to food safety and human health. The aim of this study was to determine the diversity, intensity and drug resistance of potentially pathogenic filamentous fungi isolated from the fresh raspberries (Rubus idaeus L.). A total of 50 strains belonging to genera Fusarium, Cladosporium, Alternaria, Penicillium, Mucor, Rhizopus, Aspergillus and Acremonium were tested for drug resistance against 11 antifungals by disc diffusion and gradient strips methods. The average mycological contamination in the examined samples of raspberries amounted to 4.34 log CFU/g. The Cladosporium was isolated from all tested samples, followed by Alternaria and Fusarium with a frequency of 61% and 34%, respectively. The highest level of drug resistance was observed for Acremonium genera and Fusarium strains recorded a wide variation in drug resistance as revealed by susceptibility with amphotericin B and voriconzole with MICs ranged from 0.5-4 µg/ml and posaconazole with MICs ranging from 3-8 µg/ml. All fungal strains showed 100% resistance to caspofungin, fluconazole and flucytosine with both the methods, and 100% resistance to micafungin and anidulafungin in the gradient strip method.

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.

[Assessment of fungal aerosol exposure at selected workplaces contaminated with organic dust of different origin]. / Ocena narazenia na aerozol grzybowy na wybranych stanowiskach pracy zanieczyszczonych pylem organicznym o róznym pochodzeniu.

BACKGROUND: In recent years, the number of people suffering from diseases caused by fungi has been increasing. However, knowledge of the biodiversity of fungal pathogens in the work environment is still insufficient. The aim of this work was to evaluate the exposure to fungi being disseminated in the air of workplaces contaminated with organic dust of plant and animal origin. MATERIAL AND METHODS: Bioaerosol samples were collected at 3 occupational settings (poultry farm, biomass burning power plant and wastewater treatment plant) using button samplers. Quantitative and qualitative analysis of fungal aerosol was conducted by employing macro- and microscopic methods. Selected strains were then studied by polymerase chain reaction (PCR) using srodointernal transcribed spacers (ITS): ITS1-ITS2, ITS3-ITS4 and ITS1-ITS4 primer pairs. RESULTS: Average concentrations of fungal aerosol at workplaces ranged 1.2×102-2.1×106 cfu/m3. The highest fungal concentrations were recorded in the poultry farm, while the lowest were noted at the wastewater treatment plant. Aspergillus fumigatus was a predominant component of the mycobiota in the power plant and wastewater treatment plant. Almost 100% identification agreement of this pathogen between the traditional and molecular method was noted. CONCLUSIONS: The fungal concentrations in poultry farms exceeded the Polish proposal for the threshold limit value (5×104 cfu/m3). The results of the study demonstrate a high compatibility of A. fumigatus' identification using the traditional and molecular methods. Taking into account the fact, that a long term exposure to A. fumigatus conidia at workplaces may result in numerous health complaints, the use of proper protective equipment by workers must be a standard procedure. Med Pr 2018;69(3):269-280.

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.

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.

Knowledge, protection behaviours and seroprevalence of Lyme borreliosis in inhabitants of Lublin Province, eastern Poland - evaluation of a prophylaxis programme.

INTRODUCTION AND OBJECTIVE: Lyme borreliosis (LB) is the most frequent tick-borne disease with 17,338 cases reported in Poland in 2022. Since research on a LB vaccine is still ongoing, the promotion of individual behaviours and limiting of tick exposure is one of the most effective ways to prevent LB. The aim of the study was to evaluate the effectiveness of the LB prevention programme by assessing the knowledge, practice behaviours, seroprevalence of LB and satisfaction among the population of the Lublin Province in eastern Poland. MATERIAL AND METHODS: The prevention programme was carried out among 2,920 participants who were asked about their exposure to ticks, history of LB and prevention behaviours. Awareness of knowledge was evaluated before and after training. Seroprevalence of LB was rated by ELISA and immunoblot assays. RESULTS: Over 73% of participants reported tick bites in their lifetime, without significant differences between rural and urban area inhabitants. More than 80% of individuals declared that they use protective measures (PPM), such as proper clothes and body checking; repellents were the least frequently used by participants. The diagnosis of LB but not tick bites in a lifetime influenced the more frequent use of PPM. Increase in knowledge was observed in 86% of participants after education, and the highest knowledge was noted among respondents with higher education. The seroprevalence of anti-B. burgdorferi antibodies was 37% and was higher among men than women (40% vs. 36%). CONCLUSIONS: The population of Lublin Province is highly exposed to tick bites and infection with B. burgdorferi. The high seroprevalence and increase in knowledge confirmed the effectiveness and need for preventive programmes. These results can be useful for optimizing and enhancing the effects of future prevention campaigns.

Filters of automobile air conditioning systems as in-car source of exposure to infections and toxic moulds.

The main component of an air conditioning system is air filters. Over time, the filters of an air conditioning system in cars can turn into sources of emission of microbiological hazards. The aim of this study was to quantitatively and qualitatively assess the presence of infectious and toxic fungi in the AC filters in passenger cars. The studied non-woven filters were removed from passenger cars during the "winter"/"summer" seasons. The taxonomic identification of the fungi isolated from the filters was performed using both the culture-based and molecular methods. RT-PCR was applied to assess the presence of gene fragments regulating aflatoxin biosynthesis in the isolates obtained from fungal cultures. The average fungal concentrations in the filter samples collected during the summer/winter season were 5.4 × 104 cfu/m2 and 2.4 × 104 cfu/m2, respectively. Most of the filter samples, collected in both the studied seasons, revealed the presence of Aspergillus species including A. fumigatus, A. niger, A. terreus and/or A. flavus. The recorded levels of fungal contamination of AC filters in passenger cars indicate the necessity for more frequent filter replacement in this type of vehicle. Occupational exposure to moulds and the resulting health problems that may be experienced by professional drivers should be properly recognised in order to undertake effective preventive measures.

The Consumption of Raw Goat Milk Resulted in TBE in Patients in Poland, 2022 "Case Report".

The alimentary route is the second most important route of tick-borne encephalitis infection. In Poland, the last TBE case due to the consumption of unpasteurized milk or dairy products of infected animals was recorded in 2017 as the fourth documented outbreak of TBEV infection in the country. In this study, two patients infected with TBEV through consumption of unpasteurized goat's milk from one source are described from a cluster of eight cases. In August and September 2022, a 63- and 67-year-old woman were hospitalized at the Infectious Diseases Clinic of the Institute of Rural Health (Lublin, Poland). The patients denied been recently bitten by a tick, and neither had been vaccinated against TBEV. The disease had a biphasic course. In the first case, the patient suffered from a fever, spine pain, and muscle weakness and paresis of the lower left limb. The second patient suffered from fever, vertigo, headaches, abdominal pain, and diarrhoea. The results of IgM and IgG antibodies were positive in both cases. After three weeks hospitalization, the patients were discharged in good condition. In one case, slight hearing impairment was observed. Vaccination and avoiding the consumption of unpasteurized milk remain the most effective ways to prevent tick-borne encephalitis.

Comparison of the effectiveness of various parasitological methods in detecting nematode eggs in different types of soil.

INTRODUCTION AND OBJECTIVE: Natural fertilizers, sewage sludge, digestates, as well as organic fertilizers produced on their basis, can become a source of parasitological contamination of cultivated land. High concentration of invasive forms of parasites in the soil may pose a threat to human and animal health. Therefore, it is necessary to control the hygienic condition of fertilizers and fertilized soils with particular emphasis on parasites. The aim of the study was to compare the effectiveness of methods commonly used for parasitological examination of soil with own methods which were used to develop the standards. MATERIAL AND METHODS: The study was carried out using samples of sandy soil (SS), horticultural mix soil (HS) and peat-based substrate (PS). Each sample was spiked with 100 dyed Ascaris suum eggs and examined with the use of 6 methods: Vasilkova, Dada, Quinn, and 3 methods according to the Polish Standards (PN-19000, PN- 19005, PN-19006). For each variant, 8 repetitions were made. RESULTS: The largest number of A. suum eggs were found with PN-19006 (mean number of detected eggs was 21.25, 46.50, 23.00 for HS, SS, PS, respectively. Slightly lower results were obtained using PN-19005 - the mean number eggs was 21.25, 36.00, 16.75, respectively. On the other hand, the mean number of A. suum eggs found with the Dada method was about 2-3 times lower than with the PN-19006 - 15.75, 22.50, 6.50 for HS, SS, PS soil, respectively. Other methods were much less effective. CONCLUSIONS: PN-19006 method turned out to be the most effective in detecting A. suum eggs. This method can be used for parasitological examination of soils and can be the basis for developing a system of methods dedicated to testing different types of soils for the presence of nematode eggs.

Gelatin medium for preserving of Trichinella spp. for quality control in laboratories - estimation of larvae viability.

INTRODUCTION AND OBJECTIVE: Official food control laboratories ensure food safety using reliable, validated methods. Council Regulations (EC) No. 853/2004, 854/2004 and 882/2004 of the European Parliament established hygiene rules the production of food of animal origin, together with requirements for official controls. This leads to detailed requirements for Trichinella control set out in Commission Implementing Regulation (EU) 2015/1375 of 10 August 2015. These regulations require the laboratory to participate in proficiency testing (PT) to confirm their competence and improve the quality of testing, and require the PT Organizer to use methods for the preparation and preservation of parasite larvae in order to evaluate and improve detection. Traditional methods of preparing such larvae expose them to rapid degradation, making it necessary to simultaneously isolate the larvae and place them in meatballs to ensure quality. MATERIAL AND METHODS: We developed a technique for preserving of Trichinella spp. for quality control such as PT sample preparation. The procedure protects larvae against toxic oxygen activity and bacterial destruction via a gelatin barrier. To estimate the viability of larvae preserved by this method, gelatin capsules with 10 larvae of T. spiralis in each were stored (4-8 °C) during 45 days of an experiment. Samples were tested at 2 day intervals (3 samples each day of testing). RESULTS: In total, 75 samples were tested. Larvae remained alive up to 3 weeks. The number of living larvae diminished after 27 days through day 43, after which no living larvae were observed. CONCLUSIONS: The gelatin medium procedure facilitated easy, high-throughput sample preparation and supported 100% recovery for 3 weeks. The method allows fast, efficient and accurate PT sample preparation.

Parasitological contamination of arable soil in selected regions of Poland - preliminary study.

INTRODUCTION AND OBJECTIVE: The hygienic status of arable soils in most developed countries has been unknown. In the presented study, a preliminary investigation was undertaken to determine the contamination with eggs of parasitic nematodes in the soil of arable fields in Poland. The aim of the study was to determine whether such contamination is common enough to constitute a significant problem and what factors may influence it. MATERIAL AND METHODS: The study was conducted in 5 Polish provinces from autumn 2021 to spring 2022. The provinces differed significantly in terms of the area of agricultural land, agricultural suitability, type of soil, scale of cattle and pig breeding, production of manure and slurry, and the use of manures and organic fertilizers for fertilization. A total of 133 soil samples were collected. Parasitological examination of soil samples was carried out using the PN-Z-19006 method [1], with confirmed high sensitivity. RESULTS: Parasite eggs were found in a total of 67 samples, of which 56 samples contained eggs of roundworms of the genus Ascaris (an average of 3.29 eggs/100 g of soil), 23 contained eggs of whipworms (an average of 1.22 eggs/100 g), and 3 contained eggs of Toxocara (1 egg/100 g). CONCLUSIONS: Differences in the percentage of positive samples were found depending on the period in which the samples were taken. The percentage of positive samples collected in autumn (53.57%) was higher than the percentage of positive samples collected in spring (48.05%). Similarly, the average number of eggs of in positive samples collected in autumn (3.43 eggs/100 g) was higher than the average number of eggs in samples collected in spring (2.90 eggs/100 g). Differences in the percentage of positive samples were also found depending on the region of origin of the samples.

Molecular confirmation of the systemic toxoplasmosis in cat.

INTRODUCTION AND OBJECTIVE: Systemic toxoplasmosis with tissue-spread parasites occurring in intermediate hosts may also occur in immunocompromised cats (e.g., infected with FLV or FIV). To the best of our knowledge, no reports have been published on the detection and genotyping of T. gondii DNA in cats with extraintestinal toxoplasmosis in Poland. The article describes the case of the sudden death of 3 out of 4 cats in a cattery, and the detection and molecular characterization of T. gondii DNA detected in the tissues of one of the dead cats. MATERIAL AND METHODS: Samples of brain, lungs, heart, and liver of the cat that died suddenly were examined for the presence of T. gondii DNA (B1 gene) by nested PCR and real-time PCR. DNA positive samples were also genotyped at 12 genetic markers using multiplex multilocus nested PCR-RFLP (Mn-PCR-RFLP) and multilocus sequence typing (MLST). RESULTS: A total of 9 out of the 20 DNA samples were successfully amplified with nested and/or Real-time PCR. DNA from 3 out of 5 types of tested samples were genotyped (brain, heart and muscle). Mn-PCR-RFLP and MLST results revealed type II (and II/III at SAG1) alleles at almost all loci, except a clonal type I allele at the APICO locus. This profile corresponds to the ToxoDB#3 genotype, commonly identified amongst cats in Central Europe. CONCLUSIONS: To the best of our knowledge, this is the first study describing the genetic characteristics of T. gondii population determined in a cat in Poland. These data confirm the importance of this host as a reservoir for this pathogen, and demonstrate the genotypic variation of this parasite. Veterinarians should take into account that cats may develop disseminated toxoplasmosis, and that it is a systemic disease which may lead to the death of the cat, and to transmission of the pathogen to other domestic animals and to humans.

Polymorphisms of apolipoprotein E gene and cognitive functions of postmenopausal women, measured by battery of computer tests - Central Nervous System Vital Signs.

OBJECTIVES: Apolipoprotein E (APOE) gene belongs to the group of genes increasing the risk of the Alzheimer's disease (AD) development. The purpose of the study was the analysis of cognitive functions in postmenopausal women having different polymorphisms of APOE gene; battery of computer tests - Central Nervous System Vital Signs (CNS-VS) were employed. METHODS: The women were qualified into the examined group on the grounds of clinical symptoms (minimum 2 years after the last menstruation), as well as on the basis of FSH concentration. At the qualification stage, a short test - Montreal Scale of Cognitive Function Assessment (MoCA) was conducted. The assessment of cognitive functions was made with the use of diagnostic CNS-Vital Signs equipment. Genomic DNA isolation was extracted from human whole blood. Multiplex PCR reactions have been performed in a single reaction tube with six (6) primers, consisting of 2 common primers and 4 specific primers [2 - for each of 2 single nucleotide polymorphism (SNP) sites]. RESULTS AND CONCLUSIONS: About half of the examined postmenopausal women were placed below average in the majority of the examined cognitive functions. The biggest impairments occurred in the field of processing speed, and the smallest - in the field of verbal and visual memory. Polymorphisms of APOE gene were considerably linked with the level of results of the majority of cognitive functions among postmenopausal women, as measured by battery of computer tests - CNS-VS. The presence of ε2/ε3 polymorphism of APOE gene impacted positively the obtained results of cognitive functions, whereas the presence of ε3/ε4, or ε4/ε4 polymorphisms worsened the obtained results.