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. Med. Pr. 2014;65(5):575–58

Microbiological characterization of vegetables and their rhizosphere soil in Eastern Poland

The aim of this study was to investigate the bacteriological quality of 5 kinds of vegetables (lettuce, dill, radish, beetroot, carrot) and their rhizosphere soil, originating from conventional farms located in the Lublin Province of Eastern Poland. A total number of 35 samples of fresh vegetables (FV) taken immediately from soil, 35 samples of soil from rhizosphere of these vegetables (SR) and 35 samples of vegetables sold at retail in the markets in Lublin (VR) were examined. The samples were analysed for the content of: aerobic mesophilic bacteria (AMB) grown at 30°C and 37°C, Gram-negative bacteria of Enterobacteriaceae family, faecal coliform (FC) bacteria, Salmonella spp., and Clostridium perfringens. Median AMB values determined at 30°C for FV, SR and VR were 5.27, 5.00, and 5.00 log 10 CFU g -1 , respectively, being significantly greater compared to those recorded at 37°C. The exceeding of the threshold value of 6.0 log 10 CFU g -1 proposed by Gelosa (1998) was noted only in 5 FV samples grown at 30°C (14.3%), and in 3 FV samples grown at 37°C (8.6%). The threshold value was never exceeded in SR and VR samples. Median concentrations of Enterobacteriaceae determined for FV, SR and VR were 4.03, 3.87, and 3.04 log 10 CFU g -1 , respectively. Eleven species of Enterobacteriaceae were identified in the FV, SR and VR samples. The percent of samples containing Escherichia coli was greatest for VR (22.9%), smaller for FV (17.1%) and smallest for SR (5.7%). The median concentrations of the faecal coliform bacteria (FC), determined by culture at 44°C, were low, amounting to 1.000 log 10 CFU g -1 for FV and SR and 0.00 for VR. All examined vegetable and soil samples tested negative for the presence of Salmonella. The median concentrations of Clostridium perfringens were low, amounting to 0.00 log 10 CFU g -1 for all categories of samples. This bacterium was relatively common in soil samples with the prevalence of 40.0%, but very rare in vegetable samples (occurring in 5.7% of FV and in none of VR samples). In conclusion, the results of the present study generally indicate that the microbiological quality of Polish vegetables grown on conventional farms is satisfactory and safe for consumers

Comparison of the efficiency of two commercial kits – ELFA and Western blot in estimating the phase of <i>Toxoplasma gondii</i> infection in pregnant women

Sera of 89 pregnant women were selected according to the results of ELFA IgM, IgG and avidity IgG, and tested with commercial tests IgM, IgG and avidity IgG Western Blot (WB) to compare the efficacy of both techniques in determining the phase of T. gondii infection. In total, 81 of 89 tested sera (91.0%) were classified as positive, both in the ELFA and WB tests for the presence of anti- Toxoplasma antibodies of class IgG, indicating a past infection, while the prevalence of anti- Toxoplasma positive reactions associated with the antibodies of class IgM indicating a recent infection was much lower – 31.5% and 20.2%, respectively. Sera of 81 women were also tested in the ELFA and WB tests for avidity, e.g. ability of forming high-molecular IgG antibody complexes. Low or medium results in these tests (in this study all classified as low), indicating a recent infection, were detected by ELFA and WB in 22.2% and 45.7% of the total examined samples, respectively. The Spearman’s rank test for correlation, performed for recognition of quantitative data of the ELFA and WB tests (index, units or points), revealed a highly significant correlation between the ELFA and WB tests for homologous classes of antibodies, both for IgM and IgG (p0.05), except for the WB test for IgM antibodies, which showed a significant correlation with the ELFA test for IgG antibodies (p<0.01). A highly significant negative correlation between the ELFA and WB test for IgM antibodies and ELFA and WB tests for IgG avidity was demonstrated (p<0.01), except for a relationship between the WB test for IgM and WB for avidity, which was not significant. Such negative correlations are theoretically expected, as strong complexes with the participation of IgG antibodies are absent in the early phase of toxoplasmosis when early antibodies of IgM class are present. Summarizing, this study indicates the high usefulness of the commercial ELFA and WB tests in serodiagnostics of toxoplasmosis in pregnant women. Special attention should be paid to parallel detection of IgM antibodies and low values in the ELFA and WB tests for IgG avidity, which indicates a recent infection which may be associated with a clinical form of congenital toxoplasmosis and damage to the foetus

Prevalence of infections and co-infections with 6 pathogens in <i>Dermacentor reticulatus</i> ticks collected in eastern Poland

Occurrence of co-infections with various pathogens in ixodid ticks creates a risk of increased severity of tick-borne diseases in humans and animals exposed to bite of the ticks carrying multiple pathogens. Accordingly, co-infections in ticks were subject of numerous analyses, but almost exclusively with regard to Ixodes ricinus complex whereas potential tick vectors belonging to other genera were much less studied. Taking into consideration the role of Dermacentor reticulatus in the transmission of various pathogens, we carried out for the first time the comprehensive statistical analysis of co-infections occurring in this tick species. An attempt was made to determine the significance of the associations between 6 different pathogens occurring in D. reticulatus (Tick-borne encephalitis virus = TBEV, Anaplasma phagocytophilum , Rickettsia raoultii , Borrelia burgdorferi s. l., Babesia spp., Toxoplasma gondii ), using 2 statistical methods: determination of Odds Ratios (ORs) and the Fisher’s exact test. 634 questing Dermacentor reticulatus ticks (370 females and 264 males) were collected in 2011– 2013 by flagging the lower vegetation in 3 localities in the area of Łęczyńsko-Włodawskie Lakeland, situated in the Lublin region of eastern Poland. The presence of individual pathogens was detected by PCR. Ticks were infected most often with Rickettsia raoultii (43.8%), less with TBEV (8.5%), and much less with Babesia spp., Toxoplasma gondii , Borrelia burgdorferi s.l., and Anaplasma phagocytophilum (2.5%, 2.1%, 1.6% and 1.1%, respectively). The locality-dependent variability proved to be significant for TBEV (χ 2 =11.063; P=0.004) and Toxoplasma gondii (χ 2 =11.298; P=0.0035), but not for other pathogens. Two hundred seventy (42.6%) of the examined ticks were infected only with a single pathogen, and 54 (8.5%) showed the presence of dual co-infections, each with 2 pathogens. The most common were dual infections with participation of Rickettsia raoultii (7.41%); next, those with participation of the TBEV (5.21%), Toxoplasma gondii (1.58%), Borrelia burgdorferi s.l. (1.26%), Anaplasma phagocytophilum (0.95%), and Babesia spp. (0.63%). On the total number of 15 possible associations, in 9 cases co-infections occurred whereas in 6 cases they were not detected. The most noteworthy were positive co-infections with the participation of TBEV, which proved to be weakly significant (0.05&lt;P&lt;0.1) in associations with Toxoplasma gondii and Anaplasma phagocytophilum , with Odds Ratios over 3.3 and 4.4, respectively. The values of Odds Ratios exceeded 3.0 also at the co-infections of Rickettsia raoultii with B. burgdorferi s.l., and T. gondii with Babesia spp., but these associations did not attain a significance level. The co-infections of Rickettsia raoultii with Babesia spp. appeared not to be significant (0.05&lt;P&lt;0.1) with OR below 0.3. In conclusion, co-infections with various pathogens in D. reticulatus ticks seem to be relatively rare and mostly not significant

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. Med. Pr. 2014;65(5):575–58

Optimization of flotation, DNA extraction and PCR methods for detection of Toxoplasma gondii oocysts in cat faeces

Introduction and objective The aim of the study was to compare the effectiveness of selected oocysts concentration methods, DNA extraction protocols and PCR assays targeting the B1 gene, for the development of procedures which would be effective and useful in laboratory practice for the detection of T. gondii in faecal samples from cats. Material and methods In order to compare the influence of the flotation fluids on microscopy and PCR detection of T. gondii , saturated solutions of saccharose, MgSO 4 , ZnSO 4 and NaNO 3 were used. To determine the sensitivity of PCR tests used: Real time PCR (RT) and nested PCR, water samples spiked with T. gondii tachyzoites and oocysts were tested. DNA was extracted using DNeasy Blood and Tissue Kit (Qiagen) (K1). The same PCR tests were used to assess the efficacy of T. gondii DNA detection in samples of cat faeces spiked with oocysts, using DNA extraction by a K1 set and a K2 set (QIamp DNA Stool Mini Kit, (Qiagen). Results The initial results showed that NaNO 3 was most useful as a flotation fluid due to the lack negative effect on the oocysts and amplification efficacy in PCR. The level of detection for water samples (100 µl) was determined as 100 tachyzoites and 1–50 oocysts in RT, and 2–20 oocysts in nested PCR. The limit of detection (LD) for stool samples (250 mg) spiked with oocysts, where the K1 set was used, determined as 250 and 5 oocysts in RT and nested PCR, respectively. For samples extracted with the K2 set, LD in RT was determined as 1–50 oocysts (depending on the variant) and 50 oocysts in nested PCR. Conclusions The most effective methods for detection of T. gondii in cat faeces seem to be centrifugal flotation with NaNO 3 , followed by DNA extraction with removing of inhibitors (K2 set) and Real Time PCR targeting B1 gene

Study on Giardia duodenalis and Cryptosporidium spp. infection in veterinarians in Poland

The risk of exposure to zoonotic factors among veterinarians comprises still underestimated problem. Many etiological factors of infectious diseases are so far poorly known, including the way of their transmission from environment to humans and their impact for health. The main aim of the study was to determine the risk of two selected zoonosis infections caused by Giardia duodenalis and Cryptosporidium spp. among occupational group of veterinarians in Poland. Two hundred ninety seven samples of stool were tested for the presence of Giardia cysts and Cryptosporidium oocysts using Direct Fluorescent Assay (DFA). There were no positive results for Cryptosporidium . The presence of Giardia cysts was found in two samples of faeces (0.67%), confirmed by polymerase chain reaction and sequencing. The risk with regard to the parasites Giardia duodenalis and Cryptosporidium spp. seems to be low among the group of veterinarians

Microbiological characterization of vegetables and their rhizosphere soil in Eastern Poland

The aim of this study was to investigate the bacteriological quality of 5 kinds of vegetables (lettuce, dill, radish, beetroot, carrot) and their rhizosphere soil, originating from conventional farms located in the Lublin Province of Eastern Poland. A total number of 35 samples of fresh vegetables (FV) taken immediately from soil, 35 samples of soil from rhizosphere of these vegetables (SR) and 35 samples of vegetables sold at retail in the markets in Lublin (VR) were examined. The samples were analysed for the content of: aerobic mesophilic bacteria (AMB) grown at 30°C and 37°C, Gram-negative bacteria of Enterobacteriaceae family, faecal coliform (FC) bacteria, Salmonella spp., and Clostridium perfringens. Median AMB values determined at 30°C for FV, SR and VR were 5.27, 5.00, and 5.00 log 10 CFU g -1 , respectively, being significantly greater compared to those recorded at 37°C. The exceeding of the threshold value of 6.0 log 10 CFU g -1 proposed by Gelosa (1998) was noted only in 5 FV samples grown at 30°C (14.3%), and in 3 FV samples grown at 37°C (8.6%). The threshold value was never exceeded in SR and VR samples. Median concentrations of Enterobacteriaceae determined for FV, SR and VR were 4.03, 3.87, and 3.04 log 10 CFU g -1 , respectively. Eleven species of Enterobacteriaceae were identified in the FV, SR and VR samples. The percent of samples containing Escherichia coli was greatest for VR (22.9%), smaller for FV (17.1%) and smallest for SR (5.7%). The median concentrations of the faecal coliform bacteria (FC), determined by culture at 44°C, were low, amounting to 1.000 log 10 CFU g -1 for FV and SR and 0.00 for VR. All examined vegetable and soil samples tested negative for the presence of Salmonella. The median concentrations of Clostridium perfringens were low, amounting to 0.00 log 10 CFU g -1 for all categories of samples. This bacterium was relatively common in soil samples with the prevalence of 40.0%, but very rare in vegetable samples (occurring in 5.7% of FV and in none of VR samples). In conclusion, the results of the present study generally indicate that the microbiological quality of Polish vegetables grown on conventional farms is satisfactory and safe for consumers