Streptococcus suis: a re-emerging pathogen associated with occupational exposure to pigs or pork products. Part II - Pathogenesis.

Streptococcus suis is a re-emerging zoonotic pathogen that may cause severe disease, mostly meningitis, in pigs and in humans having occupational contact with pigs and pork, such as farmers, slaughterhose workers and butchers. The first stage of the pathogenic process, similar in pigs and humans, is adherence to and colonisation of mucosal and/or epithelial surface(s) of the host. The second stage is invasion into deeper tissue and extracellular translocation of bacterium in the bloodstream, either free in circulation or attached to the surface of monocytes. If S. suis present in blood fails to cause fatal septicaemia, it is able to progress into the third stage comprising penetration into host's organs, mostly by crossing the blood-brain barrier and/or blood-cerebrospinal fluid barrier to gain access to the central nervous system (CNS) and cause meningitis. The fourth stage is inflammation that plays a key role in the pathogen esis of both systemic and CNS infections caused by S. suis. The pathogen may induce the overproduction of pro-inflammatory cytokines that cause septic shock and/or the recruitment and activation of different leukocyte populations, causing acute inflammation of the CNS. Streptococcus suis can also evoke - through activation of microglial cells, astrocytes and possibly other cell types - a fulminant inflammatory reaction of the brain which leads to intracranial complications, including brain oedema, increased intracranial pressure, cerebrovascular insults, and deafness, as a result of cochlear sepsis. In all stages of the pathogenic process, S. suis interacts with many types of immunocompetent host's cells, such as polymorphonuclear leukocytes, mononuclear macrophages, lymphocytes, dendritic cells and microglia, using a range of versatile virulence factors for evasion of the innate and adaptive immune defence of the host, and for overcoming environmental stress. It is estimated that S. suis produces more than 100 different virulence factors that could be classified into 4 groups: surface components or secreted elements, enzymes, transcription factors or regulatory systems and transporter factors or secretion systems. A major virulence factor is capsular polysaccharide (CPS) that protects bacteria from phagocytosis. However, it hampers adhesion to and invasion of host's cells, release of inflammatory cytokines and formation of the resistant biofilm which, in many cases, is vital for the persistence of bacteria. It has been demonstrated that the arising by mutation unencapsulated S. suis clones, which are more successful in penetration to and propagation within the host's cells, may coexist in the organism of a single host together with those that are encapsulated. Both 'complementary' clones assist each other in the successful colonization of host's tissues and persistence therein. S. suis has an open pan-genome characterized by a frequent gene transfer and a large diversity. Of the genetic determinants of S. suis pathogenicity, the most important are pathogenicity islands (PAI), in particular, a novel DNA segment of 89 kb length with evident pathogenic traits that has been designated as 89K PAI. It has been estimated that more than one-third of the S. suis virulence factors is associated with this PAI. It has been proved that the virulent S. suis strains possess smaller genomes, compared to avirulent ones, but more genes associated with virulence. Overall, the evolution of the species most probably aims towards increased pathogenicity, and hence the most significant task of the current research is an elaboration of a vaccine, efficient both for humans and pigs.

Streptococcus suis: a re-emerging pathogen associated with occupational exposure to pigs or pork products. Part I - Epidemiology.

Streptococcus suis (ex Elliot 1966, Kilpper-Bälz & Schleifer 1987) is a facultatively anaerobic Gram-positive ovoid or coccal bacterium surrounded by a polysaccharide capsule. Based on the antigenic diversity of the capsule, S. suis strains are classified serologically into 35 serotypes. Streptococcus suis is a commensal of pigs, commonly colonizing their tonsils and nasal cavities, mostly in weaning piglets between 4-10 weeks of age. This species occurs also in cattle and other mammals, in birds and in humans. Some strains, mostly those belonging to serotype 2, are also pathogenic for pigs, as well as for other animals and humans. Meningitis is the primary disease syndrome caused by S. suis, both in pigs and in humans. It is estimated that meningitis accounted for 68.0% of all cases of human disease reported until the end of 2012, followed by septicaemia (including life-threatening condition described as 'streptococcal toxic shock-like syndrome' - STSLS), arthritis, endocarditis, and endophthalmitis. Hearing loss and/or ves tibular dysfunction are the most common sequelae after recovery from meningitis caused by S. suis, occurring in more than 50% of patients. In the last two decades, the number of reported human cases due to S. suis has dramatically increased, mostly due to epidemics recorded in China in 1998 and 2005, and the fulminant increase in morbidity in the countries of south-eastern Asia, mostly Vietnam and Thailand. Out of 1,642 cases of S. suis infections identified between 2002-2013 worldwide in humans, 90.2% occurred in Asia, 8.5% in Europe and 1.3% in other parts of the globe. The human disease has mostly a zoonotic and occupational origin and occurs in pig breeders, abattoir workers, butchers and workers of meat processing facilities, veterinarians and meat inspectors. Bacteria are transmitted to workers by close contact with pigs or pig products, usually through contamination of minor cuts or abrasions on skin of hands and/or arms, or by pig bite. A different epidemiologic situation occurs in the Southeast Asian countries where most people become infected by habitual consumption of raw or undercooked pork, blood and offal products in the form of traditional dishes. Prevention of S. suis infections in pigs includes vaccination, improvement in pig-raising conditions, disinfection and/or fumigation of animal houses, and isolation of sick animals at the outbreak of disease. Prevention of human infections comprises: protection of skin from pig bite or injury with sharp tools by people occupationally exposed to pigs and pig products, prompt disinfection and dressing of wounds and abrasions at work, protection of the respiratory tract by wearing appropriate masks or repirators, consulting a doctor in the case of febrile illness after exposure to pigs or pork meat, avoidance of occupations associated with exposure to pigs and pork by immunocompomised people, avoidance of consumption of raw pork or pig blood, adequate cooking of pork, and health education.

Prevalence of infections and co-infections with 6 pathogens in Dermacentor reticulatus 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 Leczynsko-Wlodawskie 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 (c2=11.063; P=0.004) and Toxoplasma gondii (c2=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

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.

Comparison of the efficiency of two commercial kits - ELFA and Western blot in estimating the phase of Toxoplasma gondii 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 (p<0.00001). In contrast, the ELFA and WB tests for detection of anti-Toxoplasma IgM antibodies were not correlated with the ELFA and WB tests for detection of anti-Toxoplasma IgG antibodies (p>0.05), 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.

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.

Spiroplasma - an emerging arthropod-borne pathogen?

Spiroplasma is a genus of wall-less, low-GC, small Gram-positive bacteria of the internal contractile cytoskeleton, with helical morphology and motility. The genus is classified within the class Mollicutes. Spiroplasma / host interactions can be classified as commensal, pathogenic or mutualist. The majority of spiroplasmas are found to be commensals of insects, arachnids, crustaceans or plants, whereas a small number of species are pathogens of plants, insects, and crustaceans. Insects are particularly rich sources of spiroplasmas. The bacteria are common in haematophagous arthropods: deerflies, horseflies, mosquitoes, and in ticks, where they may occur abundantly in salivary glands. The ability of spiroplasmas to propagate in rodents was experimentally proven, and Spiroplasma infections have been reported recently in humans. Some authors have purported an etiological role of Spiroplasma in causing transmissible spongiform encephalopathies (TSEs), but convincing proof is lacking. The possibility for humans and other vertebrates to be infected with Spiroplasma spp. in natural conditions is largely unknown, as well as the possibility of the transmission of these bacteria by ticks and haematophagous insects. Nevertheless, in the light of new data, such possibilities cannot be excluded.

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.

Evaluation of reactivity to Echinococcus spp. among rural inhabitants in Poland.

A group of 172 rural inhabitants from eastern Poland (68 males and 104 females, mean age 49.0 ± 12.0 years) was examined for the presence of antibodies against Echinococcus granulosus and Echinococcus multilocularis. A population of 38 healthy urban dwellers from the city of Lublin (17 males and 21 females, mean age 36.2 ± 9.6 years) were examined as a control group. Sera of 22 rural inhabitants (12.8%) reacted positively to Echinococcus granulosus hydatid fluid antigen in the screening test. A cross-reactivity was observed with two serum samples that tested positive in ELISA for E. granulosus. Three serum samples were tested positive for E. multilocularis using the Em2plus ELISA assay and also positive for Western blot. None of the members of control group showed the presence of a seropositive reaction to Echinococcus spp. The reactivity to Echinococcus spp. among rural inhabitants decreased with age and this correlation was statistically significant (R = -0.197151, p = 0.009535). The percentage of positive findings was the highest (50.0%) in the youngest age group (14-20). No significant correlations were found between responses to interview questions (possession of domestic and farm animals, contact with wild animals, eating unwashed berries, drinking unboiled water) and the presence of seropositive reactions to Echinococcus spp. The presented results seem to indicate that echinococcosis is still a current problem in Poland that should not be neglected and, moreover, indicates the need for improvement in the routine laboratory diagnostics of Echinococcus spp. by standardizing the ELISA and Western blot tests.

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.

Occurrence of Francisella spp. in Dermacentor reticulatus and Ixodes ricinus ticks collected in eastern Poland.

A total of 530 questing Dermacentor reticulatus ticks and 861 questing Ixodes ricinus ticks were collected from Lublin province (eastern Poland) and examined for the presence of Francisella by PCR for 16S rRNA (rrs) and tul4 genes. Only one female D. reticulatus tick out of 530 examined (0.2%) was infected with Francisella tularensis subspecies holarctica, as determined by PCR of the rrs gene. None of 861 I. ricinus ticks were infected with F. tularensis. In contrast, the presence of Francisella-like endosymbionts (FLEs) was detected in more than half of the D. reticulatus ticks (50.4%) and 0.8% of the I. ricinus ticks. The nucleotide sequences of the FLEs detected in D. reticulatus exhibited 100% homology with the nucleotide sequence of the FLE strain FDrH detected in Hungary in D. reticulatus. In conclusion, our results suggest a low contribution of D. reticulatus and I. ricinus ticks to the circulation of F. tularensis in eastern Poland. This finding, however, needs to be confirmed by further studies in other areas. Our study confirmed the common infection of D. reticulatus with Francisella-like endosymbionts (FLEs) of unknown pathogenic potential and revealed, for the first time, a low grade of infection of I. ricinus with FLEs.

Occurrence of Leptospira DNA in water and soil samples collected in eastern Poland.

Leptospira is an important re-emerging zoonotic human pathogen, disseminated by sick and carrier animals, water and soil. Weather calamities, such as flooding or cyclones favour the spreading of these bacteria. To check a potential role of natural water and soil in the persistence and spread of Leptospira on the territory of eastern Poland, 40 samples of natural water and 40 samples of soil were collected from areas exposed to flooding, and 64 samples of natural water and 68 samples of soil were collected from areas not exposed to flooding. Samples of water were taken from various reservoirs (rivers, natural lakes, artificial lakes, canals, ponds, farm wells) and samples of soils were taken at the distance of 1-3 meters from the edge of the reservoirs. The samples were examined for the presence of Leptospira DNA by nested-PCR. Two out of 40 samples of water (5.0%) collected from the area exposed to flooding showed the presence of Leptospira DNA, while all 40 samples of soil from this area were negative. All samples of water and soil (64 and 68, respectively) collected from the areas not exposed to flooding were negative. No significant difference were found between the results obtained in the areas exposed and not exposed to flooding. In conclusion, these results suggest that water and soil have only limited significance in the persistence and dissemination of Leptospira in eastern Poland.

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.

A small scale survey of Leptospira in mammals from eastern Poland.

Samples of 30 dead small mammals each were collected on area 'A' located in eastern Poland and exposed to floods by the Vistula river, and on area 'B', also located in eastern Poland, but not exposed to floods. Kidneys and livers of the mammals were examined by the PCR and nested PCR methods for the presence of Leptospira DNA. From 7 species of small mammals examined, the presence of Leptospira DNA was detected in 2 of them. The prevalence of positive results was greatest in Apodemus agrarius which was the mostly numerous mammal species (14 out of total 39 specimens, 35.9%). The presence of Leptospira DNA was also found in Microtus arvalis (1 out of 1 specimen, 100%), whereas the remaining 5 species (Apodemus flavicollis , Apodemus sylvaticus, Microtus agrestis, Myodes glareolus, Sorex araneus) were negative. No significant difference in the prevalence of positive findings was found between the small mammals from areas 'A' exposed to flooding, compared to those from area 'B' not exposed to flooding (20.0% vs. 30.0%, p=0.3748). The overall positivity of the examined small mammals population from areas 'A' and 'B' was 25.0%. The prevalence of dual positivity (leptospiral DNA found both in kidney and liver) was greater in the mammals from areas exposed to flooding compared to those from areas not exposed to flooding (16.7% vs. 6.7%), but this dependence was also not significant (p=0.2382).

Study on tick-borne rickettsiae in eastern Poland. I. Prevalence in Dermacentor reticulatus (Acari: Amblyommidae).

Rickettsia spp. transmitted by ticks are classified mostly in the Spotted Fever Group Rickettsiae (SFGR). Numerous species of this group have been identified in Eurasia as emerging pathogens, but still little is known about their occurrence, effects on human health, and co-incidence with other tick-borne pathogens. The aim of the presented study was to determine the prevalence of Rickettsia spp. in adult Dermacentor reticulatus (Acari: Amblyommidae) ticks collected in Lublin province of eastern Poland using the PCR method. The infection rate of D. reticulatus with Rickettsia spp. was 53.0%. All except one rickettsial isolates showed 100% homology with Rickettsia raoultii. A high prevalence of R. raoultii in Dermacentor reticulatus ticks from eastern Poland suggests that the SFGR species should be considered as potential causative agents of tick-borne diseases in this area.

Study on tick-borne rickettsiae in eastern Poland. II. Serological response of the occupationally exposed populations.

A group of 150 persons living in the Lublin province of eastern Poland and occupationally exposed to tick bite were examined by the immunoenzymatic ELISA test for the presence of antibodies against tick-borne Spotted Fever Group (SFG) rickettsiae. The group consisted of 75 forestry workers employed in 3 forest inspectorates and 75 agricultural workers living in 2 villages. As a control group, 43 urban dwellers living in the city of Lublin and not occupationally exposed to tick bite were examined. Among 150 persons occupationally exposed to tick bite, the presence of antibodies against SFG rickettsiae was found in 54 (36.0% of the total). In the control group, the frequency of positive findings was only 4.7%, being significantly smaller compared to the exposed group (p=0.0001). Within the exposed group, the percentage of positive results in forestry workers (50.7%) was greater than in agricultural workers (21.3%); the difference was statistically significant (p=0.0002). Also within this group, the frequency of positive findings in males(46.5%) was significantly greater than in females (21.9%) (p=0.0029). In the exposed group, the positive results tended to increase with the age of the examined persons. However, a significant relationship between age and positive findings was found only in forestry workers (χ(2) =14.207, p=0.00264), but not in agricultural workers and total exposed workers. The frequencies of positive results in forestry workers varied significantly depending on place of work (χ(2) =11.271, p=0.00357). Similarly, the difference between the positive reactions in agricultural workers living in 2 villages proved to be significant (34.2% vs. 8.1%; p=0.0074). The obtained results indicate that people occupationally exposed to tick bite and living in the area of eastern Poland where over half of Dermacentor reticulatus ticks harbour SFG rickettsiae, are under significantly increased risk of infection with these rickettsiae.

A small-scale survey of hantavirus in mammals from eastern Poland.

Samples of 30 dead small mammals each were collected on area 'A' located in eastern Poland which is exposed to flooding by the Vistula river, and on the area 'B', also located in eastern Poland but not exposed to flooding. Kidneys and livers of the mammals were examined by the PCR and nested PCR methods for the presence of hantavirus RNA. Out of 7 species of small mammals examined, the presence of hantaviruses was detected in 4 of them. Hantavirus prevalence was low in Apodemus agrarius (2.6%), the most numerous mammal species, whereas in the remaining 3 positive species (Microtus agrestis, Myodes glareolus, Sorex araneus) this was 12.5-100%. The presence of hantaviruses was detected only in the animals found on area 'A' exposed to flooding, and their prevalence was statistically greater compared to area 'B' not exposed to flooding (16.7% vs. 0%, p=0.0345). The overall positivity of the examined small mammals population from the areas 'A' and 'B' was 8.3%. The sequence analysis of the samples positive for hantavirus proved that the amplified products showed 77-86% homology with the L segment sequence of hantavirus Fusong-Mf-731 isolated from Microtus fortis in China. The presented study is the first to demonstrate the occurrence of hantavirus infection in small mammals from eastern Poland, and the first to demonstrate the significant relationship between flooding and the prevalence of hantaviruses in small mammals.

Risk of Lyme disease at various sites and workplaces of forestry workers in eastern Poland.

Knowledge about the seasonal activity of various stages of the Ixodes ricinus tick is of great importance while developing models of the circulation of pathogens transmitted by ticks in a given environment, as well as while evaluating the risk of infection with these pathogens among individuals performing work in this environment. The objectives of the study was determination of the relative activity of Ixodes ricinus ticks occurring at 4 different workplaces of workers employed in one randomly selected forestry inspectorate, and the comparison of this activity to the Borrelia burgdorferi prevalence in ticks collected from the above-mentioned working stands. Ticks were collected by dragging a woollen flag over lower vegetation and litter along the paths and edges of a forest in July and September 2011 at the following sites and workplaces: acquisition of timber, growing of forest, forest cultivation and forest protection. The relative activity (density) of ticks was determined by means of a combined method of single sample and area sampling. A forest area of approximately 100 m(2) was brushed with a flag. The isolates obtained from Ixodes ricinus ticks were examined for the presence of Borrelia burgdorferi sensu lato DNA by polymerase chain reaction, using primers FLA1 and FLA2 specific for the fragment of fla gene sequence. In the statistical analysis, the Mann-Whitney test, χ(2) test and Spearman test were applied. The differences between Ixodes ricinus activity at individual places of work where various biotopes were observed, were not statistically significant. A statistically significant variation in the tick infection rate, depending on work stands, was found for all the tick stages, i.e. for nymphs (χ(2)=76.516, p<0.000001), females (χ(2)=18.832, p<0.000292) and males (χ(2)=69.257, p=0.000001). Nevertheless, the statistical analysis showed the significant difference in total infection rates only between timber acquisition and growing of forest sites (p=0.049), and between growing of forest and forest cultivation sites (p=0.049). The study showed also the lack of relationship between the ticks' activity and Borrelia burgdorferi infection of ticks at individual places and sites of work of forestry workers. In conclusion, forestry workers employed at such stands of work as timber acquisition, growing of forest, forest cultivation and forest protection are nearly at the same risk of Borrelia burgdorferi infection.

Risk of tick-borne diseases in various categories of employment among forestry workers in eastern Poland.

Among the zoonotic agents causing occupational diseases, those transmitted by ticks are very important, in particular the spirochetes Borrelia burgdorferi which are the common cause of occupational Lyme borreliosis in forestry and agricultural workers. The objective of this study was an evaluation of the exposure of forestry workers employed at individual workplaces to infection with tick-borne pathogens (especially Borrelia burgdorferi spirochetes), based on epidemiological investigation and serologic tests. Epidemiological studies covered 111 forestry employees from eastern Poland employed in 4 randomly-selected forest inspectorates which replied to questions in the area of epidemiology and prophylaxis of diseases transmitted by ticks. Eighty-two forestry workers employed in one forest inspectorate were examined for the presence of specific anti-Borrelia burgdorferi antibodies. The correlation between individual items of the questionnaire was assessed by Spearman's test. Results of serological tests were assessed by Mann-Whitney test. Statistical analysis of the results indicated that the workers performing manual jobs in the forest are at the greatest risk of tick bite and contraction of tick-borne disease. They are aware of the risk, but use the improper method of removal of ticks with the fingers. Comparisons of the relationship between job category and the results of serologic study, expressed in BBU/ml, revealed that the serologic response was significantly greater in manual workers than in administrative workers (p=0.019). All other comparisons did not produce significant results. Therefore, providing a simple tweezer-like device to forest inspectorates seems to be an effective mean of protection against Lyme borreliosis and other tick-borne diseases.

Seroprevalence of leptospirosis in rural populations inhabiting areas exposed and not exposed to floods in eastern Poland.

Blood serum samples collected from randomly selected groups of 100 persons inhabiting rural community 'A' located in eastern Poland and exposed to floods by the Vistula river, and 98 persons inhabiting rural community 'B', also located in eastern Poland, but in the area not exposed to floods were examined by the microscopic agglutination test (MAT) for the presence of antibodies against 18 Leptospira serovars. Positive results showed 3% of serum samples collected from community 'A', while the seroprevalence in community 'B' was 9.2%, being insignificantly higher than in community 'A'. For both examined communities (n=198), a significant positive correlation was found between the prevalence and titer of seropositive response and age of examined people (r=0.145, p=0.042). No significant differences were found between the prevalence of positive reactions in males and females (p>0.05). The reactions with 10 serovars of Leptospira (Australis, Autumnalis, Hebdomadis, Hardjo, Sejroe, Zanoni, Bataviae, Bratislava, Canicola and Grippotyphosa), belonging to 3 species (L. interrogans, L. borgpetersenii, L. kirschneri), were found in the examined communities. From both communities, of 12 persons demonstrating positive results in MAT, 9 showed reaction with one serovar, 2 with two serovars and 1 with three serovars. The highest titers found during the examination did not exceeded 800. In conclusion, our results suggest that there is only a slight, if any, hazard of an leptospirosis epidemic after the flood that afflicted eastern Poland in the year 2010 and the general epidemiological situation of leptospirosis in eastern Poland. Although there does not seem to be any cause for concern, further research is needed.