Emergence and genetic variability of Anaplasma species in small ruminants and ticks from Central Europe.

Anaplasmoses are common tick-borne zoonotic bacterial diseases of livestock and free-living ungulates from the genus Anaplasma that are recently emerging in Central Europe. The main aim of this study was to analyze the prevalence and genetic variability of Anaplasma phagocytophilum and Anaplasma ovis in small ruminants and questing ticks from six different sites in Slovakia and the Czech Republic using the PCR of the msp4 gene followed by the sequence analysis. At two farms from southeastern Slovakia, 66.1% small ruminants were infected with A. ovis in contrast to one positive animal from both sites in northern Slovakia. It was represented by two different genotypes. A. phagocytophilum was present in all tested flocks with the infection prevalence ranging from 0.9% to 5.7%. None of the tested questing ticks carried A. ovis. A. phagocytophilum was detected in 1.1% and 7.8% of questing Ixodes ricinus ticks collected around the farms located in southeastern and northern Slovakia, respectively. A. phagocytophilum revealed higher intraspecific diversity than A. ovis.

Serological evidence of Borrelia burgdorferi sensu lato in horses and cattle from Poland and diagnostic problems of Lyme borreliosis.

In the course of epizootological research on Lyme borreliosis in animals, the serological evidence of this zoonosis in horses and cattle from different voivodships of Poland was screened. We also discussed some diagnostic problems of Lyme borreliosis resulting from, in addition to other factors, genetic and geographical heterogeneity isolates B. burgdorferi s.l. used as antigens. Using ELISA from 395 sera of horses the total mean seroprevalence for anti-Borrelia IgG antibodies 25.6% was observed. In the respective years, significant differences in the mean seroprevalence were not recorded. In the voivodships, the total mean seroprevalence and mean seroprevalence for the respective years varied from 16.6-66.6%. An analysis of seroprevalence depending on the age showed a significant difference between 0-2 year-old horses compared to older horses. The total seroprevalence in the set of 98 serum samples was lower with the strain of B. garinii (25.5%) compared to a mixture of B. burgdorferi sensu stricto with B. afzelii (36.7%) and B. afzelii (42.8%). The highest correlation of findings was reached comparing the strains of B. afzelii (South Poland) and a mixture of B. burgdorferi s. s.+B. afzelii (East Slovakia). Lower correlation was between B. garinii and mixture of B. burgdorferi s. s.+B. afzelii. On the contrary, the lowest correlation of findings was observed between the Slovak strain of B. garinii and Polish B. afzelii. In a group of 26 cow sera, the mean seroprevalence for anti-Borrelia IgG antibodies was 26.9%. In the remaining clinical signs the seroprevalence was 28.5-66.6%. In Western blot, out of 25 examined sera of horses 15 (60.0%) were positive, out of 6 cows 5 (83.3%) were positive (2 lameness, 2 phlebitis, 1 clinically healthy). The horses and cows sera recognised the proteins: 93 (MEP)-, 83-, 75-, 66-, 55-, 43-, 45-, 41 (flagellin)-, 39-, 34-, 35 (OspB) and 25-, 28 (OspC)-kDa. These results alert veterinarians to take into account the aetiology of Lyme disease in differential diagnoses.

Evaluation of recombinant line immunoblot for detection of Lyme disease in Slovakia: comparison with two other immunoassays.

In the present study the sensitivity and the specificity of three serological tests (enzyme-linked immunosorbent assay [ELISA], indirect fluorescent antibody test [IFA], and recombinant line immunoblot) were compared by examining 74 sera from patients diagnosed with Lyme disease in Eastern Slovakia. In addition, the reactivity to each of the recombinant proteins in the immunoblot was examined in order to evaluate their diagnostic value. Generally, the immunoblot (93.2%) and the ELISA (90.5%) were significantly more sensitive than the IFA (64.9%; df = 1; p < or = 0.001). Correlation between results of the ELISA, IFA, and immunoblot for IgM or IgG, when two tests were always compared, one to the other, ranged from r(s) = 0.673 to r(s) = 0.905. In the immunoblot, the highest sensitivity was observed in DbpA and VlsE proteins (76.9% and 84.6%, respectively) in IgG testing of the sera from the patient group of Lyme arthritis. VlsE proteins, together with OspC proteins, were also shown to be useful for IgM antibody detection in erythema migrans patients (up to 44.4% and 53.7% sensitivity, respectively). Our results indicate that both the ELISA and the recombinant immunoblot test were more satisfactory for seroconfirmation of Lyme disease than IFA. Moreover, the reseach confirmed diagnostic value of the in-vivo expressed proteins (VlsE and DbpA), which might have the potential to play an important role in improving whole-cell antigen-based testing.

Immune complexes in early Lyme disease.

The study investigated the presence of Borrelia-specific antibodies captured in immune complexes (ICs) in patients with early Lyme disease manifested by erythema migrans. Out of 18 patients, 15 (83.3%) tested positive for polyethylene glycol-precipitated ICs containing IgM antibodies, while only 4 (22.2%) were IgG positive. These results are in accordance with our findings obtained by standard ELISA and recombinant blot, which indicated that ICs might be used for serological diagnosis of the early disease.

Occurrence of antibodies against Chlamydophila abortus in sheep and goats in the Slovak Republic.

Chlamydophila abortus is one of the most important causative agents of enzootic abortion and other chlamydial infections of sheep and goats. The presence of specific serum antibodies to Chlamydophila abortus was studied in sheep and goats breeding in the Slovak Republic by the complement fixation test. 22,040 sheep and goats were examined during 5 years. Specific anti-Ch. abortus antibodies were found in 2,360 out of 20,878 sheep sera examined (11.7%), and in 85 out of 1,162 examined goats (7.7%). The occurrence of antichlamydial antibodies indicates the importance of performing screening examinations in commercial breeding with the aim of reducing the spread of this disease between animals, and also interrupting the spread and transmission from animals to human.

Heterogeneity of Borrelia burgdorferi sensu lato and their reflection on immune response.

Geographically different strains of Borrelia burgdorferi sensu lato (B. burgdorferi sensu stricto Ir 105, B. burgdorferi s.s. + B. afzelii V 123, B. garinii Ir 112 - isolates from eastern Slovakia, B. garinii K24 - isolate from western Slovakia and B. burgdorferi s.s. B 31 - American strain) were compared as antigens for serological study of Lyme borreliosis by IgG ELISA on a group of horses from eastern Slovakia. In a set of 101 horse serum samples, positivity with the use of Ir 105 strain was 53 (52.4%), with V 123 51 (51.49%), with Ir 112 48 (47.5%), with K 24 47 (46.5%) and with B 31 only 25 (24.7%). The seroprevalence between strains B 31 and Ir 105, B 31 and V 123, B 31 and Ir 112, B 31 and K 24 differed statistically significantly (test chi2, p<0.05); however, the differences between strains Ir 105, V 123, Ir 112 and K24 were insignificant. Consistency of positive and negative findings between American and Slovak strains ranged from 50.5-62.4%. Comparison of Slovak strains (Ir105, V 123, Ir 112 and K 24) consistency of positive and negative findings was higher from 79.2-95.04%. The highest consistency of findings was reached comparing strains Ir 112 and K 24, and the same high agreement of results was observed between the strains Ir 105 and V 123 and also Ir 112 and Ir 105. Higher consistency of findings of serologically examined horses with geographically close trains is in accordance with greater similarity of protein profiles of Slovak strains compared to the American strain.

Small mammals (Insectivora, Rodentia) as a potential source of chlamydial infection in East Slovakia.

The presence of antibodies against Chlamydophila (Chlamydia) psittaci in small mammals (Insectivora, Rodentia) in the region of East Slovakia are presented. The hosts were caught in several areas of Slovakia in habitats with different levels of anthropogenic disturbance. Research was carried out during 2000-2002. The authors examined 1,947 sera coming from 4 insectivore and 10 rodent species. Each serum was examined by micromethod of complement binding reactions using antigen Chlamydophila (Chlamydia) psittaci. Chlamydial infections were found in 251 individuals (prevalence 12.9 %) of 8 mammal species. The antichlamydial antibodies were proved at levels ranging from 1:32-1:1024. The highest prevalence of antibodies was detected in the most abundant rodent species Apodemus microps (14.8 %), Apodemus agrarius (13.9 %), Apodemus flavicolis (12.4 %), Microtus arvalis (12 %), and Clethrionomys glareolus (10.9 %). Positive hosts were registered in all studied localities. Testing of prevalence values in the individual research years confirmed significant changes. Our results showed that small mammals probably play an important role in the circulation of chlamydiae in nature.

Anti-Borrelia antibodies in rodents: important hosts in ecology of Lyme disease.

Data presented in this study focuses on the presence of anti-Borrelia antibodies in small mammals from Eastern Slovakia during 2000-2003. The total seropositivity observed was 18.78% in rodents. Amongst all species, the total seroprevalence in Apodemus flavicolis was the highest (20.87%), followed by Apodemus agrarius (19.58%) and Clethrionomys glareolus (11.11%). However, the prevalence in Apodemus flavicolis during the year 2000-2001 was higher (26.72%), which reduced to 10.60% in 2002-2003. To compare the year range of seroprevalence in other small mammals was not feasible due to the small sample number. Area-wise distribution of anti-Borrelia antibodies was even (18.75% to 20%) in this study, except in the Bot'any province (0%). This confirms the equal distribution of Borrelia spirochetes in the other 3 localities. Prevalence of anti-Borrelia antibodies during summer was significantly higher than during autumn and early spring. The overall study also reviews the importance of small mammals in Lyme disease ecology.

Molecular evidence for Anaplasma phagocytophilum and Borrelia burgdorferi sensu lato in Ixodes ricinus ticks from eastern Slovakia.

Ixodes ricinus ticks (20 males, 20 females and 20 nymphs) collected in Kosice, Slovakia were examined for the presence of Anaplasma phagocytophilum and Borrelia burgdorferi sensu lato (s.l.) by PCR. 38.3 % of the tested ticks carried single infection of B. burgdorferi s.l. and 8.3 % were infected with A. phagocytophilum. Double infection of both pathogens was detected in 5 % of tested ticks. These results indicate that both B. burgdorferi s.l. and A. phagocytophilum co-circulate in the enzootic sites of Eastern Slovakia and may cause co-infection in humans.

Seroprevalence of anti-Borrelia burgdorferi antibodies in sheep and goats from mountainous areas of Slovakia.

In the present study, domestic animals such as sheep and goats from eastern Slovakia were screened for the presence of anti-Borrelia antibodies. Seroprevalence in 181 sheep and 65 goats were carried out in 1999 and 2000. Modified ELISA method was used for detection of anti-Borrelia IgG antibodies. Seroprevalence obtained was 15.8% and 17.5% in 1999 and 2000 respectively in sheep, whereas in goats it was 17.2% and 19.4% respectively. The results suggest that these domestic species have potential to transmit the disease to other animals. Though the role of sheep and goats in Lyme disease has not yet been documented, there is great possibility of transmission of the causative agent via co-feeding to human beings.