Comparative study of binding of ovine complement factor H with different Borrelia genospecies.

This study presents the binding of ovine factor H (fH) by various serotypes of Borrelia and simultaneously correlates their complement resistance to sheep serum. Affinity ligand binding assay was employed to study the binding of borrelial proteins to ovine recombinant fH and its truncated forms (short consensus repeat, SCR 7 and SCRs 19-20). From a repertoire of 17 borrelial strains, only two strains showed affinity to sheep fH. A ~28-kDa protein of Borrelia burgdorferi sensu stricto (B. burgdorferi s.s., strain SKT-2) bound full-length fH as well as SCRs 19-20. This fH-binding protein was further identified as complement regulator-acquiring surface protein of B. burgdorferi (BbCRASP-1) by MALDI-TOF analysis. Surprisingly, a ~26-kDa protein of Borrelia bissettii (DN127) showed affinity to full-length fH but not to SCR 7 and SCRs19-20. In complement sensitivity assay, both strains-SKT-2 and DN127-were resistant to normal sheep serum. Significant complement resistance of two Borrelia garinii strains (G117 and T25) was also observed; however, none of those strains was able to bind sheep fH. Our study underscores the need of further exploration of fH-mediated evasion of complement system by Borrelia in domestic animals.

Host-dependent differential expression of factor H binding proteins, their affinity to factor H and complement evasion by Lyme and relapsing fever borreliae.

Binding of complement factor H is crucial for the resistance of Borrelia to complement-mediated lysis. This study was aimed to assess the correlation between the expression of fH binding proteins (FHBPs) during the early phase of infection (48 h after the entry of Borrelia into the blood circulation) and complement resistance of the Borrelia genus. As expected, B. afzelii, B. burgdorferi sensu stricto and B. garinii (Serotype 4, PBi) showed resistance to complement mediated lysis when incubated with human and dog complement, which coincided with the significantly higher expression (P<0.05) of the FHBPs. Similarly, B. coriaceae showed resistance to cattle complement. In non-reservoir hosts borreliae failed to induce expression of FHBPs within 48 h of complement challenge, and did not survive. It is important to note that not only the expression of FHBP but also their binding to fH is required for borrelial resistance to the complement. fH binding may depend on the coiled-coil (CC) motifs observed in the FHBPs, especially at the C terminus. A loss of the C-terminal CC motif in BgCRASP-1 of SKT-1 strain was found in in-silico CC prediction, and may be coupled with SKT-1's inability to bind factor H and evade complement-mediated attack. In contrast, the C-terminal CC motif was observed (P - 1.0) in BgCRASP-1 of PBi that may contributed to its factor H binding and human complement resistance.

Application of a Reverse Line Blot hybridisation assay for the species-specific identification of cyathostomins (Nematoda, Strongylida) from benzimidazole-treated horses in the Slovak Republic.

Five horse farms located in eastern Slovakia were investigated for the presence of benzimidazole-resistant strongyles by faecal egg count reduction test and egg hatch assay. Coprocultures were prepared for each farm from faecal samples taken pre- and post-treatment and harvested larvae were molecularly examined with a Reverse Line Blot assay. Faecal egg count reduction values ranged from 0 to 52.5% and all farms were positive for benzimidazole-resistant cyathostomins. Seven benzimidazole-resistant cyathostomin species were molecularly identified on farms before and also after treatment. These data demonstrate that resistance to benzimidazoles is well established in cyathostomin populations from horse farms in the Slovak Republic and that the molecular assay was able to determine the species-specific distribution of resistant cyathostomins under field conditions.

Worm control practices on sheep farms in the Slovak Republic.

A questionnaire to obtain information on worm control practices and sheep management was performed on 49 sheep farms in 2003 and 2004. The majority of Slovak farms kept native breeds Tsigai (22 farms) and Improved Valachian (14 farms). Farms were divided according to their altitude to lowland, upland and lower highland farms. Sizes of pastures and stocking rates for lowland, upland and lower highland farms were 81.5, 269.2, and 316.7 ha and 6.3, 2.6, and 2.9 sheep/ha, respectively. One third of farmers (33.3%) used permanent pastures and two thirds of breeders (66.7%) rotated sheep between pastures. Mean drenching rate for lambs and yearlings/adults was 1.76 and 1.70, respectively. The most frequently used drugs during period from 1999 to 2004 were albendazole and ivermectin. On 13 farms benzimidazole drugs were applied in spring before turn out and macrocyclic lactones in autumn before turn in. Benzimidazoles and macrocyclic lactones were used almost exclusively on 7 and 9 farms, respectively. Visual appraisal was the most common practice to determine weight of animals (87.8% of farmers). Weights of the heaviest animals were used for determination of anthelmintic doses only on 16.7% of farms. Coprological examinations were performed on 47.9% of farms, usually in frequency once per year (75%).

Use of two in vitro methods for the detection of anthelmintic resistant nematode parasites on Slovak sheep farms.

A survey of the prevalence of anthelmintic resistant nematode populations was conducted on 32 sheep farms in the Slovak Republic. In vitro egg hatch test and larval development tests were used for the detection of resistance to benzimidazole anthelmintics and they were compared with in vivo faecal egg count reduction tests. There was agreement in the declaration of resistance between the faecal egg count reduction test and both in vitro tests. The presence of resistant populations was determined on two farms using egg hatch test. In both farms, the LD(50) values were higher than 0.1 microg TBZ/ml, indicating resistance. By using LD(99) values it might be possible to reveal relatively small proportion of resistant larvae in the population. The prevalence of benzimidazole resistance has not change on Slovak sheep farms during last decade.