Detection of Mycobacterium avium subsp. paratuberculosis in Slovakian wildlife.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of infectious enteritis called paratuberculosis that has a high economic impact on the worldwide livestock production. A central important question arises: Can wildlife animals serve as a reservoir for transmission of MAP to domestic ruminants? With this in mind, we devised a study to detect MAP in various Slovakian wildlife species found in the areas where paratuberculosis had been documented in domestic ruminants. The samples of parenchymatous organs (intestines, ileocecal valve and mesenteric lymphatic nodes) from 83 wildlife animals representing 13 species, inclu- ding 7 herbivorous, 5 carnivorous and 1 omnivorous species were collected during a four-year period. The clinical and pathological examinations failed to demonstrate any manifestations of paratuberculosis in any of the wildlife samples. The detection of MAP was done by widely used tests, i.e. cultivation and the PCR analysis. The bacterial cultures revealed the growth of Mycobacterium spp. colonies in 58 (70%) of all of the wild animals, but the PCR testing demonstrated paratuberculosis only in one (7.69%) of the roe deer population.

Toll-like receptors TLR1, TLR2 and TLR4 gene mutations and natural resistance to Mycobacterium avium subsp. paratuberculosis infection in cattle.

Toll like receptors (TLRs) are a class of pattern recognition receptors belonging to the innate immune system. Mutations in the protein coding region of TLRs are associated with altered responsiveness to pathogen-associated molecular patterns (PAMPs). A search was performed for novel mutations in bovine TLR1, TLR2 and TLR4 genes associated with the Mycobacterium avium subsp. paratuberculosis (MAP) infection. The work was also focused on the assessment of linkage between well known mutations in TLR genes (TLR2: Arg677Trp, Pro681His and Arg753Gln; TLR4: Asp299Gly and Thr399Ile), and the susceptibility of cattle to MAP infection. Detection of MAP infection in cattle population (n=711) was based on IS900 PCR, which revealed 22.50% (n=160) MAP positivity. Known mutations in TLR2 and TLR4 genes were not found in cattle population. A novel mutation Val220Met was associated (Odd's ratio, OR-3.459) with increased susceptibility to MAP infection. Toll/interleukin-1 receptor (TIR) domain of TLR2 was screened for the presence of mutations, wherein a novel Ile680Val mutation was linked with MAP infection. In silico analysis of the bovine TLR4 ectodomain (ECD) revealed the polymorphic nature of the central ECD and irregularities in the central LRR motifs. LRR11 of the TLR4 showed five missense mutations possibly linked with the increased susceptibility to MAP infection. The most critical position that may alter the pathogen recognition of TLR molecule was 4th residue downstream to LRR domain. Two such missense mutations in TLR4 (Asp299Asn downstream to LRR11, and Gly389Ser downstream to LRR15) were associated with MAP infection. Briefly, the work describes novel mutations in the bovine TLRs and presents their association with the MAP infection.

Monitoring of humans and animals for the presence of various Rickettsiae and Coxiella burnetii by serological methods.

Serological examination of humans in Slovakia suspected of having rickettsial infections revealed the presence of antibodies to spotted fever group rickettsiae (R. conorii, R. slovaca, and R. typhi). Of interest is the finding of serological positivity to the newly recognized "IRS" agent. Antibodies to these rickettsiae and to C. burnetii were demonstrated also in domestic and hunting dogs and pet animals. These results confirm the occurrence and possible circulation of these rickettsiae and C. burnetii in the Slovak Republic.

Toll-like receptor gene polymorphism and its relationship with somatic cell concentration and natural bacterial infections of the mammary gland in sheep.

Possible correlation between Toll-like receptor (TLR)-gene mutations and the susceptibility of the mammary gland to bacterial infections and also the associate breed-dependent aspects of somatic cell concentration (SCC), bacterial infection and TLR-gene mutations in sheep are described. In Polish Lowland Sheep (PLS), milk samples exceeding the level of 500/microL (i.e. 5 x 10(5) per mL) of SCC were recorded almost twice more frequently than in Polish Heath Sheep (PHS) (40 and 22.3%, respectively). The frequency of bacterial infections was also found in a similar ratio (20 and 12.7%, respectively). During detection of the TLR-gene mutation we recorded 2 alleles of TLR1, 6 alleles of TLR2 and 10 alleles of TLR4 genes in PHS sheep, while PLS sheep possessed 2, 4 and 6 alleles, respectively. Statistical analyses revealed a relationship between the specified TLR alleles, SCC and the frequency of incidence of bacterial inflammations of mammary gland. The data may serve as a benchmark for further study of TLR-gene mutation-dependent predisposition of mammary gland defensive cells to recognize the pathogen properly and initiate the immunological response, and may help in identifying one of the markers of natural resistance against sheep mastitis.

Protein A/G dependent ELISA a promising diagnostic tool in Lyme disease seroprevalence in game animals and hunting dogs.

One of the major problems in serodiagnosis in wild animals is unavailability of specific antiglobulin conjugate. Our study focuses on validation of Protein A/G dependent ELISA in game animals like deer and mouflons as well as in hunting dogs. Binding ability of Protein A/G-conjugate to antibodies was the highest in dogs followed by fallow deer and mouflons. Three different whole cell Borrelia antigens were used to evaluate antigen dependent variation. In new Protein A/G-ELISA the highest sensitivities (90.50%, deer; 85.37%, mouflon & 94.29%, dog) were obtained by B. garinii antigen, with no statistically significant variation (chi(2), P>0.05) among all other antigens used. Average seroprevalences observed in deer, mouflons and dogs were 44.90%, 29.41% and 30.43%, respectively. Marked influence of age on seroprevalence was noticed. Protein A/G-ELISA proved to be sensitive and promising diagnostic tool in serodiagnosis of Lyme disease in game ungulates and it can be used effectively for serosurvey in different wild mammals.

Presensitization of microorganisms by acid treatments to low dose gamma irradiation with special reference to Bacillus cereus.

Effect of acid presensitization to low dose gamma irradiation on Bacillus cereus in sheep/goat meat stored at refrigeration temperature was assessed. Food grade organic acids (viz. propionic, lactic and acetic acids) were used as presensitizers followed by treatment with 1, 2 and 3 kGy irradiation doses (Co(60)). Two percent acetic acid plus 3 kGy irradiation elicited most effective to lower the total viable count and B. cereus count. Combination of treatments availed to reduce the dose required for elimination of radioresistant B. cereus and also masked the irradiation odour developed due to radiolysis. The study also revealed substantial increase in shelf life of mutton after combination treatment than single treatment without any adverse effect on acceptability of meat.