Several isolates of Gram

© 2014 Elsevier B.V.Salmonellosis causes significant economic losses to the pig industry and contaminated pork products are an important source of Salmonella for humans. The EU ban on the use of antibiotic growth promoters in pig production, and the emergence of antibiotic resistance has meant there is a pressing need for alternative control strategies for pathogenic bacteria such as S. Typhimurium in pigs. Here, we determined the effects of prebiotic, probiotic and synbiotic diet regimes on antibody responses to oral Salmonella challenge of pigs. The data demonstrate that the inclusion of the probiotic Lactobacillus plantarum B2984 in the diet of piglets (~1×1010cfu/animal/day) enhanced serum IgM (P<0.001), IgG (P=0.001) and IgA (P=0.039) responses to S. Typhimurium infection including cross-reacting antibodies to S. Enteritidis. Similarly, inclusion of the prebiotic lactulose at 1% (w/w) of the feed on a daily basis in the diet enhanced serum IgM (P=0.010), IgG (P=0.004) and IgA (P=0.046) responses to S. Typhimurium infection and also cross-reacting antibodies to S. Enteritidis. Inclusion of both additives in the synbiotic diet also elicited an enhanced immune response with IgM (P=0.009) and IgG (P=0.046) levels being increased, however a significant interaction of the pre and probiotics was observed when considering the immune responses to S. Typhimurium (IgM P=0.004; IgG and IgA, P<0.001 for interaction). With respect to immune responses, the effects of pre or probiotic administration were the same or reduced in the synbiotic diet compared to when used in isolation. The data support the use of Lactobacillus plantarum B2984 or lactulose as strategies to contribute to the protection of weaned piglets from zoonotic bacterial pathogens, but caution must be taken when combining dietary supplements as combinations can interact

Disease-associated PrP fragments produced upon in vitro or in vivo proteolysis can provide significant insight into the causal strain of prion disease. Here we describe a novel molecular strain typing assay that used thermolysin digestion of caudal medulla samples to produce PrP res signatures on Western blots that readily distinguished experimental sheep bovine spongiform encephalopathy (BSE) from classical scrapie. Furthermore, the accumulation of such PrP res species within the cerebellum also appeared to be dependent upon the transmissible spongiform encephalopathy (TSE) strain, allowing discrimination between two experimental strains of scrapie and grouping of natural scrapie isolates into two profiles. The occurrence of endogenously produced PrP fragments, namely, glycosylated and unglycosylated C2, within different central nervous system (CNS) regions is also described; this is the first detailed description of such scrapie-associated fragments within a natural host. The advent of C2 fragments within defined CNS regions, compared between BSE and scrapie cases and also between two experimental scrapie strains, appeared to be largely dependent upon the TSE strain. The combined analyses of C2 fragments and thermolysin-resistant PrP species within caudal medulla, cerebellum, and spinal cord samples allowed natural scrapie isolates to be separated into four distinct molecular profiles: most isolates produced C2 and PrP res in all CNS regions, a second group lacked detectable cerebellar C2 fragments, one isolate lacked both cerebellar PrP res and C2, and a further isolate lacked detectable C2 within all three CNS regions and also lacked cerebellar PrP res . This CNS region-specific deposition of disease-associated PrP species may reflect the natural heterogeneity of scrapie strains in the sheep population in the United Kingdom.

The molecular diagnosis of prion diseases almost always involves the use of a protease to distinguish PrPC from PrPSc and invariably the protease of choice is proteinase K. Here, we have applied the protease thermolysin to the diagnosis of animal prion diseases. This thermostable protease cleaves at the hydrophobic residues Leu, Ile, Phe, Val, Ala, and Met, residues that are absent from the protease accessible aminoterminal region of PrPSc. Therefore, although thermolysin readily digests PrPC into small protein fragments, full-length PrPSc is resistant to such proteolysis. This contrasts with proteinase K digestion where an aminoterminally truncated PrPSc species is produced, PrP27-30. Thermolysin was used in the diagnosis of ovine scrapie and bovine spongiform encephalopathy and produced comparable assay sensitivity to assays using proteinase K digestion. Furthermore, we demonstrated the concentration of thermolysin-resistant PrPSc using immobilized metal-affinity chromatography. The use of thermolysin to reveal a full-length PrPSc has application for the development of novel immunodiagnostics by exploiting the wide range of commercially available immunoreagents and metal affinity matrices that bind the amino-terminal region of PrP. In addition, thermolysin provides a complementary tool to proteinase K to allow the study of the contribution of the amino-terminal domain of PrPSc to disease pathogenesis.