Prion gene (PRNP) haplotype variation in United States goat breeds (Open Access publication).

Scrapie eradication efforts cost 18 million dollars annually in the United States and rely heavily upon PRNP genotyping of sheep. Genetic resistance might reduce goat scrapie and limit the risk of goats serving as a scrapie reservoir, so PRNP coding sequences were examined from 446 goats of 10 breeds, 8 of which had not been previously examined at PRNP. The 10 observed alleles were all related to one of two central haplotypes by a single amino acid substitution. At least five of these alleles (M142, R143, S146, H154, and K222) have been associated with increased incubation time or decreased odds of scrapie. To the best of our knowledge, neither S146 nor K222 has been found in any goats with scrapie, though further evaluation will be required to demonstrate true resistance. S146 was more common, present in several breeds at widely varying frequencies, while K222 was observed only in two dairy breeds at low frequency. Overall, this study provides frequency data on PRNP alleles in US goats, shows the pattern of relationships between haplotypes, and demonstrates segregation of multiple scrapieassociated alleles in several breeds not examined before at PRNP.

Myenteric neurons of the ileum that express somatostatin are a target of prion neuroinvasion in an alimentary model of sheep scrapie.

Neuroinvasion of the enteric nervous system by prions is an important step in dissemination to the brain, yet very little is known about the basic process of enteric neuroinvasion. Using an alimentary model of neonatal disease transmission, neuroinvasion by scrapie prions in the ileum of lambs was detected by immunohistochemical staining for the disease-associated form of the prion protein, PrPSc. Odds ratios (OR) were determined for the frequency of PrPSc staining within enteric somata categorized by plexus location (myenteric, submucosal) and neurochemical staining (PGP 9.5, neural nitric oxide synthase, somatostatin, substance P, and vasoactive intestinal polypeptide). PrPSc was observed in 4.48 +/- 4.26% of myenteric neurons and 2.57 +/- 1.82% of submucosal neurons in five lambs aged 208-226 days but not in a lamb aged 138 days. The relative frequency of PrPSc within enteric somata was interdependent on plexus location and neurochemical type. Interestingly, PrPSc was observed more frequently within myenteric neurons than in submucosal neurons (PGP 9.5; OR = 1.72, 95% confidence interval = 1.21-2.44), and was observed within the myenteric plexus approximately 4x (2.16-6.94) more frequently in somatostatin neurons than in the general neural population stained by PGP 9.5. Nerve fibers stained for somatostatin were present in the mucosa and near PrPSc staining within Peyer's patches. The results suggest that somatostatin-expressing enteric neurons, with fiber projections near Peyer's patches, but with somata present in greatest proportion within the myenteric plexus, are an early target for neuroinvasion by scrapie prions and could serve an important role in neural dissemination.

PrPSc accumulation in fetal cotyledons of scrapie-resistant lambs is influenced by fetus location in the uterus.

Placentae from scrapie-infected ewes have been shown to accumulate PrPSc when the genotype of the fetus is of a susceptible genotype (VRQ/VRQ, ARQ/VRQ or ARQ/ARQ). Cotyledons from fetuses of genotypes ARR/ARR, ARQ/ARR and ARQ/VRR have previously been shown to be resistant to PrPSc accumulation. By using ewes from a naturally infected scrapie flock, cotyledons from fetuses of multiple births of different genotypes were examined. PrPSc was detected in fetal cotyledons of genotype ARQ/ARQ, but not in cotyledons from their dizygotic twin of genotype ARQ/ARR. This confirms earlier reports of single fetuses of these genotypes, but is the first description of such a finding in twin fetuses, one of each genotype. It is also demonstrated that cotyledons from sibling fetuses of genotypes ARQ/VRQ and ARQ/ARQ have different patterns of PrPSc accumulation depending on whether the dam is of genotype ARQ/ARQ or ARQ/VRQ. Lastly, it is shown that cotyledons from fetuses with resistant genotypes are weakly positive for PrPSc when they have shared the same pregnant uterine horn with a fetus of a susceptible genotype with cotyledons positive for the detection of PrPSc. Additionally, a PCR product for the Sry gene, a product specific to males, was found in cotyledons from female fetuses that had shared a uterine horn with a male fetus. This indicates that some sharing of fetal blood occurs between placentomes and fetuses residing in the same uterine horn, which can result in PrPSc accumulation in cotyledons with resistant genotypes.

Fewer PrPc myeloid-based cells in sheep with the prion-resistant genotype.

Numerous sheep with the prion gene (PRNP) that encodes for 171QQ develop scrapie, a neurodegenerative prion disease of sheep. Relatively few cases of scrapie-affected sheep with PRNP genetics that encode for 171RR, however, have been found. Using flow cytometric analysis, statistically fewer PrPc-positive microglia and monocytes were observed from sheep with 171RR PRNP genetics than from sheep with 171QQ PRNP genetics (P<0.05). One possibility for the lack of PrP(Sc) accumulation in brains and lymph nodes of scrapie-exposed sheep with 171RR PRNP genetics is because of fewer PrPc-positive myeloid-derived cells available for conversion of PrPc to PrP(Sc).

Transcriptional regulation of the ospAB and ospC promoters from Borrelia burgdorferi.

OspA, OspB and OspC are the major outer surface proteins of Borrelia burgdorferi that are differentially synthesized in response to environmental conditions, including culture temperature. We found that DNA was more negatively supercoiled in B. burgdorferi cultures grown at 23 degrees C compared with cultures grown at 35-37 degrees C. We examined the regulation of ospAB and ospC transcription by temperature and DNA supercoiling. DNA supercoiling was relaxed by adding coumermycin A1, an antibiotic that inhibits DNA gyrase. Syntheses of the major outer surface proteins, expression of the ospA and ospC genes and the activities of the ospAB operon and ospC gene promoters were assayed. ospA product levels decreased, whereas ospC product levels increased after shifting from 23 degrees C to 35 degrees C or after adding coumermycin A1. In addition, OspC synthesis was higher in a gyrB mutant than in wild-type B. burgdorferi. Promoter activity was quantified using cat reporter fusions. Increasing temperature or relaxing supercoiled DNA resulted in a decrease in ospAB promoter activity in B. burgdorferi, but not in Escherichia coli, as well as an increase in ospC promoter activity in both bacteria. ospC promoter activity was increased in an E. coli gyrB mutant with an attenuated DNA supercoiling phenotype. These results suggest that B. burgdorferi senses environmental changes in temperature by altering the level of DNA supercoiling, which then affects the expression of the ospAB operon and the ospC gene. This implies that DNA supercoiling acts as a signal transducer for environmental regulation of outer surface protein synthesis.

groEL expression in gyrB mutants of Borrelia burgdorferi.

GroEL protein and groEL mRNA transcript were up-regulated in gyrB mutants of Borrelia burgdorferi, a causative agent of Lyme disease. Furthermore, the protein and transcript levels in gyrB mutants were greater than those in experimentally heat-shocked cultures of wild-type B. burgdorferi. Circular DNA in the gyrB mutants was more relaxed than in wild-type cells, although groEL is on the linear chromosome of B. burgdorferi. To our knowledge, this is the first evidence, albeit indirect, for the effect of DNA topology on gene expression from a linear DNA molecule in a bacterium.

Effect of antifungal agents on biological fitness of Lygus hesperus (Heteroptera: Miridae).

Artificial diets have become important components of rearing systems for insects that are used for research purposes and in commercial production. Because the rearing conditions for insects also provide ideal settings for mold growth, antifungal additives are often used to reduce diet contamination. However, the antifungal agents must not only be effective in mold suppression, they must also be safe to the target insects of the rearing programs. The toxicity of five commonly used antifungal agents (benzoic acid, formalin, methyl paraben, propionic acid, and sorbic acid) was tested using diet bioassays on Lygus hesperus Knight, and the effect on biological fitness was measured. Biological fitness was defined as total number of survivors, mean biomass (dry weight) accumulated per cage over the total treatment period, egg production, time to adult emergence, and time to start of egg laying. Methyl paraben and formalin were found to have significant negative effects on these measurements of biological fitness. Challenge tests to determine the ability of the antifungal agents to suppress mold growth when inoculated into the diet medium are currently in progress.