Transmissible spongiform encephalopathy in goats: is PrP rapid test sensitivity affected by genotype?

Transmissible spongiform encephalopathy (TSE) surveillance in goats relies on tests initially approved for cattle, subsequently assessed for sheep, and approval extrapolated for use in "small ruminants." The current EU-approved immunodetection tests employ antibodies against various epitopes of the prion protein PrPSc, which is encoded by the host PRNP gene. The caprine PRNP gene is polymorphic, mostly at codons different from the ovine PRNP. The EU goat population is much more heterogeneous than the sheep population, with more PRNP-related polymorphisms, and with marked breed-related differences. The ability of the current tests to detect disease-specific PrPSc generated against these different genetic backgrounds is currently assumed, rather than proven. We examined whether common polymorphisms within the goat PRNP gene might have any adverse effect on the relative performance of EU-approved rapid tests. The sample panel comprised goats from the UK, Cyprus, France, and Italy, with either experimental or naturally acquired scrapie at both the preclinical and/or unknown and clinical stages of disease. Test sensitivity was significantly lower and more variable when compared using samples from animals that were preclinical or of unknown status. However, all of the rapid tests included in our study were able to correctly identify all samples from animals in the clinical stages of disease, apart from samples from animals polymorphic for serine or aspartic acid at codon 146, in which the performance of the Bio-Rad tests was profoundly affected. Our data show that some polymorphisms may adversely affect one test and not another, as well as underline the dangers of extrapolating from other species.

Effect of Polymorphisms at Codon 146 of the Goat PRNP Gene on Susceptibility to Challenge with Classical Scrapie by Different Routes.

This report presents the results of experimental challenges of goats with scrapie by both the intracerebral (i.c.) and oral routes, exploring the effects of polymorphisms at codon 146 of the goat PRNP gene on resistance to disease. The results of these studies illustrate that while goats of all genotypes can be infected by i.c. challenge, the survival distribution of the animals homozygous for asparagine at codon 146 was significantly shorter than those of animals of all other genotypes (chi-square value, 10.8; P = 0.001). In contrast, only those animals homozygous for asparagine at codon 146 (NN animals) succumbed to oral challenge. The results also indicate that any cases of infection in non-NN animals can be detected by the current confirmatory test (immunohistochemistry), although successful detection with the rapid enzyme-linked immunosorbent assay (ELISA) was more variable and dependent on the polymorphism. Together with data from previous studies of goats exposed to infection in the field, these data support the previously reported observations that polymorphisms at this codon have a profound effect on susceptibility to disease. It is concluded that only animals homozygous for asparagine at codon 146 succumb to scrapie under natural conditions.IMPORTANCE In goats, like in sheep, there are PRNP polymorphisms that are associated with susceptibility or resistance to scrapie. However, in contrast to the polymorphisms in sheep, they are more numerous in goats and may be restricted to certain breeds or geographical regions. Therefore, eradication programs must be specifically designed depending on the identification of suitable polymorphisms. An initial analysis of surveillance data suggested that such a polymorphism in Cypriot goats may lie in codon 146. In this study, we demonstrate experimentally that NN animals are highly susceptible after i.c. inoculation. The presence of a D or S residue prolonged incubation periods significantly, and prions were detected in peripheral tissues only in NN animals. In oral challenges, prions were detected only in NN animals, and the presence of a D or S residue at this position conferred resistance to the disease. This study provides an experimental transmission model for assessing the genetic susceptibility of goats to scrapie.

Biochemical and histological contributions to textural changes in watermelon fruit modulated by grafting.

Increased watermelon fruit flesh firmness is systematically incurred with grafting on Cucurbita hybrid rootstocks (heterografting). Possible differences in mesocarp cell wall constitution and histology between heterografted, homeografted (self-grafted) and non-grafted watermelon were examined, as well as their contributions to fruit texture. Firmness correlated positively (r=0.78, p<0.001) with cell density (cellsmm-2) which was higher in heterografts (5.83) than homeografts (4.64) and non-grafted controls (4.69). Mean cell size was smallest in heterografts and correlated negatively (r=-0.75, p<0.001) with firmness. Cell wall material, particularly the water-insoluble pectin fractions associated with firmness, were highest in heterografts. No associations with firmness were found for cell wall neutral sugars and membrane permeability. Higher parenchymatic cell density with higher content of alcohol insoluble residue and more abundant water-insoluble pectin fractions underscore enhanced firmness in heterografts. Possible implication of osmolytes in rootstock-mediated cell pressure regulation warrants further investigation.

Effects of air embolism size and location on porcine hepatic microcirculation in machine perfusion.

The handling of donor organs frequently introduces air into the microvasculature, but little is known about the extent of the damage caused as a function of the embolism size and distribution. Here we introduced embolisms of different sizes into the portal vein, the hepatic artery, or both during the flushing stage of porcine liver procurement. The outcomes were evaluated during 3 hours of machine perfusion and were compared to the outcomes of livers with no embolisms. Dynamic contrast-enhanced ultrasound (DCEUS) was used to assess the perfusion quality, and it demonstrated that embolisms tended to flow mostly into the left lobe, occasionally into the right lobe, and rarely into the caudate lobe. Major embolisms could disrupt the flow entirely, whereas minor embolisms resulted in reduced or heterogeneous flow. Embolisms occasionally migrated to different regions of the same lobe and, regardless of their size, caused a general deterioration in the flow over time. Histological damage resulted primarily when both vessels of the liver were compromised, whereas bile production was diminished in livers that had arterial embolisms. Air embolisms produced a dose-dependent increase in vascular resistance and a decline in oxygen consumption. This is the first article to quantify the impact of air embolisms on microcirculation in an experimental model, and it demonstrates that air embolisms have the capacity to degrade the integrity of donor organs. The extent of organ damage is strongly dependent on the size and distribution of air embolisms. The diagnosis of embolism severity can be safely and easily made with DCEUS.