[Show abstract][Hide abstract] ABSTRACT: Prion protein (PrP) is encoded by the PRNP gene, which is highly polymorphic in goats, with polymorphisms encoding amino acid substitutions at the protein level. In the current study, the reactivity of monoclonal antibody (mAb) F99/97.6.1 in binding PrP from goats polymorphic at PRNP codon 222 was investigated. Nervous tissue from 30 scrapie-negative goats with 3 different genotypes (222Q/Q, 222Q/K, and 222K/K) was analyzed by Western blot using mAbs P4 and F99/97.6.1. Although PrP was detected in all 30 samples by mAb P4, detection of PrP by mAb F99/97.6.1 was limited to 222Q/Q (12/12). No PrP was detected by mAb F99/97.6.1 in the 222K/K samples (n = 6), and the signal intensity of mAb F99/97.6.1 for PrP was lower for the 222Q/K samples (12/12 samples). To further investigate these results, additional Western blot analyses were performed, and the PrP signals detected by mAbs F99/97.6.1 and SAF84 were then quantified. The mean F99/SAF84 ratio (± standard deviation) calculated for the 222Q/Q group was 0.73 ± 1.26, and the mean for the 222Q/K group was 0.27 ± 1.31. Statistical analysis of these values evidenced statistically significant differences between the 222Q/Q and 222Q/K samples. The results of the study thus revealed an inhibition by lysine at position 222 on the binding of mAb F99/97.6.1 to goat PrP. This has implications for the use of mAb F99/97.6.1 for diagnostic purposes. Because the 222K allele could be a target for genetic selection in goats, the differential reactivity of mAb F99/97.6.1 could be exploited with a genotyping test setup.
Journal of veterinary diagnostic investigation: official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 09/2012; 24(5):971-5. · 1.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: ABSTRACT:
Three distinct forms of bovine spongiform encephalopathy (BSE), defined as classical (C-), low (L-) or high (H-) type, have been detected through ongoing active and passive surveillance systems for the disease.The aim of the present study was to compare the ability of two sets of immunohistochemical (IHC) and Western blot (WB) BSE confirmatory protocols to detect C- and atypical (L- and H-type) BSE forms.Obex samples from cases of United States and Italian C-type BSE, a U.S. H-type and an Italian L-type BSE case were tested in parallel using the two IHC sets and WB methods.
The two IHC techniques proved equivalent in identifying and differentiating between C-type, L-type and H-type BSE. The IHC protocols appeared consistent in the identification of PrPSc distribution and deposition patterns in relation to the BSE type examined. Both IHC methods evidenced three distinct PrPSc phenotypes for each type of BSE: prevailing granular and linear tracts pattern in the C-type; intraglial and intraneuronal deposits in the H-type; plaques in the L-type.Also, the two techniques gave comparable results for PrPSc staining intensity on the C- and L-type BSE samples, whereas a higher amount of intraglial and intraneuronal PrPSc deposition on the H-type BSE case was revealed by the method based on a stronger demasking step.Both WB methods were consistent in identifying classical and atypical BSE forms and in differentiating the specific PrPSc molecular weight and glycoform ratios of each form.
The study showed that the IHC and WB BSE confirmatory methods were equally able to recognize C-, L- and H-type BSE forms and to discriminate between their different immunohistochemical and molecular phenotypes. Of note is that for the first time one of the two sets of BSE confirmatory protocols proved effective in identifying the L-type BSE form. This finding helps to validate the suitability of the BSE confirmatory tests for BSE surveillance currently in place.
[Show abstract][Hide abstract] ABSTRACT: Nor98 is an atypical scrapie strain characterized by a molecular pattern and brain distribution of the pathological prion protein (PrP(Sc)) different from classical scrapie. In Italy, 69 atypical cases have been identified so far and all were characterized as Nor98 strain. In this paper we report an unusual case in a sheep which showed immunohistochemical and molecular features of PrP(Sc) different from the other atypical cases. The sheep was from an outbreak where the index and the other four cases were affected by classical scrapie. Histopathological, immunohistochemical and Western blot analyses on the brain of the unusual case revealed the simultaneous presence of pathological features characteristic of Nor98 and classical scrapie. Interestingly, the prevalent disease phenotype in the brainstem was classical scrapie-like, while in the cerebral cortex and cerebellum the Nor98 phenotype was dominant. The sub-mandibular lymph node was positive and showed a PrP(Sc) molecular pattern referable to classical scrapie. The PrP genotype was AL(141)RQ/AF(141)RQ. Taken together, the occurrence of classical scrapie in the outbreak, the PrP genotype, the involvement of different cellular targets in the brain and the pathological and molecular PrP(Sc) features observed suggest that this unusual case may result from the co-existence of Nor98 and classical scrapie.
Research in Veterinary Science 06/2010; 88(3):478-85. · 1.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To evaluate further the reactivity of prion-specific monoclonal antibodies containing the 89-112 or 136-158 prion protein (PrP) polypeptides, immunoprecipitations were performed on brain extracts from Italian bovines, sheep and goats with transmissible spongiform encephalopathies. No binding of IgG 89-112 or IgG 136-158 to PrP in normal brain extracts was detected. Conversely, both reagents immunoprecipitated PrP from bovine and bovine amyloidotic spongiform encephalopathies, and from typical and atypical scrapie brain extracts. The immunoprecipitated PrP bands mirrored the Western blot (WB) profile of the different prion strains, indicating universal affinity of two independent PrP regions for disease-associated PrP conformers regardless of species source and strain properties. Immunoprecipitation with motif-grafted antibodies increased the sensitivity of conventional detection methods based on centrifugation followed by WB, which was confirmed by assay of diluted samples using both methods. These reagents or derivative molecules may thus find broad applications in prion detection and research.
Journal of General Virology 03/2009; 90(Pt 4):1048-53. · 3.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The olfactory system (OS) is involved in many infectious and neurodegenerative diseases, both human and animal, and it has recently been investigated in regard to transmissible spongiform encephalopathies. Previous assessments of nasal mucosa infection by prions following intracerebral challenge suggested a potential centrifugal spread along the olfactory nerve fibers of the pathological prion protein (PrP(Sc)). Whether the nasal cavity may be a route for centripetal prion infection to the brain has also been experimentally studied. With the present study, we wanted to determine whether prion deposition in the OS occurs also under field conditions and what type of anatomical localization PrP(Sc) might display there. We report here on detection by different techniques of PrP(Sc) in the nasal mucosa and in the OS-related brain areas of sheep affected by natural scrapie. PrP(Sc) was detected in the perineurium of the olfactory nerve bundles in the medial nasal concha and in nasal-associated lymphoid tissue. Olfactory receptor neurons did not show PrP(Sc) immunostaining. PrP(Sc) deposition was found in the brain areas of olfactory fiber projection, chiefly in the olfactory bulb and the olfactory cortex. The prevalent PrP(Sc) deposition patterns were subependymal, perivascular, and submeningeal. This finding, together with the discovery of an intense PrP(Sc) immunostaining in the meningeal layer of the olfactory nerve perineurium, at the border with the subdural space extension surrounding the nerve rootlets, strongly suggests a probable role of cerebrospinal fluid in conveying prion infectivity to the nasal submucosa.
Journal of Virology 02/2009; 83(8):3657-67. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Bovine spongiform encephalopathy (BSE), the prion disease in cattle, was widely believed to be caused by only one strain, BSE-C. BSE-C causes the fatal prion disease named new variant Creutzfeldt-Jacob disease in humans. Two atypical BSE strains, bovine amyloidotic spongiform encephalopathy (BASE, also named BSE-L) and BSE-H, have been discovered in several countries since 2004; their transmissibility and phenotypes in humans are unknown. We investigated the infectivity and human phenotype of BASE strains by inoculating transgenic (Tg) mice expressing the human prion protein with brain homogenates from two BASE strain-infected cattle. Sixty percent of the inoculated Tg mice became infected after 20 to 22 months of incubation, a transmission rate higher than those reported for BSE-C. A quarter of BASE strain-infected Tg mice, but none of the Tg mice infected with prions causing a sporadic human prion disease, showed the presence of pathogenic prion protein isoforms in the spleen, indicating that the BASE prion is intrinsically lymphotropic. The pathological prion protein isoforms in BASE strain-infected humanized Tg mouse brains are different from those from the original cattle BASE or sporadic human prion disease. Minimal brain spongiosis and long incubation times are observed for the BASE strain-infected Tg mice. These results suggest that in humans, the BASE strain is a more virulent BSE strain and likely lymphotropic.
Journal of Virology 05/2008; 82(7):3697-701. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Susceptibility to scrapie in sheep depends on the host PrP genotype. No data about the linkage of the rare ARK allele to differential scrapie susceptibility are currently available. Several tissues isolated from sheep from an Italian scrapie outbreak and carrying the ARK allele were examined for the presence of the pathological prion protein. A weak positivity was detected only by Western blot in the brainstem of one ARK/ARH sheep. This result shows that the ARK allele does not confer full resistance against scrapie and that the allele needs to be studied further before it can be considered for breeding purposes.
Archives of Virology 10/2006; 151(9):1875-80. · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tongue involvement by prion spreading was shown to be a common outcome after oral or intracranial experimental challenge with scrapie and transmissible mink encephalopathy sources in rodent models. It is also known that bovine spongiform encephalopathy, which is pathogenic for humans, is experimentally transmissible to sheep and can lead to a disease indistinguishable from scrapie. A recent European Food Safety Authority opinion recommended research into PrPsc accumulation in the tongues of ruminants. We report on the detection of PrPsc in the tongues of seven scrapie-infected sheep by immunohistochemistry and Western blotting.
Journal of Virology 06/2005; 79(9):5847-9. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In accordance with EU Regulation 999/2001, rapid tests already adopted for bovine spongiform encephalopathy (BSE; Prionics Check Western, Platelia-BSE and Enfer TSE) are to be applied in all European countries to a sub-population of over 18-month-old slaughtered or dead sheep and goats to improve Scrapie surveillance and to determine the possible presence of BSE in sheep; however, the three tests have thus far been evaluated only for BSE and no official data are available about their performances on Scrapie. We evaluated the accuracy of these methods for TSE diagnosis in Italian sheep and goats, using a pre-homogenisation protocol on brain-stem samples to obtain comparable data from the three tests. Our results show that the tests can be considered reliable tools for active surveillance in the small ruminants population.
[Show abstract][Hide abstract] ABSTRACT: ABSTRACT: Susceptibility of sheep to scrapie, a transmissible spongiform encephalopathy of small ruminants, is strongly influenced by polymorphisms of the prion protein gene (PRNP). Breeding programs have been implemented to increase scrapie resistance in sheep populations; though desirable, a similar approach has not yet been applied in goats. European studies have now suggested that several polymorphisms can modulate scrapie susceptibility in goats: in particular, PRNP variant K222 has been associated with resistance in case-control studies in Italy, France and Greece. In this study we investigated the resistance conferred by this variant using a natural Italian goat scrapie isolate to intracerebrally challenge five goats carrying genotype Q/Q 222 (wild type) and five goats carrying genotype Q/K 222. By the end of the study, all five Q/Q 222 goats had died of scrapie after a mean incubation period of 19 months; one of the five Q/K 222 goats died after 24 months, while the other four were alive and apparently healthy up to the end of the study at 4.5 years post-challenge. All five of these animals were found to be scrapie negative. Statistical analysis showed that the probability of survival of the Q/K 222 goats versus the Q/Q 222 goats was significantly higher (p = 0.002). Our study shows that PRNP gene mutation K222 is strongly associated with resistance to classical scrapie also in experimental conditions, making it a potentially positive target for selection in the frame of breeding programs for resistance to classical scrapie in goats.