Evaluation and validation of an alternative method to detect Campylobacter spp. in dairy products.

Foods implicated in human campylobacteriosis include raw or undercooked poultry and raw dairy products. Because Campylobacter spp. are the most frequently reported cause of bacterial infection in the European Union and because conventional methods are cumbersome, rapid methods for Campylobacter detection and quantification in food are needed. With this study we sought to validate, according to the standard procedure (UNI EN ISO 16140:2003), an alternative to the reference analytical method (UNI EN ISO 10272-1:2006) for official controls of Campylobacter spp. in raw milk and dairy products. Milk samples collected from 16 milk vending machines located throughout the Genoa metropolitan area were analyzed using two different methods, an enzyme-linked fluorescent assay (ELFA) and a real-time PCR assay, and evaluated in parallel against the reference method. In addition, a total of 460 samples of raw milk collected from milk vending machines were analyzed by ELFA. Results obtained with ELFA showed it was compliant with UNI EN ISO 10272-1:2006 criteria and that the immunoassay had 100% sensitivity, specificity, and accuracy. Regarding samples of milk vending machines, 5.0% (23/460) tested positive at ELFA screening and were subsequently confirmed as C. jejuni. Validation according to UNI EN ISO 16140:2003 of the ELFA method suggests it may be a useful alternative to conventional methods for detecting Campylobacter spp. in official controls.

Resistance to classical scrapie in experimentally challenged goats carrying mutation K222 of the prion protein gene.

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.

Evaluation of two sets of immunohistochemical and Western blot confirmatory methods in the detection of typical and atypical BSE cases.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Olfactory system involvement in natural scrapie disease.

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.