Innovative and Highly Sensitive Detection of Clostridium perfringens Enterotoxin Based on Receptor Interaction and Monoclonal Antibodies

Clostridium perfringens enterotoxin (CPE) regularly causes food poisoning and antibioticassociated diarrhea; therefore, reliable toxin detection is crucial. To this aim, we explored stationary and mobile strategies to detect CPE either exclusively by monoclonal antibodies (mAbs) or, alternatively, by toxin-enrichment via the cellular receptor of CPE, claudin-4, and mAb detection. Among the newly generated mAbs, we identified nine CPE-specific mAbs targeting five distinct epitopes, among them mAbs recognizing CPE bound to claudin-4 or neutralizing CPE activity in vitro. In surface plasmon resonance experiments, all mAbs and claudin-4 revealed excellent affinities towards CPE, ranging from 0.05 to 2.3 nM. Integrated into sandwich enzyme-linked immunosorbent assays (ELISAs), the most sensitive mAb/mAb and claudin-4/mAb combinations achieved similar detection limits of 0.3 pg/mL and 1.0 pg/mL, respectively, specifically detecting recombinant CPE from spiked feces and native CPE from 30 different C. perfringens culture supernatants. The implementation of mAb- and receptor-based ELISAs into a mobile detection platform enabled the fast detection of CPE, which will be helpful in clinical laboratories to diagnose diarrhea of assumed bacterial origin. In conclusion, we successfully employed an endogenous receptor and novel high affinity mAbs for highly sensitive and specific CPE-detection. These tools will be useful for both basic and applied research.Peer Reviewe

Prions in Milk from Ewes Incubating Natural Scrapie

Since prion infectivity had never been reported in milk, dairy products originating from transmissible spongiform encephalopathy (TSE)-affected ruminant flocks currently enter unrestricted into the animal and human food chain. However, a recently published study brought the first evidence of the presence of prions in mammary secretions from scrapie-affected ewes. Here we report the detection of consistent levels of infectivity in colostrum and milk from sheep incubating natural scrapie, several months prior to clinical onset. Additionally, abnormal PrP was detected, by immunohistochemistry and PET blot, in lacteal ducts and mammary acini. This PrPSc accumulation was detected only in ewes harbouring mammary ectopic lymphoid follicles that developed consequent to Maedi lentivirus infection. However, bioassay revealed that prion infectivity was present in milk and colostrum, not only from ewes with such lympho-proliferative chronic mastitis, but also from those displaying lesion-free mammary glands. In milk and colostrum, infectivity could be recovered in the cellular, cream, and casein-whey fractions. In our samples, using a Tg 338 mouse model, the highest per ml infectious titre measured was found to be equivalent to that contained in 6 µg of a posterior brain stem from a terminally scrapie-affected ewe. These findings indicate that both colostrum and milk from small ruminants incubating TSE could contribute to the animal TSE transmission process, either directly or through the presence of milk-derived material in animal feedstuffs. It also raises some concern with regard to the risk to humans of TSE exposure associated with milk products from ovine and other TSE-susceptible dairy species

Top-Down Mass Spectrometry for Trace Level Quantification of Staphylococcal Enterotoxin A Variants

International audienc

Quantitative Determination of Staphylococcus aureus Enterotoxins Types A to I and Variants in Dairy Food Products by Multiplex Immuno-LC-MS/MS

International audienceStaphylococcal enterotoxins (SEs) are responsible for frequent food poisoning outbreaks worldwide. Specific identification of SEs is crucial for confirmation of food poisoning, tracking of the incriminated foods or food ingredients, and removal from the food chain. Here, we report on a new food testing protocol addressing the challenge of low abundance of SEs in contaminated food and high sequence heterogeneity. Multiplex ability of targeted high-resolution mass spectrometry was succesfully applied to the simultaneous and quantitative determination of the eight most frequent SEs including sequence variants. In this aim, between three and eight proteotypic peptides of each SE were selected by carefully considering amino acid variations within each type, and sequence homology between types. Quantification of trace levels of SEs directly in food samples was reached by immunoaffinity enrichment and optimized analytical conditions. The assay was validated in dairy food products with a lower limit of quantification down to 0.1 ng/g (in milk), and quantification of SEs was successfully demonstrated in real-life samples collected during staphylococcal food poisoning outbreaks. Importantly, the ability of the method to detect diverse sequence variants was also illustrated. By enabling for the first time the simultaneous quantification of the eight most frequent SEs, the new mass spectrometry-based assay would facilitate the laboratory confirmation of positive samples in situation of food poisoning outbreaks

From Foodborne Disease Outbreak (FBDO) to Investigation: The Plant Toxin Trap, Brittany, France, 2018

International audienceOn 6 July 2018, the Center for Epidemiology and Public Health of the French Armed Forces was informed of an outbreak of acute gastroenteritis among customers of a dining facility at a military base in Brittany, France. A total of 200 patients were reported out of a population of 1700 (attack rate: 12%). The symptoms were mainly lower digestive tract disorders and occurred rapidly after lunch on 5 July (median incubation period: 3.3 h), suggesting a toxin-like pathogenic process. A case–control survey was carried out (92 cases and 113 controls). Statistical analysis pointed to the chili con carne served at lunch on 5 July as the very likely source of poisoning. Phytohaemagglutinin, a plant lectin, was found in the chili con carne at a concentration above the potentially toxic dose (400 HAU/gram). The raw kidney beans incorporated in the chili con carne presented a high haemagglutination activity (66,667 HAU/gram). They were undercooked, and the phytohaemagglutinin was not completely destroyed. FBDOs due to PHA are poorly documented. This study highlights the need to develop methods for routine testing of plant toxins in food matrices. Improved diagnostic capabilities would likely lead to better documentation, epidemiology, and prevention of food-borne illnesses caused by plant toxins

Estimation of infectious titre in colostrum and milk from scrapie incubating ewes with apparently healthy mammary glands or lymphoproliferative mastitis (consecutive to Maedi infection).

<p>For each fraction (cell pellet, casein whey, cream) the quantity of the material submitted to immunoprecipitation process is detailed and linked to the initial volume of colostrum or milk from which it was prepared. In samples for which a 100% attack rate was observed, mean incubation period were used to estimate the infectious titre (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000238#ppat-1000238-g003" target="_blank">Figure 3E</a>). For each considered fraction the infectious titre per ml of starting material was calculated. The global infectious titre per ml of colostrum and milk was finally obtained by adding the value corresponding to each fraction.</p><p>N.A: not available at the moment of writing. *Infectivity was estimated from the only those fractions for which results are available. Consequently the calculated infectious titre/ml of milk is certainly underestimated.</p

Immunoprecipitation of PrP in milk and colostrum.

<p>(A) PrP in milk (▵) and colostrum (○), from a negative control animal and three scrapie incubating sheep (casein whey protein extract following NP40/DOC – 10 min at 37°C treatment). PRP levels were measured before (black symbols) and after (white symbols) immunoprecipitation with antibodies (SHa31, SAF-34, and βS-36). The dosage was performed using a two-site sandwich immunoassay (capture antibody 11C6, tracer antibody Bar-224). The positive threshold of the test (0.040 absorbance units) is symbolised by the dotted line. (B–E) PrP contained in different fractions was immunoprecipitated with Sha31/SAF-34/BS36 immunobeads. After washings, PK in PBS (0 to 10 µg in 50 µL) was added to the beads for 10 min at 37°C. Samples were denatured in laemmli's buffer (25 µL), without β-mercaptoethanol, for 5 min at 100°C. Supernatants were then analysed by western blot. (B) 1.4 mL of casein whey, prepared from colostrum (left four lanes) or milk (right four lanes of the gel), from a scrapie incubating ewe (0942 see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000238#ppat-1000238-t004" target="_blank">Table 4</a>), (C) 1.4 mL of casein whey prepared from a TSE free control milk, (D) 100 µl of scrapie positive 2% brain homogenate or (E) 100 µl of scrapie negative 2% brain homogenate.</p

Infectivity testing in a reference brain sample and colostrum/milk fractions from scrapie incubating ewes.

<p>(A,B) Survival curve in Tg338 mice (transgenic mice over-expressing ovine VRQ PRP allele) intracerebrally inoculated with colostrum (A) and milk (B), collected from ewes incubating scrapie. Samples were first fractionated into cellular pellet (▵), cream (▿), and casein whey (○). An immunoprecipitation of PrP on magnetic beads coated with anti-PrP antibodies was then carried out. Beads from each fraction were inoculated into five or six Tg338 mice. (A) Colostrum fractions from a ewe harbouring mammary ectopic lymphoid follicles associated with Maedi lesions (white symbols) and from a ewe with a healthy mammary gland (black symbols). (B) Milk fractions from the same ewes as in A (black symbols and white symbols) and of the cellular fraction from a second scrapie incubating ewe with a healthy mammary gland (grey symbols). The experiment was terminated after 900 days (normal Tg338 mouse lifespan). Incubation periods have to be compared to those of successive 1/10 dilutions of brain (obex- vertical dotted lines) material from a sheep clinically affected with scrapie. The start point (neat) corresponds to the inoculation of 2.5 µg of brain tissue per mice. (C) Western-blotting (anti-PrP SHa31 antibody) of without (lane 1) and with (lane 2) PK treatment of brain material from a Tg338 mouse inoculated with scrapie positive brain (10⁻³ diluted); (lanes 2–6) PK digested brain material from mice inoculated with milk and colostrum cellular fraction – (lane 3) milk from a ewe with a healthy mammary gland – (lane 4) colostrum from a ewe with a healthy mammary gland – (lane 5) milk from TSE free control – (lane 6) colostrum from a Maedi affected (ectopic lymphoid follicle) ewe. (D) Intracerebral end point titration of a 12.5% obex homogenate, prepared from a terminally scrapie affected sheep (Langlade isolate), in a Tg338 mouse model. This titration allowed the determination of the infectious dose 50 (ID₅₀) of the brain sample (10^6.8 ID₅₀/g), see the text. (E) Variation of the incubation period as a function of the infectious dose inoculated intracerebrally in Tg338 mice (obex – Langlade isolate), see the text.</p