The molecular fingerprints of 1,349 isolates of Mycobacterium bovis received between 1979 and August 2000 at Agence Française de Sécurité Sanitaire des Aliments (Afssa) have been obtained by spoligotyping. The majority of the isolates (1,266) were obtained from cattle living in France. An apparently high level of heterogeneity was observed between isolates. One hundred sixty-one spoligotypes were observed in total, of which 153 were from French isolates. The two predominant spoligotypes, designated BCG-like and GB54, accounted for 26 and 12% of the isolates, respectively. In addition, 84% of the spoligotypes were found fewer than 10 times. Analysis of the results by clustering and parsimony-based algorithms revealed that the majority of the spoligotypes were closely related. The predominant spoligotype was identical to that of the vaccine strain Mycobacterium bovis BCG, which was isolated in France at the end of the 19th century. Some spoligotypes were closely associated with restricted geographical areas. Interestingly, some spoligotypes, which were frequently observed in France, were also observed in neighboring countries. Conversely, few spoligotypes were common to France and England, and those that were shared were observed at very different frequencies. This last point illustrates the potential role for an international data bank, which could help trace the spread of M. bovis across national borders.Bovine tuberculosis (TB) was endemic in France until the 1960s, with herd prevalence rates of 25% in 1955 (9). From this time onwards, a national program for TB control based on tuberculin skin testing with control of animal movements and total slaughter of infected herds was implemented. This control strategy resulted in a dramatic decrease in bovine tuberculosis leading to a herd prevalence rate of 0.09% in 1998 (2), suggesting that cattle are the most important reservoir, or even the sole reservoir, for Mycobacterium bovis in France. Due to the success of this control strategy, France was declared "officially free of bovine TB" by the European Commission (3).The very low level of TB in cattle has resulted in the introduction of new control strategies. Consequently, there has been a progressive reduction in the use of skin testing, with an increasing emphasis on systematic sampling of suspect lesions identified at slaughterhouses for M. bovis isolate identification and molecular typing. New laboratory tools were therefore required in order to improve the traceability of the infections and identification of the origin of the outbreak (i.e

Staphylococcal food poisoning is one of the most common food-borne diseases and results from the ingestion of staphylococcal enterotoxins (SEs) preformed in food by enterotoxigenic strains of Staphylococcus aureus. To date, more than 20 SEs have been described: SEA to SElV. All SEs have superantigenic activity whereas only a few have been proved to be emetic, representing a potential hazard for consumers. Characterization of staphylococcal food poisoning outbreaks (SFPOs) has considerably progressed compared to 80 years ago, when staphylococci were simply enumerated and only five enterotoxins were known for qualitative detection. Today, SFPOs can be characterized by a number of approaches, such as the identification of S. aureus biovars, PCR and RT-PCR methods to identify the se genes involved, immunodetection of specific SEs, and absolute quantification by mass spectrometry. An integrated gene-to-protein approach for characterizing staphylococcal food poisoning is advocated.

In order to gain a better understanding of the molecular epidemiology of Mycobacterium bovis isolates in Cameroon, 75 isolates of M. bovis collected in three provinces of northern Cameroon were studied by spoligotyping. For 65 of these isolates, typing was also carried out by pulsed-field gel electrophoresis (PFGE) with DraI, and 18 of the isolates were also typed by restriction fragment length polymorphism (RFLP) analysis with probe IS6110-RHS. Molecular typing of the isolates by these techniques revealed a high degree of homogeneity, with 10 spoligotypes for 75 isolates, four PFGE profiles for 65 isolates, and three RFLP types for 18 isolates. Some types were present in the three different provinces, while some were confined to one or two areas. These results suggest that geographical mapping of M. bovis strains could be helpful for the control of bovine tuberculosis at the regional level. An interesting feature of all the spoligotypes was the absence of spacer 30, suggesting a common origin for all of the Cameroon isolates tested; an evolutionary scenario for the isolates is discussed. In addition, a comparison of the three techniques showed that for M. bovis strain differentiation in Cameroon and in surrounding countries, spoligotyping would be a more discriminating and practical tool for molecular typing than the other two techniques used in this study.Bovine tuberculosis (TB) is endemic in many African countries, but economic constraints preclude the use of skin test and slaughter control strategies, which have proved effective in the developed world. In Cameroon, the majority of cattle herds are concentrated in the north (13), which is surrounded by Nigeria, Chad, and the Central African Republic. From visible lesion data obtained in the main slaughterhouses, it would appear that the prevalence of bovine TB in Cameroon is high (7). In addition, frequent cattle movement across the different areas of the country and across frontiers favors strain dissemination. In order to reduce the transmission of bovine TB, a bill from the Ministry of Livestock, Fisheries, and Animal Industries of Cameroon (no. 76/420) was introduced in 1976 to prevent the circulation of cattle between Adamaoua and the other two areas of northern Cameroon, i.e., Extreme North and North. This action resulted in the isolation of cattle within Adamaoua.To date, few studies have been performed to determine the correct prevalence of Mycobacterium bovis infection at local and regional levels (3,16,17,19), and there are no available data regarding the variability of M. bovis isolates within Cameroon. The aim of this study was to apply a number of molecular typing techniques to M. bovis isolates from different slaughterhouses located in three different provinces of northern Cameroon-North, Extreme North, and Adamaoua-in order gain a better understanding of the geographical distribution of M. bovis strains. The typing techniques used in this study were spoligotyping (11), pulsed-field gel electrophoresis (PFGE) (14) and, for some isolates, restri...

Staphylococcal poisoning is a common food-borne disease for which immunoassays to detect enterotoxins were developed, but these assays often lead to false diagnoses due to interferences or lack of specificity. Absolute quantitative mass spectrometry was for the first time successfully applied to an investigation of a staphylococcal outbreak due to coconut pearls.Staphylococcal food poisoning (SFP) is one of the most common food-borne diseases (12,15,16), resulting from ingestion of staphylococcal enterotoxins (SEs) produced in food by enterotoxigenic strains of coagulase-positive staphylococci (CPS), mainly Staphylococcus aureus (11). The European Food Safety Authority (2) reported that SEs were involved in 4.1% of food poisoning outbreaks, but this percentage is certainly underestimated due to poor analytical performances in the detection and identification of SEs in food remnants. Most cattle carry staphylococci on their skin and mucous membranes, which can contaminate animal-derived products. S. aureus can also be transferred into food by handlers not respecting hygienic standards during manufacturing (3) or cooking (19).To date, 23 SEs have been described: SEA to SElV (21). All share superantigenic activity, whereas only few of them (SEA to SEI, SER, SES, and SET) have been proved to be emetic (16,18). Until recently, SEs were hardly distinguishable from food proteins, as SEs are small (22-to 29-kDa) proteins without any physicochemical particularities. Their detection was possible only by immunoassay-based methods, such as the enzyme-linked immunosorbent assay (ELISA). Actually, qualitative or semiquantitative commercial kits are able either to detect SEA to SEE as a whole ("total" SEs) or to differentiate and quantify six or seven types of SE (SEA, SEB, SEC 1 , SEC 2 , SEC 3 , SED, and/or SEE); none of those kits are able to detect SEG to SElV (10).Very recently, a mass spectrometry (MS)-based method enabling the detection and absolute quantification of SEA was developed (8). In this work, we compared this innovative analytical approach with the official ELISA method, the PCR and reverse transcription-PCR (RT-PCR) methods for characterization of S. aureus strains, and the conventional microbiology methods (enumeration and characterization of S. aureus strains) classically used for the characterization of SFP outbreaks.Four SFP outbreaks associated with coconut pearls successively occurred in the Ile-de-France area (France) during July 2006. Out of 14 exposed people, 11 experienced nausea, vomiting, abdominal cramps, and diarrhea 2 to 7 hours after consumption of meals. One food sample from each outbreak was subjected to CPS counting, and isolated strains were analyzed by biotyping, pulsotyping by pulsed-field gel electrophoresis, and PCR targeting genes sea to sej and 23S rRNA (14). An RT-PCR for the sea, sed, and sej genes was performed to evaluate the expression of se mRNA. All culture supernatants of isolates were also tested for production of SEA to SED by a semiquantitative SE reversed passive latex a...

Staphylococcal food poisoning is caused by enterotoxins excreted into foods by strains of staphylococci. Commission Regulation 1441/2007 specifies thresholds for the presence of these toxins in foods. In this article we report on the progress towards reference materials (RMs) for Staphylococcal enterotoxin A (SEA) in cheese. RMs are crucial to enforce legislation and to implement and safeguard reliable measurements. First, a feasibility study revealed a suitable processing procedure for cheese powders: the blank material was prepared by cutting, grinding, freeze-drying and milling. For the spiked material, a cheese-water slurry was spiked with SEA solution, freeze-dried and diluted with blank material to the desired SEA concentration. Thereafter, batches of three materials (blank; two SEA concentrations) were processed. The materials were shown to be sufficiently homogeneous, and storage at ambient temperature for 4weeks did not indicate degradation. These results provide the basis for the development of a RM for SEA in cheese.