Deliverables Development of molecular sub-typing techniques for the characterisation of Cryptosporidium Molecular methods for species differentiation and characterisation of Cryptosporidium species were developed. PCR methods were based on the amplification of the COWP or 18s rRNA genetic regions. Sub-typing methods developed included PCR followed by sequence analysis; nucleic acid based sequence analysis (NASBA); gp40/15 as a sub-typing marker gene and a multilocus microsatellite-fingerprinting assay. These techniques have allowed detailed characterisation of specific isolates and provided valuable epidemiological information. The developed methods will be of value to both research laboratories and reference laboratories in conducting epidemiological studies on Cryptosporium and in characterising isolates. Studies on the prevalence, numbers and types of Cryptosporidium in the environment, beef, salad, water and shellfish A number of food and environmental samples were examined for the incidence and number of Cryptosporidium isolates. In summary: A two year survey on and farmed shellfish (n=333) in Northern Ireland identified a total of 7 positive samples were including mussels (n=4), king scallop (n=1), oyster (n=1) and cockle (n=1). This is first report of C. parvum in shellfish. Studies on the prevalence of Cryptosporidium in slurry and dirty water samples showed the parasite to be present in 40% slurries at a concentration of 1-1500 oocysts g-1. The parasite was present in 62% of dirty water samples at a concentration of 1-32 oocysts g-1. In unwashed baby leaf lettuce, sampled directly from a farm, the parasite was detected was detected in 40% of lettuce tested at concentrations ranging from 3- 40 per 15g-1 while on whole leaf lettuce (n=43) the pathogen was detected on 16% of lettuce at a concentration of 5-45 oocysts per 100g. No Cryptosporidium was detected on retail lettuce (n=22). At a commercial beef abattoir in Ireland, Cryptosporidium oocysts were isolated from 21/288 (7.3%) faecal samples at a level of 25-37,500 g-1. The parasite was not detected on carcass meat. Cryptosporidium oocysts were present in 12/49 water samples (50L) used to wash beef carcasses (a river water supply treated by clorination and slow sand filtration) at a level of 0.08 to 9.0 oocysts per litre. The parasite was not detected in water from a borehole supply. This is the first information of this type to be reported in the literature and forms the basis of an exposure assessment on these pathogens in the food chain. Data on these studies are published in the scientific literature. Risk assessments on C. parvum in water, beef and lettuce Data on exposure assessment for C.parvum in the lettuce and beef chain were linked to data on consumption levels and on hazard characterisation (from dose response models). A semi-quantitative risk assessment was performed using @ risk software for a baseline scenario and some worst-case assumptions. The general conclusion was that the risk posed by Cryptosporidium in drinkable water is low (mode: 1 in 5 million per year) for the baseline (normal) conditions increasing to (1 in 500 per year) for worst-case scenario. The initial level of contamination is the most relevant risk factor. This can be difficult to control but the level of contamination in the raw water and the resultant risk posed can be reduced by limiting the collection of water from areas likely to be contaminated, in particular unprotected water sources where livestock are kept in the vicinity, or where animal waste or run-off water from agricultural land may contaminate the water. When water is sourced from areas of high risk for contamination (i.e. unprotected water sources) a more stringent physical treatment of the water should be implemented. Once the oocysts are in the raw water, the most important risk factors are the length of storage of water before purification (physical treatments) and the type of treatment, which the water receives. For the risk calculation on beef, it was assumed that the beef was undercooked. Even for the baseline scenario the risk was high (1 in 400). This must be regarded as an over estimation of risk as in general meat is cooked. The model demonstrated that the main risk factor was contamination of the faeces, though the transmission rate to carcass appears to be low and various operations in the meat processing such as freezing and thawing or any thermal treatment received substantially reduces the viability of the parasite and therefore the risk posed. The general conclusion was that the risk posed by Cryptosporidium on beef was low, as when products are cooked it should effectively inactivate any oocysts present. However, under-cooking or cross contamination from raw too ready to eat foods in the domestic environment is a risk, particularly because of the low infective dose. The most effective risk reduction steps are to ensure the water used in beef processing is either potable or treated to remove parasites and that HACCP (Hazard analysis critical control point systems) based procedures are in place along the meat chain. For lettuce the baseline model demonstrated that the initial contamination of the lettuce crops was mainly determined by direct contamination with animal manure from the environment (99%) and with the weather, rainfall etc. expected to have a high impact on the transfer factor. If the fertilizers are treated, their contribution is expected to be low compared to that factor (2.0 %). The mean risk of illness per serving for the baseline scenario is 1 in 23,400. The results indicated that the most important factors to control the risk posed by C. parvum on lettuce are the initial contamination of the lettuce crops. This is mainly related to direct contamination from the application of contaminated slurry or manure or water, or accidental contamination from run off water particularly in wet weather. Controls should include treatment of any animal waste or water applied to lettuce to ensure any C. parvum oocysts present are inactivated. Lettuce should be grown in an area protected from accidental incursion from grazing animals and preferably in an area not affected by agriculture run off waters. The other main risk reduction steps are washing, removing outer leafs and length and temperature of storage. These assessments have highlighted the risk factors posed in each chain and have given an indication of where control measures may be addressed. It has also highlighted the areas where further research is required. Commercial development of a pulsifier machine for the isolation of Cryptosporidium from complex matrices Pulsification is a patented process that has many advantages in the field of food sample preparation for microbiological analysis. The new Pulsifier instrument, which has been re-designed during the course of the Carafe project, delivers improved samples containing less food matrix debris that can interfere with rapid microbiology techniques. During the CARAFE project the Pulsifier instrument was compared to other food sample preparation instruments and found to deliver enhanced recovery of Cryptosporidium parvum oocysts for beef carcass and salad leaf crops. The samples prepared by the Pulsifier were easier to process and as a result higher numbers of oocysts were recovered from each Pulsified sample compared to samples prepared using traditional food samples processors. The Pulsifier can be used for traditional culture based microbiological analysis of food samples, however, the main advantage of the Pulsifier is the improved samples, containing less interfering substances for those laboratories that wish to take up test methods based on new rapid microbiology technologies. The Pulsifier is now in routine manufacture and Microgen Bioproducts has appointed distributors in the main continental markets i.e. Europe, North America and Japan. Development of methods for the isolation and detection of C. parvum Methods for the isolation and detection of C. parvum using the pulsifier machine together with filtration, centrifugation or immunomagnetic separation gace been developed. Detection was done by immunofluoresent microscopy using fluorescein isothiocyante (FITC) labelled antibodies. Routine methods were developed for the isolation and detection of Cryptosporidium from complex matrices (animal faeces, dirty water, animal slurry, lettuce and beef). The method varied depending on the sample matrice but a key feature of most of the isolation methods was the use of a pulsifier machine (a newly developed instrument that is based on a combined shock wave generator/stirrer that drives attached microorganisms from the sample into suspension without crushing the sample) (Microgen Bioproducts, UK) followed by filtration, centrifugation or immunomagnetic separation. Detection was by immunofluoresent microscopy using fluorescein isothiocyante (FITC) labelled antibodies. The developed methods will be of value to research laboratories and reference/ service laboratories in conducting studies on the prevalence of this parasite in the food chain. The methods will also enable further research on this parasite. Studies on the persistence and infectivity of C. parvum in the environment, beef and salad A number of studies were carried out on the persistence of C. parvum in the salad and beef chain providing essential information for the exposure assessment needed to conduct a quantitative risk assessment on the pathogen in both of these products. In slurry (cattle, pig); different soil types (clay, chalky, sandy) and in dirty water, the survival of C. parvum was monitored over a period of about162 days at pH 4,7, and 9 and at 4, 10 and 20?C. In general, the parasite survived best at 4?C at an acid or neutral pH (4 and 7) and under these conditions was often still viable after 162 days. It was only in pig slurry at pH 4stored at 4?C that showed infective oocysts. This may have been partly related to the low numbers remaining and the difficulty in conducting infectivity assays with low numbers of oocysts. The viability of C. parvum inoculated onto lettuce was assessed over a 10-day period at 4 and 20?C. The pathogen survived better on lettuce with curly/crinkly-textured leaves, which appeared to protect the oocysts from desiccation. The parasite survived better at 4 than at 20?C with all oocysts gone by day 3 at 20?C but ~ 10% remaining at 4?C. These results suggest that if a contaminated lettuce is stored in a domestic refrigerator oocysts may remain viable for several. The survival of C. parvum was assessed on lean and fat beef trimmings that were boxed, frozen and thawed under commercial conditions prior to burger production. After the freeze/tempering process 9.46 % of the inoculum were still viable on lean trimmings, and 7.17% of the inoculum were still viable on fat trimmings. All data will be published in scientific publications. Assessment of a number of control measures for C.parvum on farm, in water, salad chain and beef chain A number of studies were undertaken to assess the effect of current or novel control measures on the inactivation of C. parvum oocysts. As regards to the control of cryptosporidiosis at the herd level, the results from this study suggest that desiccation is the most effective way of reducing oocyst infectivity. However, calf hutches and maternity facilities need to be thoroughly cleaned and left to dry for at least one week to efficiently reduce the risk of infection. A combination of drying and either high or low temperatures would further decrease this risk. The ability of C. parvum to tolerate pH fluctuations and survive for prolonged periods in different environments may be of importance not only for transmission of cryptosporidiosis at the herd level but also to food safety, and demonstrate the need for further studies of oocyst survival in various matrices. The survival of C. parvum oocysts on lean beef (knuckle) flowing thermal treatment at 60, 75 and 82°C with a control of 25°C was assessed with a view to establishing the effect of a hot water carcass washing procedure in beef processing. The viability of oocysts was determined before and after heat treatment using a DAPI/PI vital dye assay. The results showed that following treatment at 60°C the oocyst viability dropped slowly from 83.5% after 15 sec to 64.2% after 1 minute. At 75°C, a large initial loss in viability occurred (53.7%) which continued to decrease reaching 11.2% after treatment for 1 minute. The final treatment of 82°C resulted in the survival of only 17.9% after 15 sec, dropping to a complete death following the treatment for 1 minute. A control treatment at 25°C for 60s did not significantly reduce the viability of the oocysts (97.8% viable). The effect of different washing procedures on the removal of spiked oocysts was conducted on three types of whole head lettuces (Iceberg, Romaine and Red Oak Leaf). Three conditions were investigated: unwashed (control); immersion wash (10 minutes in water); and thorough hand wash of the entire leaf surface. Physical hand washing of the leaves was more effective but a small percentage of oocysts still remained viable on each of 3 lettuce types (Iceberg, Romaine and Red Oak) ranging from about 30, 8 and 2% respectively. The results will be published in scientific publications. 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