Detection of infectious Cryptosporidium oocysts by cell culture immunofluorescence assay: applicability to environmental samples.

In the past few years many waterborne outbreaks related to Cryptosporidium have been described. Current methods for detection of Cryptosporidium in water for the most part rely on viability assays which are not informative concerning the infectivity of oocysts. However, for estimation of the risk of infection with Cryptosporidium this information is required. For environmental samples the oocyst counts are often low, and the oocysts have been exposed to unfavorable conditions. Therefore, determination of the infectivity of environmental oocysts requires an assay with a high level of sensitivity. We evaluated the applicability of in vitro cell culture immunofluorescence assays with HCT-8 and Caco-2 cells for determination of oocyst infectivity in naturally contaminated water samples. Cell culture assays were compared with other viability and infectivity assays. Experiments with Cryptosporidium oocysts from different sources revealed that there was considerable variability in infectivity, which was illustrated by variable 50% infective doses, which ranged from 40 to 614 oocysts, and the results indicated that not only relatively large numbers of fresh oocysts but also aged oocysts produced infection in cell cultures. Fifteen Dutch surface water samples were tested, and the cell culture immunofluorescence assays were not capable of determining the infectivity for the low numbers of naturally occurring Cryptosporidium oocysts present in the samples. A comparison with other viability assays, such as the vital dye exclusion assay, demonstrated that surrogate methods overestimate the number of infectious oocysts and therefore the risk of infection with Cryptosporidium. For accurate risk assessment, further improvement of the method for detection of Cryptosporidium in water is needed.

Cryptosporidium and Giardia in swimming pools in the Netherlands.

The occurrence of Cryptosporidium and Giardia in indoor swimming pools in the Netherlands was studied at five locations. The backwash water from seven pool filters was analysed for the presence of Cryptosporidium oocysts and Giardia cysts for a period of 1 year. Of the 153 samples of filter backwash water analysed, 18 (11.8%) were found positive for either Cryptosporidium (4.6%), Giardia (5.9%) or both (1.3%). Oocysts and cysts were also detected in the water of one toddler pool and one learner pool. Although most of the (oo)cysts in the filter backwash water were dead, viable and potentially infectious oocysts were detected in the learner pool. On the basis of numbers of potentially infectious (oo)cysts detected in the learner pool, and assuming one visit to an infected pool per year, risk assessment indicated an estimated risk of infection with Cryptosporidium that exceeded the generally accepted risk of one infection per 10,000 persons per year. Guidelines for pool operators on how to manage faecal accidents and public information on the importance of hygiene in swimming pool complexes are recommended tools in controlling the risk of infection.

EU Drinking Water Directive reference methods for enumeration of total coliforms and Escherichia coli compared with alternative methods.

AIMS: The reference methods for enumeration of total coliforms and Escherichia coli as stated in the European Drinking Water Directive were compared with alternative methods. METHODS AND RESULTS: Laboratories used the reference method on Lactose TTC agar (LTTC), the Colilert/18 system, Laurysulphate Agar (LSA), Chromocult Coliform Agar and the E. coli Direct Plating (DP) method. They enumerated more total coliforms on LTTC than on LSA. CONCLUSIONS: LTTC is suitable for analysis of very clean water samples only, due to heavy background growth. Colilert/18 is a good alternative but it enumerates a broader group of total coliforms, resulting in higher counts. The DP method appeared to be the best choice for enumeration of E. coli because Colilert/18 produces lower counts and false-negative results. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows the limitations of the EU reference method on LTTC due to lack of selectivity and suggests alternative methods for the enumeration of total coliforms and E. coli.

Prevalence of Campylobacter jejuni and Salmonella during pig slaughtering.

It was found that 79% of healthy pigs, slaughtered in three different slaughterhouses in the Netherlands, were intestinal carriers of Campylobacter jejuni (mean number 4000 cfu per g), and 21% of the same pigs had Salmonella in the intestinal tract (mean number 10 cfu per g). Immediately after slaughter, Campylobacter was swabbed from 9% of the carcasses and Salmonella from 13%. It is concluded from these data that most of the contamination on carcasses does not originate directly from the intestinal tracts of the animals but rather from surfaces, equipment, and utensils in the slaughter hall. It was demonstrated that Salmonella could survive in the slaughter hall, whereas Campylobacter died off, probably due to its vulnerability to drying conditions and its inability to grow at temperatures below 30 degrees C. Campylobacter was not isolated from the carcasses after cooling. It had been shown earlier that this again was caused by dry conditions, brought about by the use of forced ventilation in the cooling rooms. In an additional investigation, Campylobacter was not isolated from 248 samples of minced pork (10 g each), whereas Salmonella was found in 13% of these samples.

Origin and Prevalence of Campylobaeter jejuni in Poultry Processing.

Investigations of two chicken processing plants in The Netherlands have shown that large contamination with Campylobacter jejuni can exist on birds, equipment, hands of processing-line workers and in air samples from the processing facility. This contamination appeared only to be of intestinal origin. Intestinal contents of birds to be processed contained up to 107 C. jejuni per gram. Contamination of birds was reduced during scalding at 58°C, but this reduction was not always observed at 51.8°C. The number of C. jejuni on carcasses increased during defeathering and evisceration. Large numbers of C. jejuni were washed off the carcasses when a spinchiller was used. When air-cooling was employed, C. jejuni in some instances died off, probably due to drying. End-products from these chicken processing plants contained C. jejuni in 50% of carcasses and 75% of livers.

Campylobacter jejuni in Cattle and Raw Milk in The Netherlands.

A survey was done on the occurrence of Campylobacter jejuni in slaughtered cattle and raw milk from dairy farms in The Netherlands, In the first part of the survey, in which direct plating techniques were used, no C. jejuni was detected in any of 200 samples of caecal contents of cattle or in 200 samples of raw milk. A second series of investigations was done using a new enrichment procedure. This time C. jejuni was isolated from 11 of 200 caecal contents, but from none of 200 samples of milk. Further experiments showed that Campylobacter can survive in milk at 4°C for weeks, whether the milk was shaken with air (as occurs during the milking process) or not. Our investigations indicate that C. jejuni was not excreted with the milk. It can be concluded that cattle in The Netherlands do not play an important role in the epidemiology of C. jejuni .