Long-term study of Cryptosporidium prevalence on a lowland farm in the United Kingdom.

A longitudinal sample survey testing for Cryptosporidium in livestock and small wild mammals conducted over 6 years (1992-1997) on a lowland farm in Warwickshire, England, has shown the parasite to be endemic and persistently present in all mammalian categories. Faecal samples were taken throughout the year and oocysts concentrated by a formal ether sedimentation method for detection by immunofluorescence staining using a commercially available genus specific monoclonal antibody. Cryptosporidium parvum was identified by morphology and measurement of modified Ziehl-Neelsen stained oocysts. C. muris was rarely found in wild mammals and C. andersoni oocysts were never detected in livestock. Faecal samples from 3721 individuals gave cumulative 6-year prevalences for C. parvum as follows: bull beef, 3.6%; dairy cows, 3.5%; ewes, 6.4%; horses, 8.9%; calves (home bred), 52%; calves (bought-in) 23.2%; lambs, 12.9%; small wild mammals (rodents) living in and around farm buildings, 32.8%; small wild mammals (mainly rodents) living in areas of pasture, 29.9%. Animal categories with the highest prevalences also shed the highest average oocyst numbers per gram of faeces (ranging from 1.4 x 10(3) for bull beef to 1.1 x 10(5) for calves). Analysis of annual and seasonal data for each animal category revealed that patterns of infection were variable and sporadic; this means that short-term sampling was never likely to provide a true or representative picture. Seasonally combined data for adult livestock, young livestock and small wild mammals showed all three categories tended to have the highest Cryptosporidium prevalences in the autumn. Calves were separated from their dams within 24h of birth and yet showed high prevalence of infection in most years despite the low prevalence for the dairy herd. It is possible the coincidence of high autumn prevalence in mice with the main period for the rearing of calves contributed to the infection of the latter. The farming estate was used to teach students of agriculture and took pride in good land management and husbandry practices that produced well fed and healthy livestock. The data from this estate may represent, therefore, the baseline, the lowest possible levels to be expected, for Cryptosporidium infection and oocyst production on a lowland farm in the United Kingdom.

Occurrence of Cryptosporidium in agricultural surface waters during an annual farming cycle in lowland UK.

A 17-month survey based on weekly testing for Cryptosporidium oocysts in surface waters draining a livestock farm on a Warwickshire (UK) estate has shown that the parasite is present throughout the year, with the highest frequency of occurrence and maximum concentrations during the autumn and winter. The 190 ha farm is managed as an exemplar for a teaching institution. There were up to 800 livestock present at peak times of year in the catchment of the stream draining the estate. Oocysts were concentrated from grab samples by a flocculation procedure, stained with monoclonal antibody and detected by fluorescence microscopy. Overall, 274/418 (66%) of samples were positive for Cryptosporidium. Where the stream passed from the estate, the occurrence was 79%, with mean and median oocysts/l of 0.48 and 0.2, respectively. Highest oocyst levels coincided with calving and increased wild animal numbers following breeding. There was no correlation of oocyst levels with rainfall or slurry spreading. Cryptosporidium was also frequently found in a pond on arable land (no livestock) indicating that wild animals alone contributed oocysts to surface waters. These results from a well managed livestock farm may represent a typical natural baseline for levels of occurrence and concentration of Cryptosporidium oocysts in surface waters of the lowland agricultural environment of the UK.

The Norway rat as a reservoir host of Cryptosporidium parvum.

The potential of Norway rats (Rattus norvegicus) to spread the parasite Cryptosporidium parvum was investigated by examining parasite prevalence in relation to the structure and movements of three permanent rat populations living on farmland in Warwickshire (UK) from October 1994 to March 1997. One population lived among a group of farm buildings housing cattle, while the other two had no contact with livestock, one living around a pond and its outflowing stream and the other on a rubbish tip. Overall, parasite occurrence was 24% (n = 438), but it varied according to body weight (age) with 40% of juveniles (< or =100 g) infected decreasing to 12% for adults >400 g, suggesting that actively breeding populations are potentially more likely to spread the parasite than non-breeding populations. There was no difference in prevalence between the three populations. The parasite was detected in more males (29%) than females (19%). Seasonally, on the livestock farm, prevalence was significantly lower in autumn (10%), but varied little (31-36%) from winter to summer. In contrast, on the arable farm, prevalence peaked in summer (50%) with a trough in winter (6%). Infection in rats appeared to last <67 days. Rats living on the livestock farm had home ranges largely confined to the cattle sheds, thereby maintaining a potential source of infection for livestock if rodent control was not part of a decontamination program. Equally, rats living around the pond on the arable farm provided a source of oocysts to contaminate the pond water, as well as being able to carry the parasite to nearby farm buildings or even to neighboring farms.

The Cryptosporidium "mouse" genotype is conserved across geographic areas.

A 298-bp region of the Cryptosporidium parvum 18S rRNA gene and a 390-bp region of the acetyl coenzyme A synthetase gene were sequenced for a range of Cryptosporidium isolates from wild house mice (Mus domesticus), a bat (Myotus adversus), and cattle from different geographical areas. Previous research has identified a distinct genotype, referred to as the "mouse"-derived Cryptosporidium genotype, common to isolates from Australian mice. Comparison of a wider range of Australian mouse isolates with United Kingdom and Spanish isolates from mice and cattle and also an Australian bat-derived Cryptosporidium isolate revealed that the "mouse" genotype is conserved across geographic areas. Mice are also susceptible to infection with the "cattle" Cryptosporidium genotype, which has important implications for their role as reservoirs of infection for humans and domestic animals.

Detection of Cryptosporidium oocysts in wild mammals of mainland Britain.

This paper combines the results from a preliminary survey of occurrence of Cryptosporidium species in faecal samples from a range of wild mammal species inhabiting mainland Britain with a tabulated literature review of world-wide reports of the parasite in those British mammals. In the literature, C. parvum was reported from 11 wild mammals found in Britain and elsewhere, mainly in rodents but also in insectivores, lagomorphs and ungulates. C. muris has been reported only in wild rodents. The sample survey detected C. parvum in seven additional British species, including carnivores. Overall, 12% of 184 faecal samples tested with a genus-specific monoclonal antibody contained oocysts of C. parvum. The results further emphasise the widespread distribution of Cryptosporidium amongst wild mammals in Britain, highlight the potential for transmission between host species and warn of the possibility of direct exposure for anybody using the countryside for professional or recreational purposes (e.g. farmers and ramblers) to previously unregarded sources of infection. It seems increasingly likely that most, if not all, mammalian species can be infected with C. parvum.

Cross-reaction of an anti-Cryptosporidium monoclonal antibody with sporocysts of Monocystis species.

The non-specific cross-reaction of a fluorescently labelled anti-Cryptosporidium monoclonal antibody was observed microscopically when testing faecal specimens from small mammals. The reactive particles were identified as sporocysts of the Gregarine family Monocystidae, and indicate that considerable care should be taken so that false positives are not recorded.

The prevalence of Cryptosporidium parvum and C. muris in Mus domesticus, Apodemus sylvaticus and Clethrionomys glareolus in an agricultural system.

Wild mice and voles were tested for Cryptosporidium during a 2-year survey at an agricultural site in Warwickshire, United Kingdom. C. parvum and C. muris, the two cryptosporidial species known to infect mammals, were detected. Prevalence figures of 22%, 21% and 13% noted for C. parvum for Mus domesticus, Apodemus sylvaticus and Clethrionomys glareolus, respectively, were higher than those recorded for C. muris at 10%, 6% and 2%. C. parvum causes the sometimes severe diarrhoeal disease cryptosporidiosis in many hosts, but the wild rodents were asymptomatic. The discovery of C. muris in A. sylvaticus and C. glareolus confirms a wider distribution in wild rodents than has previously been reported. Rodents may represent a significant reservoir of Cryptosporidium with a high potential for infection of man and livestock due to cohabitation.

Cryptosporidium parvum in environmental samples in the Sligo area, Republic of Ireland: a preliminary report.

To detect Cryptosporidium in environmental specimens in the Republic of Ireland, grab samples of river water were prepared by calcium carbonate flocculation, and marine mussel tissue homogenated prior to testing with a fluorescently labelled monoclonal antibody and fluorescence microscopy. The parasite was detected in both river waters and marine mussels (Mytilus edulis). Filter feeders such as Mytilus edulis may be of value as biological monitors for the presence of cryptosporidial oocysts in sea water. The presence of Cryptosporidium in river and marine waters and, in particular, contaminating mussels used for human consumption, has obvious health implications.

Dynamics of xenobiotic metabolism by isolated rat hepatocytes using a multichannel perifusion system.

The multichannel perifusion system in recirculating and non-recirculating (single-pass) mode has been used to monitor the rate of oxidative metabolism of three model substrates--7-ethoxycoumarin, dichloronitroanisole and aldrin. With control hepatocytes, the rate of de-ethylation of 7-ethoxycoumarin derived from recirculating mode was essentially similar to the rate obtained with conventional flask-incubated cell suspensions. The formation of 7-hydroxycoumarin glucuronide and sulphate by hepatocytes exposed to 7-ethoxycoumarin demonstrated the retention of conjugative ability of cells in the perifusion system. The rate of demethylation of dichloronitroanisole to dichloronitrophenol was low whilst aldrin epoxidation to dieldrin was rapid using control hepatocytes in recirculating mode. The inductive effect of phenobarbitone on hepatic mixed-function oxidases was demonstrated by a marked increase in the rate of 7-ethoxycoumarin (nine-fold) and dichloronitroanisole (64-fold) dealkylation by hepatocytes from phenobarbitone-treated animals in recirculating mode. The rate of substrate oxidation by hepatocytes perifused in the recirculating and the single-pass mode were the same. With dichloronitroanisole as substrate and a single-pass mode, the rate of dichloronitrophenol formation declined rapidly on perifusion with substrate-free medium and rapidly re-attained steady state on re-introduction of the substrate; the presence of metyrapone effectively inhibited dichloronitroanisole metabolism. The perifusion system is recommended for the study of the dynamics of xenobiotic metabolism by isolated mammalian hepatocytes.

Metabolic capability of rat hepatocytes isolated by perfusion and tissue slice techniques.

The metabolic capability of hepatocytes prepared by the perfusion method (P cells) and the tissue slice method (S cells) has been compared using standardised procedures. Yields of P cells were four times greater than for S cells. Trypan blue exclusion viability and oxygen utilization were similar although the viability of S cells deteriorated faster with time. P cells had a lower endogenous rate of glycogenolysis and showed better glucagon stimulation than S cells. Similarly, P cells performed gluconeogenesis at a higher rate. However, there was no significant differences in the metabolism of the xenobiotic ethoxycoumarin. It is concluded that while S cells are probably satisfactory for studies of drug metabolism their use for work involving surface receptor binding and energy demanding processes should be questioned.

A multichannel perifusion system for the continuous monitoring of glycogenolysis and gluconeogenesis in isolated rat hepatocytes.

A multichannel perifusion system for isolated rat hepatocytes entrapped in a Sephadex matrix is described and criteria for the choice of matrices are discussed. This system overcomes the usual problem of clogged filters and impaired flow rates encountered in suspension perifusion systems, and is assembled from standard widely available components. Gluconeogenic capability and mitochondrial respiratory control ratios were unaltered. Decreases in trypan blue viability index and respiration rate were small when compared with flask-incubated hepatocytes. The endogenous rate of glycogenolysis was slightly higher in perifused hepatocytes but hormone response, as judged by glucagon stimulation of glycogenolysis, was unimpaired. The potential of this system is indicated by experiments monitoring glycogenolysis and gluconeogenesis in recycling and non-recycling modes.

Acid hydrolases in the suckling rat small intestine. II. On the importance of alkaline phosphatase inhibition in the histochemical localization of acid phosphatase activity.

Phosphatase activities against beta-glycerophosphate, I-naphthyl phosphate and naphthol AS-TR phosphate were investigated, at acid and aldaline pH levels, using unfixed and fixed cryostat sections of suckling rat jejunum. The use of 10 mm EDTA and 10 mm NaF as inhibitors indicated that alkaline phosphate is predominantly located in the microvillous region of the adsorptive cells, while acid phosphatase is located in small particles distributed between the brush borders and the nuclei of these cells. Alkaline phosphatase activity was found to interfere with the localization of acid phosphatase unless EDTA was included in the incubation medium. A modified Gomori medium, containing 10 mm EDTA and additional lead nitrate, is described. Latency experiemtns using this medium, with unfixed sections, indicated the lysosomal nature of particulate acid phosphatase. The discussion stresses the importance of including an aldaline phosphatase inhibitor in incubation media designed to localize extralysosomal acid phosphatase activity.

A zonal rotor method for the preparation of microperoxisomes from epithelial cells of guinea pig small intestine.

A method is described for the preparation of catalase particles from homogenates made from suspensions of epithelial cells of the small intestine of the guinea pig. Electron microscope examination of the preparations revealed the presence of small diaminobenzidine-positive particles measuring 0.1-0.3 nm in diameter and resembling the microperoxisomes observed by Novikoff and Novikoff (1972. J. Cell Biol.53:532.). Analytical data upon which the method is based are presented. The technique consisted of a rate zonal separation of microperoxisomes from large particles followed by an isopycnic separation from less dense organelles. Application of the method yielded microperoxisomes purified between 20- and 30-fold.