Concentrations of faecal coliforms, Escherichia coli, enterococci and Campylobacter spp. in equine faeces.

AIMS: To determine the concentration of Campylobacter spp. as well as faecal indicator bacteria; faecal coliforms, Escherichia coli and enterococci in the faeces of healthy adult horses in a sample of properties in the Canterbury region of New Zealand. METHODS: The faeces of healthy adult horses (n=59), including ponies, pleasure horses and Thoroughbreds, were collected from eight properties around Christchurch, New Zealand. The faeces were analysed for concentrations of Campylobacter spp and faecal indicator bacteria; faecal coliforms, Escherichia coli and enterococci. The presence of other animals on the properties sampled as well as the age, feed and health of the horses at the time of sampling was recorded. RESULTS: Enterococci and faecal coliforms were isolated from all samples, and E. coli was isolated from 58/59 samples. Mean concentrations of faecal coliforms and E. coli did not differ between properties, but there was a significant difference in mean concentration of enterococci between properties. Campylobacter spp. were detected in two faecal samples with one isolate being determined by PCR analysis to be a thermotolerant Campylobacter species, the other C. jejuni. CONCLUSIONS: This is the first known report quantifying the concentration of Campylobacter spp. present in healthy adult horses in New Zealand. The presence of equine faecal material in water could elevate concentrations of faecal bacteria and therefore needs to be considered as a source of water contamination. The access of horses to waterways and coastal environments may also need to be restricted to prevent transmission of faecal indicator bacteria and potentially zoonotic agents.

Leaching of Escherichia coli from sheep faeces during simulated rainfall events.

UNLABELLED: Sheep faeces are known to harbour to a high concentration of microbial indicators and pathogens. These can be released under rainfall and may result in contamination of waterways, potentially leading to illnesses in humans. A study was designed to determine the concentration of Escherichia coli released from fresh and aged (0-21 days old) ovine faeces. In summer and autumn, ovine faeces were subjected to simulated rainfall and the resultant run-off collected. Escherichia coli were enumerated in both the run-off and the faeces. In autumn total suspended solids (TSS) and turbidity were also monitored in the run-off. This study provides quantitative evidence that E. coli in aged sheep faeces is mobilized by rainfall events. Simulated rainfall events released between 10(3) and 10(4) CFU E. coli ml(-1) throughout the 21 days. TSS or turbidity with fresh faeces may be indicative of microbial contamination, but from aged faeces, this may not be the case. SIGNIFICANCE AND IMPACT OF THE STUDY: This study confirms that faecal bacteria can be released from fresh and aged ovine faeces under stimulated rainfall. It demonstrates that aged faeces remain a source of faecal bacteria, which under rainfall can release the bacteria and result in pollution of waterways. This study aids in our understanding of the potential impact of grazing sheep on the microbial quality of surface waters in NZ.

Survival of Escherichia coli, enterococci and Campylobacter jejuni in Canada goose faeces on pasture.

Freshly excreted Canada goose faeces pose a public health risk as they contain pathogenic microorganisms. Accordingly, a study was carried out on the growth and survival of resident indicator bacteria (enterococci and Escherichia coli) and inoculated Campylobacter jejuni in freshly excreted faeces over summer and winter. Canada goose faeces were collected, mixed thoroughly and inoculated with 108 g⁻¹ C. jejuni. The faeces were mixed again before making the Canada goose dropping. The simulated goose droppings (N = 70) were placed on pasture, and the concentrations of E. coli, enterococci and the pathogen, C. jejuni, were monitored. In summer only, the molecular marker of E. coli LacZ and the avian-associated bacteria E2 was also monitored. Results of the survival study indicated that significant growth of enterococci and E. coli occurred in summer, before concentrations decreased to less than 15% of the original concentration (day 77) for enterococci and 0.01% for E. coli. LacZ followed a similar pattern to E. coli, while the E2 marker dropped to below 0.1% of the original concentration within 4 days. In winter, enterococci grew slightly, while no growth of E. coli occurred. In both summer and winter, C. jejuni was rapidly inactivated. This research highlights the ability of bacterial indicators to replicate and survive in the environment when harboured by avian faeces, and the limited risk aged Canada goose faeces pose as an environmental source of Campylobacter spp.

A survey of enteric bacteria and protozoans in fresh bovine faeces on New Zealand dairy farms.

AIMS: To determine the counts and/or prevalence in fresh bovine faeces of Escherichia coli, enterococci, Campylobacter, Salmonella, shiga toxin-producing E. coli (STEC), Giardia and Cryptosporidium, as inputs to numerical models designed to estimate microbial loadings on pasture grazed by cattle in New Zealand. METHODS AND RESULTS: In each season over one year, samples of freshly deposited bovine faeces were collected from four New Zealand dairy farms (n = 155), and enumerated for E. coli, enterococci, Campylobacter, Giardia and Cryptosporidium. They were also tested for the presence of Salmonella and STEC. The overall median bacterial counts (g(-1) wet weight) were E. coli- 5.9 x 10(6); enterococci - 1.3 x 10(4); Campylobacter- 3.9 x 10(5). All counts were highly variable within and between samplings, and few seasonal or regional patterns emerged. However, mean Campylobacter counts were consistently higher in spring. No Salmonella spp. was detected, and only two samples were positive for STEC. Cryptosporidium and Giardia were isolated from 5.2% and 4.5% of the samples, respectively, yielding low numbers of (oo)cysts (1-25 g(-1) and 1-17 g(-1), respectively). CONCLUSIONS: Fresh bovine faeces are a significant source of E. coli, enterococci and Campylobacter on New Zealand pastures, although numbers are likely to vary markedly between faecal samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The study provides the first significant set of indicator and pathogen counts for one of the largest sources of faecal contamination of natural waters in New Zealand, and will be used to model these inputs.

Biochemical screening of type I collagen in osteogenesis imperfecta: detection of glycine substitutions in the amino end of the alpha chains requires supplementation by molecular analysis.

BACKGROUND: The biochemical test for osteogenesis imperfecta (OI) detects structural abnormalities in the helical region of type I collagen as delayed electrophoretic migration of alpha chains on SDS-urea-PAGE. Sensitivity of this test is based on overmodification of alpha chains in helices with a glycine substitution or other structural defect. The limits of detectability have not been reported. METHODS: We compared the collagen electrophoretic migration of 30 probands (types III or IV OI) with known mutations in the amino half of the alpha1(I) and alpha2(I) chains. Differences in sensitivity were examined by 5% and 6% SDS-urea-PAGE, and with respect to alpha chain, location along the chain, and substituting amino acid. RESULTS: Sensitivity was enhanced on 5% gels, and by examination of intracellular and secreted collagen. In alpha1(I), substitutions in the first 100 residues were not detectable; 7% of cases in the current Mutation Consortium database are in this region. alpha1(I) substitutions between residues 100 and 230 were variably detectable, while those after residue 232 were all detected. In alpha2(I), variability of electrophoretic detection extended through residue 436. About a third of cases in the Consortium database are located in the combined variable detection region. Biochemical sensitivity did not correlate with substituting residue. CONCLUSIONS: Complete testing of probands with normal type I collagen biochemical results requires supplementation by molecular analysis of cDNA or gDNA in the amino third of alpha1(I) and amino half of alpha2(I). Mutation detection in OI is important for counselling, reproductive decisions, exclusion of child abuse, and genotype-phenotype correlations.

The prevalence and characterisation of Cryptosporidium spp. in beef abattoir water supplies.

The prevalence of Cryptosporidium spp. in 50 l samples of water used to wash beef carcasses at (a) an abattoir with a borehole water (BH) supply (n = 46) and (b) an abattoir with a river water (RW) supply (n = 48) was determined. In addition, a 100 l water sample and post-wash carcass samples (n = 24) were collected from the RW supply on a single day in July. Cryptosporidium spp. was detected in 0% and 26.1% of samples from the BH and RW supply abattoirs, respectively, with oocyst concentrations ranging from 0.02 to 8.6/l. Cryptosporidium spp. was not isolated from post-wash beef carcasses, while it was detected in water samples from that day at a concentration of 0.06 oocysts/l. The species of 3/5 isolates were identified as C. parvum, and the remaining were C. andersoni. This study has demonstrated that water used to wash beef carcasses can be contaminated with Cryptosporidium of human health importance and is a potential source of carcass contamination.

Prevalence and characterisation of Cryptosporidium species in cattle faeces and on beef carcases at slaughter.

Cattle are known reservoirs and asymptomatic excretors of Cryptosporidium, a protozoan parasite that causes severe and protracted diarrhoea in people. The incidence of Cryptosporidium was investigated in 288 matched samples taken from beef carcases of 1 g samples of faeces retrieved immediately after de-legging, 25 cm2 samples of beef excised from the rump of uneviscerated carcases, and 25 cm2 samples of beef excised from the brisket area of eviscerated carcases. Cryptosporidium species were detected in 21 of the faecal samples after salt flotation and immunofluorescent microscopy. The species isolated from the positive samples were identified by restriction fragment length polymorphism and PCR as Cryptosporidium andersoni (54.5 per cent) and Cryptosporidium parvum genotype 2 (45.5 per cent). In the faecal samples, there was a significantly higher prevalence of the parasite in samples taken in summer (May to July) and winter (November to January) than in spring or autumn. No Cryptosporidium species were recovered from any of the beef samples.

The effect of thermal treatments on the viability and infectivity of Cryptosporidium parvum on beef surfaces.

AIMS: The aim of this research was to examine the effect of thermal treatments on the viability and infectivity of Cryptosporidium parvum oocysts attached to a beef surface. METHODS AND RESULTS: This study examined the effects of heat treatment (60 or 75 degrees C) on the viability of C. parvum oocysts inoculated onto the surface of beef muscle estimated by vital dye assay. The infectivity of the oocysts was assessed against monolayers of HCT-8 cells. At 60 degrees C viability of the oocysts decreased from 100% at T0 to 64.2% at T60. At 75 degrees C the viability of the oocysts decreased from 100% at T0 to 53.7% at T15 and finally to 11.2% at T60. Oocysts were rendered noninfective against monolayers of HCT-8 cells following treatments of 60 degrees C/45 s and 75 degrees C/20 s. CONCLUSION: The washing of carcasses with hot water and standard thermal treatments is sufficient to kill C. parvum on beef. SIGNIFICANCE AND IMPACT OF THE STUDY: This study found that relatively mild heat, currently used to decontaminate and heat treat beef carcasses and to cook meat products, is capable of inactivating C. parvum.

Effect of a commercial freeze/tempering process on the viability of Cryptosporidium parvum oocysts on lean and fat beef trimmings.

Lean and fat beef trimmings (25 cm(-2)) were inoculated with approximately 250,000 Cryptosporidium parvum oocysts, placed in commercial packages (28 kg boxes) and subjected to normal commercial processes i.e. blast frozen (to -20 °C within 60 h), stored (-20 °C, 21 days), tempered (48 h at -3 °C), and held at 0 °C for 10 h. Inoculated areas were then excised, pulsified (30 s in 50 ml PBST), and centrifuged (2500×g, 15 min). The resultant pellet was resuspended in 10 ml water and subjected to immunomagnetic separation and viability dye assay. Following the commercial freeze/tempering process the viability of the oocysts had decreased from 90.6% viable in the working stock suspension to 7.17% and 9.46% viable on lean and fat trimmings, respectively. The results of this study indicate that if C. parvum oocysts were present on beef trimmings their viability would be substantially reduced as a result of the freeze/tempering process.

Sperm storage in the oviduct of the internal fertilizing frog Ascaphus truei.

This study provides the first descriptions of sperm storage at the tissue and cellular levels in a female frog or toad. Oviducal anatomy was studied by light and electron microscopy in Ascaphus truei from north coastal California. Ascaphus truei is one of the few species of anurans in which fertilization is internal. Unlike other anurans with internal fertilization, however, mating in A. truei consists of a unique combination of amplectic and copulatory mechanisms that we term "copulexus." Posterior to a short, aglandular infundibular region, the oviduct possesses: 1) a proximal, convoluted ampullary region where intrinsic tubular glands secrete gelatinous envelopes around eggs; 2) a middle ovisac region where fertilization occurs; and 3) a distal oviducal sinus formed by medial junction of the ovisacs. Sperm storage tubules (SSTs) occur in the anterior portions of the ovisacs and consist of simple tubular glands. SSTs and the rest of the oviducal lining stain positively with the periodic acid-Schiff's procedure for neutral carbohydrates and this reaction is especially intense in reproductively active females. Sperm were found in the SSTs of gravid females as well as some nonvitellogenic females. The sperm are in orderly bundles in the SSTs, and although occasionally sperm nuclei were embedded in the epithelium, no evidence for spermiophagy was found. Oviducal sperm storage in A. truei is homoplastic, with closest structural similarities to squamate reptiles. Oviduct/sperm design constraints appear to limit the options for expression of features associated with oviducal sperm storage.

Sperm storage in females of the smooth newt (Triturus v. vulgaris L.): I. Ultrastructure of the spermathecae during the breeding season.

Sperm storage in cloacal spermathecae was studied in females of Triturus v. vulgaris collected early in the breeding season in southern England. Females collected in terrestrial situations, presumably unmated, were mated in the laboratory, and the ultrastructure of the transferred sperm and the spermathecae was observed at various intervals after mating. Sperm from a spermatophore cap lodged in a female's cloacal orifice can migrate into spermathecae within 1 hr after mating. Spherical structures on the axial fibers of some sperm in the cap could indicate immaturity. Disorderly clusters of sperm from the cap are still present in the cloacal chamber 12 hr after mating but are absent 24 hr after mating. During storage, sperm often are in tangled masses in the spermathecal tubules. The sperm are coated with spermathecal secretions, and some sperm nuclei were observed embedded in the spermathecal epithelium. Little evidence for spermiophagy early in the breeding season was found. During oviposition, mazes of sperm occur external to the spermathecal orifices, and sperm may be released in this condition onto eggs as they pass through the cloaca. The tangled clusters in which sperm are found from pick-up to oviposition are hypothesized as an adaptation to reduce the effectiveness of sperm competition from the ejaculates of rival males. Additional studies, using the same protocol and covering the entire cycle of sperm storage, are necessary to enable interspecific comparisons leading to phylogenetic hypotheses.

Infusional cisplatin and etoposide in the treatment of stage III-B and IV non-small cell lung cancer: a well tolerated regimen.

Twenty-eight evaluable patients were treated with an infusion of cisplatin and etoposide for advanced non-small cell lung cancer. A response was demonstrated in 43%, although only two patients had documented partial responses. The regimen was surprisingly low in toxicity, both acute and chronic, and is suitable for palliation of patients who are elderly or suffer from chronic illnesses which preclude more agressive therapy.