Enteric viruses in New Zealand drinking-water sources.

This study determined whether human pathogenic viruses are present in two New Zealand surface waters that are used as drinking-water sources. Enteric viruses were concentrated using hollow-fibre ultrafiltration and detected using PCR for adenovirus (AdV), and reverse transcription PCR for norovirus (NOV) genogroups I-III, enterovirus, rotavirus (RoV) and hepatitis E virus (HEV). Target viruses were detected in 106/109 (97%) samples, with 67/109 (61%) samples positive for three or more viral types at any one time. AdV, NoV and ROV were detected the most frequently, and HEV the least frequently. Human NoV was not usually associated with animal NOV. Our results suggest that New Zealand would be well served by assessing the ability of drinking-water treatment plants to remove viruses from the source waters, and that this assessment could be based on the viral concentration of AdV-NoV-RoV. The long-term aim of our work is to use this information to estimate the risk of waterborne viral infection.

Preliminary development and validation of a real-time reverse transcription PCR assay for the semi-quantification of viable Campylobacter jejuni in water samples.

A TaqMan-based real-time reverse transcription PCR (RT-PCR) assay was developed for semi-quantification of viable Campylobacter jejuni in water samples. This preliminary assay is based on measuring the heat-shock induction of groEL messenger RNA (mRNA); the logic being that only viable cells can synthesise new mRNA. A 128-bp C. jejuni groEL DNA fragment was cloned and used as a positive control standard in TaqMan runs. The assay could detect as few as 13 genome equivalent copies of groEL plasmid DNA and 1-2 colony forming unit (CFU) of viable C. jejuni. A multi-step concentration technique was developed for collecting C. jejuni from large volumes of water samples. An average recovery of 20% (range: 8-47%) was obtained at spiking levels of 750-6,500 CFU per 10-litre of river water. Starting from concentration, the enumeration of viable C. jejuni in river water samples was achieved in approximately 12 hours. Quantification of viable C. jejuni in natural river water samples by this method showed similar trends to a culture-based double enrichment most probable number (MPN) -PCR method. There is potential to apply this fast, specific and sensitive semi-quantitative method to monitor a range of water samples for viable C. jejuni.

Survival of Campylobacter spp. in bovine faeces on pasture.

AIMS: To determine the survival on pasture of Campylobacter spp. naturally present in bovine faeces and compare this with a previously published study using laboratory-cultured Campylobacter spp. METHODS AND RESULTS: Ten freshly collected cow pats were deposited on pasture during summer, and Campylobacter spp. were enumerated by enrichment broth culture. The counts in three pats were below detection limits. Counts of Campylobacter spp. in the other seven pats fell below detection limits within 14 days. The geometric means of the counts up to 7 days produced a T(90) of 2.2 days. Characterization of Campylobacter spp. by PCR and pulsed field gel electrophoresis indicated the presence of at least six genotypes of Campylobacter jejuni, Campylobacter coli and Campylobacter lari. CONCLUSIONS: Campylobacter spp. naturally present in cow faeces exhibited a similar survival rate to that previously determined using laboratory-cultured strains. The highly variable counts of naturally occurring Campylobacter spp., and the predominance of lower counts, also support the earlier decision to use laboratory-cultured strains in survival experiments. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reaffirms the short survival of Campylobacter spp. in cow faeces deposited on pasture. This information will be incorporated into a 'reservoir model' for Campylobacter spp. in cow pats on New Zealand pastures.

Comparison of Campylobacter jejuni genotypes from dairy cattle and human sources from the Matamata-Piako District of New Zealand.

AIMS: To identify the prevalence and types of Campylobacter jejuni carried by dairy cattle and the extent of overlap of these types with those causing disease in humans. METHODS AND RESULTS: Faecal samples from 410 dairy cattle were collected from 36 farms in the Matamata-Piako district in New Zealand. Campylobacter jejuni was isolated on all 36 farms, with a prevalence of 51% (95% CI 45-57) in dairy cattle and 65% (95% CI 58-72) in calves. Eighty-nine of these isolates were typed using Penner serotyping and pulsed field gel electrophoresis and were compared with 58 human C. jejuni isolates from people resident within this study area. CONCLUSIONS: Campylobacter jejuni were found in the faeces of over half of the dairy cows and calves examined. Twenty-one per cent of the bovine isolates and 43% of the human isolates formed indistinguishable clusters of at least one bovine and one human isolate. SIGNIFICANCE AND IMPACT OF THE STUDY: While a direct link between bovine isolates and human cases was not demonstrated, the finding of indistinguishable genotypes among C. jejuni isolates from bovine and human sources confirms that dairy cows and calves are a potential source of human campylobacteriosis. Barriers to separate bovine faecal material from the general public are therefore important public health measures.

The transmission of thermotolerant Campylobacter spp. to people living or working on dairy farms in New Zealand.

New Zealand has one of the highest rates of campylobacteriosis in the developed world with an incidence rate of 383.5 cases per 100,000 in 2006. Dairy farming has been suggested as a potential source of campylobacteriosis. To explore this connection, seven farm investigations were undertaken at dairy farms on which a campylobacteriosis case had been notified. Campylobacter spp. were isolated from a range of sources on the farm (including 66% of bovine faecal samples) and genotypes compared with that of the clinical isolate of the index case. In depth, epidemiological questionnaires were also administered to determine exposure risks from a wide range of possible sources. Contact with dairy cow faeces was the most likely source of infection in four of the seven cases investigated, and occurred exclusively in new farm workers and children. In one of the cases investigated, infection was likely to have been acquired from non-dairy related sources, and in two cases the source could not be determined. The relative risk of dairy farm worker being notified with campylobacteriosis was estimated to be 1.88 (95% confidence interval=1.6-2.2).

Statistical comparison of Campylobacter jejuni subtypes from human cases and environmental sources.

AIM: To analyse Campylobacter jejuni typing data to define statistically which potential reservoirs and transmission sources contain isolates that are most similar to one another and to isolates from human infections. METHODS AND RESULTS: Serotyping and SmaI macrorestriction profiling data for C. jejuni isolates from human campylobacteriosis cases, chicken carcass rinses, duck, sheep, dairy and beef cattle faeces, river water, and sheep, beef and pork offal obtained from a defined rural area of New Zealand were compared using the Czekanowski proportional similarity index. Subtypes of isolates from ruminant animals, whether derived from their faeces or offals, were generally similar to one another. The spectrum of isolate subtypes from human cases was more similar to that from ruminant faeces than the other matrices considered. Isolate subtypes from chicken rinses, pork offal, water and duck faeces were not highly similar to those from other matrices. CONCLUSIONS: Results from a combination of phenotypic and genotypic approaches suggest that, for this rural population, exposures associated with a rural lifestyle may be significant sources of human campylobacteriosis. SIGNIFICANCE AND IMPACT OF THE STUDY: The Czekanowski index was applied to subtyping data and supported the greater importance of contact with livestock in campylobacteriosis cases associated with a rural setting, in comparison with urban studies that have identified poultry-related factors.

The occurrence of Campylobacter subtypes in environmental reservoirs and potential transmission routes.

AIM: To identify potential reservoirs and transmission routes of human pathogenic Campylobacter spp. METHODS AND RESULTS: An enrichment PCR method for the detection and identification of Campylobacter jejuni and/or Campylobacter coli in faecal, food and river water samples was applied to 1450 samples of 12 matrix types obtained from a defined geographical area. PCR-positive samples were cultured to yield isolates for typing, and the data for 616 C. jejuni isolates obtained. Serotyping and SmaI macrorestriction profiling using pulsed field gel electrophoresis revealed a high level of diversity within the isolates from each matrix. Campylobacter jejuni and C. coli subtypes indistinguishable from those obtained from human cases were detected in most of the matrices examined. No Campylobacter isolates were isolated from possum faeces. CONCLUSIONS: Ten of the 12 matrices examined may be involved in the transmission of human campylobacteriosis as they contained Campylobacter subtypes also isolated from clinical cases. SIGNIFICANCE AND IMPACT OF THE STUDY: Results indicate that, for this rural population, a range of potential transmission routes that could lead to campylobacteriosis exist. Their relative importance needs to be assessed from an exposure assessment standpoint.

The use of chemical and molecular microbial indicators for faecal source identification.

Identifying the source of faecal pollution is important to enable appropriate management of faecal pollution of water. We are developing and evaluating a combination of these microbial and chemical indicators better able to identify the source of faecal pollution. These assays make use of a combination of direct PCR, culturing, and colony hybridisation to identify source specific species of Bifidobacterium, Rhodococcus and Bacteroides. In conjunction with assays for (a) fluorescent whitening agents and (b) faecal sterols and stanols, these indicators were able to identify human derived faecal pollution in river water containing inputs from septic tanks, municipal oxidation ponds, farmed animals and feral animals. Differentiating amongst the animal sources was more difficult and will require development of molecular assays for organisms specific to each animal group.

Application of polymerase chain reaction (PCR) and TaqMan PCR techniques to the detection and identification of Rhodococcus coprophilus in faecal samples.

Rhodococcus coprophilus, a natural inhabitant of herbivore faeces, has been suggested as a good indicator of animal (as opposed to human) faecal contamination of aquatic environments. However, conventional detection methods limit its use for this as they require up to 21 days to obtain a result. In this paper an optimised method for extracting R. coprophilus DNA from faecal samples is described. PCR and 5'-nuclease (TaqMan) PCR methods were developed to allow the detection and enumeration of R. coprophilus in faecal samples within 2-3 days. Both PCR methods targeted the 16S rRNA gene, producing an amplicon of 443 bp which was specific for R. coprophilus. Sixty cells were required to produce an amplification product by conventional PCR, while as little as one cell was required for the TaqMan PCR method. The latter approach gave a linear quantitative response over at least four log units with both bacterial cells and DNA. Successful amplification by PCR was achieved using DNA extracted from cow, sheep, horse and deer faeces but was negative for samples from humans, pig, possum, duck and rabbit. These PCR methods enhance the feasibility of using R. coprophilus to distinguish faecal pollution of farmed herbivores from human pollution.

Enumeration of Campylobacter in New Zealand recreational and drinking waters.

AIMS: To use a published polymerase chain reaction (PCR) method for the detection and identification of thermotolerant Campylobacter species (Camp. jejuni, Camp. coli and Camp. lari) in tandem with a Most Probable Number (MPN) technique to enumerate these species in water samples. METHODS AND RESULTS: An initial study of 42 river water samples compared the use of conventional culture and PCR methods for the detection of Campylobacter in MPN enrichment tubes. It was found that all samples positive by culture were also positive by PCR. Thirty-seven percent more MPN tubes were positive by PCR compared with culture. The MPN/PCR technique was subsequently applied to 96 additional samples collected from rivers, drinking, roof and shallow ground water. Campylobacter was especially prevalent in river water (60% positive) and shallow ground water (75% positive) samples. Drinking water (29.2% positive) and roof water (37.5% positive) also contained Campylobacter, but the numbers detected were very low (maximum 0.3 and 0.56 MPN 100 ml-1, respectively). CONCLUSION: River waters contained Campylobacter at higher levels than any other water type and in a high percentage of the samples. Although Campylobacter was present in treated drinking water, the levels detected were low. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest that water may act as a significant transmission route for human campylobacteriosis.

Rapid detection of Listeria monocytogenes in ham samples using immunomagnetic separation followed by polymerase chain reaction.

AIMS: To develop a 24-h system for the detection of Listeria monocytogenes in ham. METHODS AND RESULTS: An immunomagnetic separation (IMS) of bacteria directly from ham followed by extraction of DNA and detection using a new multiplex polymerase chain reaction (PCR) was used. The PCR method used one primer pair targeted at the listeriolysin O gene of L. monocytogenes and the other pair for a region of the 23S rRNA genes of Listeria, giving products of 706 and 239 bp, respectively. The combined IMS/PCR was calculated to be capable of detecting as few as 1.1 L. monocytogenes cells g-1 in a 25-g ham sample. CONCLUSION: The process produced acceptable results, but the IMS step is the main barrier to further improvement of sensitivity. The DNA isolation was the most time-consuming step in the process. SIGNIFICANCE AND IMPACT OF THE STUDY: A 24-h test for the presence of L. monocytogenes will be useful to the food industry and significantly assist in the timely investigation of outbreaks.

Detection and determination of surface levels of poloxamer and PVA surfactant on biodegradable nanospheres using SSIMS and XPS.

The surface chemical characterisation of sub-200 nm poly(DL-lactide co-glycolide) nanospheres has been carried out using the complementary analytical techniques of static secondary ion mass spectrometry (SSIMS) and X-ray photoelectron spectroscopy (XPS). The nanospheres, which are of interest for site-specific drug delivery, were prepared using an emulsification-solvent evaporation technique with poly(vinyl alcohol), Poloxamer 407 and Poloxamine 908 respectively as stabilisers. The presence of surfactant molecules on the surface of cleaned biodegradable colloids was confirmed and identified on a qualitative molecular level (SSIMS) and from a quantitative elemental and functional group analysis (XPS) perspective. SSIMS and XPS data were also used in combination with electron microscopy to monitor the effectiveness of cleaning procedures in removing poorly bound surfactant molecules from the surface of nanospheres. The findings are discussed with respect to the development of nanoparticle delivery systems, particularly the composition of the surface for extending blood circulation times and achieving site-specific deposition.

Resorbable polymeric microspheres for drug delivery--production and simultaneous surface modification using PEO-PPO surfactants.

Poly(oxyethylene)-poly(oxypropylene) (PEO-PPO) co-polymers have been used as surfactants to produce resorbable poly(DL-lactide co-glycolide) (PLG) microspheres in the 500 nm-1 micron size range by an emulsification/solvent evaporation technique based on acetone-dichloromethane mixtures. The high polydispersity of microspheres could be reduced by using low PLG concentrations of 1% (w/v). Surface analysis by static secondary ion mass spectroscopy revealed the presence of PEO-PPO at the microsphere surface after cleaning by centrifuging and resuspension in water, and after further cleaning by dialysis. Physical entrapment of PEO-PPO chains in the particle surface is indicated due to rapid collapse of the solvent swollen PLG network as acetone is extracted from the suspended droplets. Opportunities are presented for simultaneous manufacture and surface modification of microspheres.

Antagonists of bombesin/gastrin releasing peptide based on [D-Ala24]GRP(20-26)-heptapeptide. Modifications leading to potent analogues with prolonged duration of action.

Analogues of gastrin releasing peptide (GRP) and bombesin based on His-Trp-Ala-Val-D-Ala-His-Leu, the 20-26 heptapeptide sequence of [D-Ala24]GRP, have been synthesized and tested in vitro for their ability to inhibit GRP (18-27)-induced mitogenesis in Swiss 3T3 cells. Compounds identified as potent antagonists in this test system were also tested in vivo for their ability to inhibit bombesin-induced amylase secretion in rats. The Trp-Ala-Val sequence was found to be a very important feature of the antagonist activity; most substitutions in this region led either to much less potent or inactive analogues. In contrast, amino acid replacements in other parts of the molecule were more tolerated and sometimes led to marked increases in the in vitro and in vivo activity. The most potent analogues were obtained by replacing Leu26 by MeLeu and His25 by Lys(X) where X = Z, PhCO, PhCH2CO or Ph(CH2)2CO. Thus 4-pyridylcarbonyl-His-Trp-Ala-Val-D-Ala-Lys(CO-CH2-CH2-Ph)-Leu- NHMe (86) and 4-pyridylcarbonyl-His-Trp-Ala-Val-D-Ala-Lys(Z)-MeLeu-OMe (87) had IC50 values of less than 20 micrograms/kg s.c. in vivo, and their effects lasted for more than 3 hr.

N-isobutyryl-His-Trp-Ala-Val-D-Ala-His-Leu-NHMe (ICI 216140) a potent in vivo antaconist analogue of bombesin/gastrin releasing peptide (BN/GRP) derived from the C-terminal sequence lacking the final methionine residue.

The GRP receptor mediated growth response in Swiss 3T3 cells has been used to identify BN/GRP antagonists. Analysis of bombesin antagonism by substance P analogues and by truncated GRP analogues revealed that deletion of the C-terminal methionine residue was important for antagonism. Des-Met analogues showing potent antagonist activity in the in vitro 3T3 system (IC50 approximately 2nM) were synthesized. Further structural modification of these peptides led to the identification of (CH3)2CHCO-His-Trp-Ala-Val-D-Ala-His-Leu-NHCH3 (ICI 216140) which reduced bombesin-stimulated rat pancreatic amylase secretion to basal levels when administered subcutaneously at 2.0 mg per kg.

The H+/ATP stoichiometry of the (H+ +K+)-ATPase of dog gastric microsomes.

Gastric microsomal vesicles isolated from dog fundic mucosa were shown to be relatively ion tight and have a low level of proton permeability. The H+ translocase, basal ATPase and K+-activated ATPase activities of these vesicles were measured and the H+/ATP stoichiometry calculated using either the total K+-ATPase or the K+-stimulatable component (total K+-ATPase--basal ATPase). The former estimations consistently gave stoichiometric of approximately one, whereas the use of only the K+-stimulatable component gave widely differing values. Measurement of the dephosphorylation of the enzyme under basal conditions revealed both a labile and a stable phosphoenzyme component. The rate of decay of the labile component completely accounted for the basal ATPase activity observed. We conclude that the basal ATPase associated with our preparations is a spontaneous dephosphorylation of the phosphoenzyme occurring in the absence of K+ and that the H+/ATP stoichiometry of the gastric ATPase is one.

The localization of a histamine H2-receptor adenylate cyclase system in canine parietal cells and its inhibition by prostaglandins.

A method is described for the preparation of parietal cell-enriched suspensions from dog gastric mucosa. Histamine and E type prostaglandins produce an elevation of cyclic AMP concentration in mixed cell preparations. Parietal cell-rich fractions respond to histamine but only weakly to prostaglandins whilst in fractions virtually free from parietal cells the converse is observed. Prostaglandins which are good antisecretory agents, PGE1, PGE2 and 16,16 dimethyl PGE2 are potent inhibitors of the histamine elevations of cyclic AMP in parietal cell-rich fractions, whilst PFG2alpha shows 1% of their potency. The experiments described support the view that histamine stimulates gastric acid secretion by excitation of an H2-receptor adenylate cyclase system in the plasma membrane of the parietal cell and that acid secretory inhibition by prostaglandins is a result of inhibition of that system.