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Recreational water quality of rivers is typically assessed by microbial analysis of indicator organisms such as Escherichia coli and enterococci in samples collected directly from the water column, implying that risk to public health comes from free-floating microorganisms. In New Zealand, the Recreational Water Quality Guidelines apply certain E. coli concentration thresholds to categorize results into ‘meet guidelines’ (≤ 260 cfu E. coli 100 mL− 1), ‘alert level’ and ‘action level’ (≥ 550 cfu E. coli 100 mL− 1). The impairment of water quality due to resuspended riverbed sediment under flood conditions has been widely recognized. However, the potential risks posed by sediment-linked microorganisms associated with river recreational activities under base flow are less appreciated. We evaluated the water quality of 30 rivers in Canterbury, New Zealand, during base flow conditions prior to and after artificially stirring the sediment. Bacterial and protozoan indicators, pathogens, water quality and land use in surrounding areas were examined. While 71% of water samples (n = 64) met New Zealand Recreational Water guidelines prior to stirring, only 39% of samples (n = 35) met the guidelines after stirring. E. coli levels increased by 110% or more following sediment stirring. Campylobacter were detected in every river at least once, and increases were observed following stirring. We conclude that sediment-associated microbes become suspended by recreational activities and may pose a considerable risk to river users. To ensure public health is protected authorities should assess microorganisms in both sediment and water samples to rate the microbiological risk of recreational water use.

To compare detection of SARS-CoV-2 from paired nasopharyngeal swabs (NPS) and saliva using molecular methods in common use for testing swabs in New Zealand.Samples from individuals testing positive for SARS-CoV-2 in Auckland, Wellington and Dunedin were tested at the local laboratories using methods previously established for these sample types.One hundred and ninety-six paired samples from unique individuals were tested, with 46 (23%) positive from either sample type, of which 43/46 (93%) tested positive from NPS, and 42/46 (91%) from saliva, indicating no significant difference in performance between sample types (p=0.69). The average Δ Ct between saliva and nasopharyngeal swabs overall across the sample set was 0.22 cycles, indicating excellent concordance; however, the difference between NPS and saliva collected from the same individual was quite variable with up to 19 cycles difference between the sample types.We found that saliva is an equivalent sample type to nasopharyngeal swab for the detection of SARS-CoV-2 in our laboratories using multiple assay combinations and is suitable for use as a diagnostic and surveillance test for selected groups of individuals.

Description, Analyses of ~2000 genomes of Listeria monocytogenes main clinical clone reveal its global spread and dynamics., The bacterial foodborne pathogen Listeria monocytogenes clonal complex 1 (Lm-CC1) is the most prevalent clonal group associated with human listeriosis and is strongly associated with cattle and dairy products. Here, we analyze 2021 isolates collected from 40 countries, covering Lm-CC1 first isolation to present days, to define its evolutionary history and population dynamics. We show that Lm-CC1 spread worldwide from North America following the Industrial Revolution through two waves of expansion, coinciding with the transatlantic livestock trade in the second half of the 19th century and the rapid growth of cattle farming and food industrialization in the 20th century. In sharp contrast to its global spread over the past century, transmission chains are now mostly local, with limited inter- and intra-country spread. This study provides an unprecedented insight into L. monocytogenes phylogeography and population dynamics and highlights the importance of genome analyses for a better control of pathogen transmission.

This study describes the epidemiology of listeriosis in New Zealand between 1999 and 2018 as well as the retrospective whole-genome sequencing (WGS) of 453 Listeria monocytogenes isolates corresponding to 95% of the human cases within this period. The average notified rate of listeriosis was 0.5 cases per 100,000 population, and non-pregnancy-associated cases were more prevalent than pregnancy-associated cases (averages of 19 and 5 cases per annum, respectively). WGS data was assessed using multilocus sequencing typing (MLST), including core-genome and whole-genome MLST (cgMLST and wgMLST, respectively) and single-nucleotide polymorphism (SNP) analysis. Thirty-nine sequence types (STs) were identified, with the most common being ST1 (21.9%), ST4 (13.2%), ST2 (11.3%), ST120 (6.1%), and ST155 (6.4%). A total of 291 different cgMLST types were identified, with the majority (n = 243) of types observed as a single isolate, consistent with the observation that listeriosis is predominately sporadic. Among the 49 cgMLST types containing two or more isolates, 18 cgMLST types were found with 2 to 4 isolates each (50 isolates in total, including three outbreak-associated isolates) that shared low genetic diversity (0 to 2 whole-genome alleles), some of which were dispersed in time or geographical regions. SNP analysis also produced results comparable to those from wgMLST. The low genetic diversity within these clusters suggests a potential common source, but incomplete epidemiological data impaired retrospective epidemiological investigations. Prospective use of WGS analysis together with thorough exposure information from cases could potentially identify future outbreaks more rapidly, including those that may have been undetected for some time over different geographical regions.

In rural environments, the sources of fecal contamination in freshwater environments are often diffuse and a mix of fresh and aged fecal sources. It is important for water monitoring purposes, therefore, to understand the impacts of weathering on detection of the fecal source markers available for mobilization from livestock sources. This study targets the impacts of rainfall events on the mobilization of fecal source tracking (FST) markers from simulated cowpats decomposing in situ for five-and-a-half-months. The FST markers analysed were Escherichia coli, microbial source tracking (MST) markers, fecal steroids and a fecal ageing ratio based on the ratio between counts of river microflora and total coliforms. There was a substantial concentration of E. coli (10

Recent studies have suggested that poikilothermic animals, such as fish, may represent a previously overlooked source of the faecal indicator bacteria (FIB) used for the assessment of water quality...

Listeria monocytogenes is a foodborne human pathogen that causes systemic infection, fetal-placental infection in pregnant women causing abortion and stillbirth and meningoencephalitis in elderly and immunocompromised individuals. This study aimed to analyse L. monocytogenes from different sources from New Zealand (NZ) and to compare them with international strains. We used pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and whole-genome single nucleotide polymorphisms (SNP) to study the population structure of the NZ L. monocytogenes isolates and their relationship with the international strains. The NZ isolates formed unique clusters in PFGE, MLST and whole-genome SNP comparisons compared to the international isolates for which data were available. PFGE identified 31 AscI and 29 ApaI PFGE patterns with indistinguishable pulsotypes being present in seafood, horticultural products and environmental samples. Apart from the Asc0002:Apa0002 pulsotype which was distributed across different sources, other pulsotypes were site or factory associated. Wholegenome analysis of 200 randomly selected L. monocytogenes isolates revealed that lineage II dominated the NZ L. monocytogenes populations. MLST comparison of international and NZ isolates with lineage II accounted for 89% (177 of 200) of the total L. monocytogenes population, while the international representation was 45.3% (1674 of 3473). Rarefaction analysis showed that sequence type richness was greater in NZ isolates compared to international trend, however, it should be noted that NZ isolates predominantly came from seafood, horticulture and their respective processing environments or factories, unlike international isolates where there was a good mixture of clinical, food and environmental isolates.

The intensification of dairy farming on the agricultural landscape in New Zealand has raised concerns about pollution sources from dairy faecal runoff into waterways. The transport of faecal pollution from farms into waterways is facilitated by overland flow, which can result from rain and flood events, poorly designed irrigation practices and the washing down of milking sheds.An important step for mitigation of pollution is the identification of the source(s) of faecal contamination. When elevated levels of faecal indicator bacteria (FIB) such as Escherichia coli are identified in a waterway, faecal source tracking (FST) tools such as microbial source tracking (MST) using quantitative polymerase chain reaction (qPCR), and faecal steroids (for example, cholesterol) provide information about the sources of faecal contamination. The understanding of the fate (degradation/persistence) and transport of these FST markers in the environment is recognised as an important requirement for the interpretation of water quality monitoring in aquatic environments.This study investigated the effects of faecal decomposition on bovine faecal indicators (E. coli and FST markers: bovine-associated qPCR markers and ten faecal steroids) by monitoring the effect of flood and rainfall events on simulated cowpats over a five and a half month period under field conditions. Two separate spring/summer trials were conducted to evaluate: Trial 1) the mobilisation under simulated flood conditions of the faecal indicators from irrigated versus non-irrigated cowpats, Trial 2) the mobilisation of faecal indicators from non-irrigated cowpat flood runoff versus runoff after simulated rainfall onto non-irrigated cowpats.The microbial community changes within the decomposing cowpat (as illustrated by amplicon-based metagenomic analysis) were expected to impact on the survival/persistence of the bacterial targets of the MST markers, and also alter the ratio between faecal sterols and their biodegradation products, the stanols. It was hypothesised, therefore, that there would be:Changes over time in the concentration of E. coli and the bovine-associated MST markers mobilised into the cowpat runoff Alterations in the FST ratio signature of the ten measured faecal steroids, resulting in a change from a bovine faecal steroid signature in fresh cowpat runoff to other animal faecal signatures in the runoff from decomposing cowpats A difference in the mobilisation decline rates of all FST and microbial markers within a treatment regime and between treatments. Linear regression analysis was undertaken to establish mobilisation decline rates for each of the analytes in the mobilisable phase from the cowpat runoff treatments, with calculation of the time taken in days for reduction in 90% of the concentration (T90), and statistical comparison of the regression coefficients (slopes) of all analytes. The results will include a discussion of the impacts of the study’s observations on the interpretation of faecal indicator assessments for water quality monitoring in waterways influenced by sources of faecal contamination.

A pilot survey was performed to determine the prevalence of Campylobacter jejuni and Campylobacter coli on three age classes (lamb, hogget, and mutton) of ovine carcass trim postdressing and prechill. Sampling of hogget carcasses was undertaken 6 months before sampling of lamb and mutton carcasses. A total of 120 trim samples were collected from 11 processing plants across New Zealand. All samples were enriched and screened using PCR for the presence of C. jejuni and C. coli, and isolation was attempted for all screen-positive samples. Enumeration of Campylobacter from lamb trim samples showed that Campylobacter bacteria were present in very low numbers (

Background We describe the investigation of a Campylobacter outbreak linked to contamination of an untreated, groundwater derived drinking water supply. Methods We analysed epidemiological data collected from clinician-confirmed diarrheal cases and estimated the total burden of Havelock North cases using an age-adjusted cross-sectional telephone survey. Campylobacter isolates from case fecal specimens, groundwater samples, and sheep fecal specimens from paddocks adjacent to the drinking water source were whole genome sequenced. Findings We estimate between 6260 and 8320 cases of illness including up to 2230 who lived outside the reticulation area, were linked to the contaminated water supply. Of these, 953 cases were physician reported, 42 were hospitalized, three developed Guillain-Barre syndrome, and Campylobacter infection contributed to at least four deaths. Of the 12 genotypes observed in cases, four were also observed in water, three were also observed in sheep and one was also observed in both water and sheep. Interpretation The contamination of the untreated reticulated water supply occurred following a very heavy rainfall event which caused drainage of sheep feces into a shallow aquifer. The existence of a routine clinical surveillance system for campylobacteriosis facilitated identification of the outbreak, recovery of clinical isolates, and early testing of the water for pathogens. Genotyping of the Campylobacter jejuni helped define the source of the outbreak and confirm outbreak periods and cases. Expected increases in heavy rainfall events and intensification of agriculture mean that additional safeguards are needed to protect populations from such drinking water outbreaks. Funding NZ Ministry of Health, Health Research Council, ESR SSIF, Royal Society.

In New Zealand, there is substantial potential for microbial contaminants from agricultural fecal sources to be transported into waterways. The flow and transport pathways for fecal contaminants vary at a range of scales and is dependent on chemical, physical and biological attributes of pathways, soils, microorganisms and landscape characteristics. Understanding contaminant transport pathways from catchment to stream can aid water management strategies. It is not practical, however to conduct direct field measurement for all catchments on the fate and transport of fecal pathogens due to constraints on time, personnel, and material resources. To overcome this problem, fecal source tracking can be utilised to link catchment characteristics to fecal signatures identifying critical sources. In this article, we have reviewed approaches to identifying critical sources and pathways for fecal microorganisms from agricultural sources, and make recommendations for the appropriate use of these fecal source tracking (FST) tools.

Four cases of listeriosis in a hospital (A) in New Zealand were identified in 2012. Pulsed-field gel electrophoresis (PFGE) used at the time identified four pulsotypes amongst the clinical isolates. Two of the pulsotypes matched to Listeria monocytogenes isolates obtained from ready-to-eat (RTE) meat samples from a RTE producer tested during a nationwide microbiological survey the month prior. The outbreak investigation confirmed that the RTE producer had supplied product to the hospital and additional testing confirmed the presence of L. monocytogenes in RTE meats from the hospital kitchen. Two further listeriosis cases presented in another hospital (B) with one clinical isolate identified as the same pulsotype as identified for one case in hospital A, but the epidemiology information concluded that the clinical cases from hospital B were not linked to the outbreak. Retrospective whole-genome sequencing confirmed that epidemiologically linked isolates belonging to three different genotypes for clinical cases from hospital A and RTE meats samples from the hospital kitchen differed by 0-1 core-genome locus or single nucleotide polymorphisms (SNP). The use of core-genome multilocus sequence typing and SNP analysis provided a greater degree of discrimination between isolates compared to PFGE. Significance and Impact of the Study This study describes a listeriosis outbreak associated with a hospital in New Zealand and attributed to contaminated ready-to-eat (RTE) meat supplied to the hospital by a single producer. Retrospective whole-genome sequence analysis of outbreak isolates was found to provide a greater degree of discrimination between isolates compared to pulsed-field gel electrophoresis and supported the conclusions made at the time of the outbreak. The multiple genotypes identified from clinical cases and the RTE meats obtained during the outbreak highlight the importance of epidemiological concordance alongside genotyping.

The objective of this study was to undertake a microbiological survey of foods, animal faeces and wastewater samples for Clostridium difficile, and determine the genotypes and antimicrobial susceptibilities of isolates. A total of 211 samples were tested for C. difficile using culture methods. Thirteen toxigenic C. difficile isolates were obtained; ten from wastewater samples, one each from pig and duck faeces and another from a raw meat product. Eight PCR-ribotypes (RTs) were identified, including two novel RTs (878 and 879). Single-nucleotide polymorphism analysis using WGS data for all isolates provided greater discrimination between C. difficile isolates within the same RT and multilocus sequence typing (MLST) profiles. All C. difficile isolates were found to be susceptible to the first-line human antimicrobials used to treat C. difficile infection. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to report the isolation of Clostridium difficile from animals, food and wastewater in New Zealand (NZ) and provides important data with respect to ribotypes and multilocus sequence typing profiles, whole genome sequence and antimicrobial susceptibilities. The results highlight the need for further investigations into the epidemiology of C. difficile in NZ and to elucidate the role of the environmental and food sources as transmission routes of human infection.

Intensive deer farming can cause environmental issues, mainly by its impact on soils and water quality. In particular, there is a risk to the microbial quality of water, as high quantities of suspended sediment and fecal bacteria can enter into water systems. The feces of farmed red deer (Cervus elaphus, n = 206) from Canterbury and Southland, New Zealand, were analyzed with regard to the presence of Campylobacter spp., Escherichia coli, enterococci, and Yersinia spp. Enterococci and E. coli were isolated from all samples, with mean concentrations of 4.5 x 105 (95% CI 3.5 x 10(3), 5.6 10(7)) and 1.3 x 10(8) (95% CI 1.1 x 10(6), 1.5 x 10(10)) per gram of dry feces, respectively. Campylobacter spp. were isolated from 27 fecal samples, giving an overall prevalence of 13.1%. Campylobacter isolation rates were variable within and between regions (Canterbury 7.95% [95% CI 2-14%], Southland 16.95% [95% CI 10-24%]). Five out of 42 composite samples were positive for Yersinia enterocolitica, and one sample for Y. pseudotuberculosis. The overall prevalence ranges on a per-animal basis were therefore 2.43 to 11.17% and 0.49 to 2.91%, respectively. This study is the first to quantify the concentration of Campylobacter spp. present in healthy deer farmed in New Zealand. Deer feces are a potential source of human campylobacteriosis, with all genotypes isolated also previously observed among human cases. The fecal outputs from deer should be regarded as potentially pathogenic to humans and therefore be appropriately managed.

The intensification of dairy farming on the agricultural landscape in New Zealand has raised concerns about pollution sources from dairy fecal runoff into waterways. An important step for mitigation of pollution is the identification of the sources of fecal contamination. An amplicon-based metagenomic assay of the bacterial community mobilised into runoff from decomposing cowpats revealed shifts in the community composition over five and a half months of on-field decomposition. Mobilised fractions from the cowpats showed major bacterial community shifts from the anaerobic bacteria (Clostridiales and Bacteroidales) that dominate the cow rumen and fresh cowpat, to the bacterial Orders of the Actinomycetales, Sphingobacteriales and Flavobacteriales. These bacterial community shifts were in conjunction with summertime conditions of low rainfall and high sunshine hours coupled with reduced water availability. The impacts of these bacterial shifts is discussed in terms of their effects on the indicators used for water quality monitoring: the fecal indicator bacterium, Escherichia coli, and fecal source tracking (FST) markers (host-associated quantitative polymerase chain reaction (qPCR) markers and fecal steroids). The hypothesis that these bacterial community shifts would impact on the steroid ratios used as FST signatures was disproved. The fecal steroids were, therefore, reliable indicators of fecal sources from cowpats, which had been decomposing for up to five and a half months post-deposition. Consistent with the noted bacterial community changes, there were, however, concentration decreases in the bacterial groups targeted by the faecal source qPCR markers, and the E. coli mobilised from decomposing cowpats.

This study explores the relationships between faecal source tracking (FST) markers (quantitative Polymerase Chain Reaction (qPCR) markers and steroids), microbial indicators, the faecal ageing ratio of atypical colonies/total coliforms (AC/TC) and potential human pathogens (Giardia, Cryptosporidium and Campylobacter). Faecal source PCR markers tested were GenBac3, HumM3, HumBac (HF183-Bac708R); Bifidobacterium adolescentis, wildfowl and canine-associated markers. Sediment and water samples from the Avon River were collected during and post-discharge of untreated human sewage inputs, following a series of earthquakes, which severely damaged the Christchurch sewerage system. Significant, positive Spearman Ranks (rs) correlations were observed between human-associated qPCR markers and steroid FST markers and Escherichia coli and F-specific RNA bacteriophage (rs 0.57 to 0.84, p Low levels of fluorescent whitening agents (FWA) (mean 0.06 μg/L, range 0.01–0.40 μg/L) were observed in water throughout the study, but steroids and FWA appeared to be retained in river sediments, months after continuous sewage discharges had ceased. No relationship was observed between chemical FST markers in sediments and the overlying water, and few correlations in sediment between chemical FST markers and target microorganisms. The low values observed for the faecal ageing ratio, AC/TC in water, were significantly, negatively correlated with increasing pathogen detection. This study provides support for the use of the AC/TC ratio, and qPCR and steroid FST markers as indicators of health risks associated with the discharge of raw human sewage into a freshwater system.

This study evaluated the sources of fecal contamination in different river catchments, using a combination of microbial source tracking tools, for human, ruminant, ovine and bovine livestock, in order to define appropriate water management strategies. Every source of waterway pollution was evaluated in river water samples from one urban river catchment and two important farming regions in New Zealand. Fecal pollution was initially measured by testing Escherichia coli and evaluating the presence of human- and ruminant-associated DNA markers of Bacteroidales (BiAdo, BacHum-UCD, BacH, and BacR) and human and ruminant fecal sterols/stanols ratios. Then specific fecal pollution sources were assessed with previously reported quantitative PCR assays targeting human-, bovine-, and ovine-specific viruses: human adenoviruses (HAdV), human JC polyomaviruses, bovine polyomaviruses (BPyV), and ovine polyomaviruses (OPyV). High level of ruminant fecal contamination was detected all over the farming areas, whereas no ruminant sources were identified in the urban river sampling sites. BacR was the most frequently observed ruminant marker and OPyV and BPyV allowed the identification of ovine and bovine fecal sources. The human fecal viral marker (HAdV) was the most frequently observed human marker, highly abundant in the urban sites, and also present in farming areas. This is the first study using simultaneously the ovine and the bovine viral markers to identify and quantify both bovine and ovine fecal pollution.

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.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.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.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.

Intensive deer farming can cause environmental issues, mainly by its impact on soils and water quality. In particular, there is a risk to the microbial quality of water, as high quantities of suspended sediment and fecal bacteria can enter into water systems. The feces of farmed red deer (, = 206) from Canterbury and Southland, New Zealand, were analyzed with regard to the presence of spp., , enterococci, and spp.. Enterococci and were isolated from all samples, with mean concentrations of 4.5 × 10 (95% CI 3.5 × 10, 5.6 10) and 1.3 × 10 (95% CI 1.1 × 10, 1.5 × 10) per gram of dry feces, respectively. spp. were isolated from 27 fecal samples, giving an overall prevalence of 13.1%. isolation rates were variable within and between regions (Canterbury 7.95% [95% CI 2-14%], Southland 16.95% [95% CI 10-24%]). Five out of 42 composite samples were positive for , and one sample for The overall prevalence ranges on a per-animal basis were therefore 2.43 to 11.17% and 0.49 to 2.91%, respectively. This study is the first to quantify the concentration of spp. present in healthy deer farmed in New Zealand. Deer feces are a potential source of human campylobacteriosis, with all genotypes isolated also previously observed among human cases. The fecal outputs from deer should be regarded as potentially pathogenic to humans and therefore be appropriately managed.

FWD-NEXT Expert Panel - Portugal/INSA - Vítor Borges (Instituto Nacional de Saúde Doutor Ricardo Jorge, Lisbon, Portugal) PulseNet International is a global network dedicated to laboratory-based surveillance for food-borne diseases. The network comprises the national and regional laboratory networks of Africa, Asia Pacific, Canada, Europe, Latin America and the Caribbean, the Middle East, and the United States. The PulseNet International vision is the standardised use of whole genome sequencing (WGS) to identify and subtype food-borne bacterial pathogens worldwide, replacing traditional methods to strengthen preparedness and response, reduce global social and economic disease burden, and save lives. To meet the needs of real-time surveillance, the PulseNet International network will standardise subtyping via WGS using whole genome multilocus sequence typing (wgMLST), which delivers sufficiently high resolution and epidemiological concordance, plus unambiguous nomenclature for the purposes of surveillance. Standardised protocols, validation studies, quality control programmes, database and nomenclature development, and training should support the implementation and decentralisation of WGS. Ideally, WGS data collected for surveillance purposes should be publicly available, in real time where possible, respecting data protection policies. WGS data are suitable for surveillance and outbreak purposes and for answering scientific questions pertaining to source attribution, antimicrobial resistance, transmission patterns, and virulence, which will further enable the protection and improvement of public health with respect to food-borne disease. info:eu-repo/semantics/publishedVersion

Shigella is a major foodborne pathogen that infects humans and nonhuman primates and is the major cause of dysentery and reactive arthritis worldwide. This is the initial public release of 16 Shigella genome sequences from four species sequenced as part of the 100K Pathogen Genome Project.

Campylobacter is a food-associated bacterium and a leading cause of foodborne illness worldwide, being associated with poultry in the food supply. This is the initial public release of 202 Campylobacter genome sequences as part of the 100K Pathogen Genome Project. These isolates represent global genomic diversity in the Campylobacter genus.

A series of large earthquakes struck the city of Christchurch, New Zealand in 2010–2011. Major damage sustained by the sewerage infrastructure required direct discharge of up to 38,000 m3/day of raw sewage into the Avon River of Christchurch for approximately six months. This allowed evaluation of the relationship between concentrations of indicator microorganisms (Escherichia coli, Clostridium perfringens and F-RNA phage) and pathogens (Campylobacter, Giardia and Cryptosporidium) in recreational water and sediment both during and post-cessation of sewage discharges. Giardia was the pathogen found most frequently in river water and sediment, although Campylobacter was found at higher levels in water samples. E. coli levels in water above 550 CFU/100 mL were associated with increased likelihood of detection of Campylobacter, Giardia and Cryptosporidium, supporting the use of E. coli as a reliable indicator for public health risk. The strength of the correlation of microbial indicators with pathogen detection in water decreased in the following order: E. coli > F-RNA phage > C. perfringens. All the microorganisms assayed in this study could be recovered from sediments. C. perfringens was observed to accumulate in sediments, which may have confounded its usefulness as an indicator of fresh sewage discharge. F-RNA phage, however, did not appear to accumulate in sediment and in conjunction with E. coli, may have potential as an indicator of recent human sewage discharge in freshwater. There is evidence to support the low-level persistence of Cryptosporidium and Giardia, but not Campylobacter, in river sediments after cessation of sewage discharges. In the event of disturbances of the sediment, it is highly probable that there could be re-mobilisation of microorganisms beyond the sediment–water exchange processes occurring under base flow conditions. Re-suspension events do, therefore, increase the potential risk to human health for those who participate in recreational and work-related activities in the river environment.

Outbreaks of waterborne gastroenteritis continue to occur in developed countries. Darfield, a rural town in the South Island of New Zealand experienced an outbreak of campylobacteriosis following a transgression of Escherichia coli on 16 August 2012. A descriptive outbreak investigation was performed. As a result, 29 cases had a laboratory-confirmed diagnosis of campylobacteriosis and 138 were identified as probable cases. Heavy rains, contamination of water with animal effluent from nearby paddocks and failures in the treatment of drinking water led to pathogens being distributed through the town's water supply. A multi-barrier approach is advocated to ensure the quality of water and many countries have legislation or programmes to address this. Although legislation for water safety plans based on a multi-barrier approach is in place in New Zealand, at the time of the outbreak it was not a requirement for the Darfield water supply. In addition, despite the awareness of the importance of a multi-barrier approach, competing interests, including those from the agricultural industry and financial restraints on water suppliers, can prevent it from being implemented. Governments need to be more willing to enforce legislation and standards to protect the public from waterborne disease.

Specificity testing of two published polymerase chain reaction (PCR) markers for the detection of human faecal pollution, revealed 100% false-positive rates to brush-tailed possum faeces (n = 10), but low false-positive rates against other potential pollution sources. Cross-reaction with possums could be a problem with other human-specific markers; therefore, a possum PCR marker was developed for use in conjunction with human PCR markers. The possum PCR marker was based on Bacteroidales 16S ribosomal ribonucleic acid sequences, and was tested on 233 individual faecal samples from 11 other animal species. Sensitivity of the possum marker in possum faeces (n = 36) was high at 83.3%. Cross-reactivity of the possum marker was limited to black swan (7/20 samples), human (2/48 samples) and rabbit (1/10) faecal samples, all at marker concentrations at least four orders of magnitude lower than possum faeces. The possum marker was not detected in human sewage or the faeces of other animal species. Specificity of the possum PCR marker, therefore, was high at 95.7%. To exclude the possibility that only possum pollution is being detected, additional testing by other faecal source tracking methods is required where the water sample is positive for both human and possum markers.

In environmental microbial forensics, as in other pursuits, statistical calculations are sometimes inappropriately applied, giving rise to the appearance of support for a particular conclusion or failing to support an innately obvious conclusion. This is a reflection of issues related to dealing with sample sizes, the methodologies involved, and the difficulty of communicating uncertainties. In this brief review, we attempt to illustrate ways to minimize such problems. In doing so, we consider one of the most common applications of environmental microbial forensics—the use of genotyping in food and water and disease investigations. We explore three important questions. (i) Do hypothesis tests’ P values serve as adequate metrics of evidence? (ii) How can we quantify the value of the evidence? (iii) Can we turn a value-of-evidence metric into attribution probabilities? Our general conclusions are as follows. (i) P values have the unfortunate property of regularly detecting trivial effects when sample sizes are large. (ii) Likelihood ratios, rather than any kind of probability, are the better strength-of-evidence metric, addressing the question “what do these data say?” (iii) Attribution probabilities, addressing the question “what should I believe?,” can be calculated using Bayesian methods, relying in part on likelihood ratios but also invoking prior beliefs which therefore can be quite subjective. In legal settings a Bayesian analysis may be required, but the choice and sensitivity of prior assumptions should be made clear.

Thirty on-site greywater systems were sampled to determine greywater characteristics and practices in the field. Kitchen greywater was present at eight sites and urine was included at seven sites. These non-traditional sources resulted in significantly higher concentrations of enterococci and 5-day biochemical oxygen demand (BOD5) in greywater. Even with the removal of these sources, the concentrations of microbial indicators indicated high levels of contamination could occur across all greywater sources, including "light" greywater. Using multiple microbial indicators showed that all samples had the potential for faecal contamination. Bacteroidales markers were confirmed in treated greywater and in each greywater source, highlighting the potential for human faecal contamination. Although Escherichia coli was absent in treated greywater recycled to the house, other microbial indicators were present; hence, caution is required in using E. coli concentrations as the sole indicator of microbiological water quality. High BOD5 or total suspended solid concentrations exceeded the levels recommended for effective disinfection. Subsurface irrigation, which is assumed to provide a five-log reduction in exposure, is a suitable reuse option for non-disinfected greywater. Only half the occupants had a good understanding of their greywater systems and 25 % of systems were poorly maintained. Elevated microbial indicator contamination of greywater sludge is a potential hazard during maintenance.

Summary 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−1C. 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.

Freshly excreted droppings from Canada geese (n=80), black swans (n=80), ducks (n=80) and gulls (n=80) were collected from sites around New Zealand. The droppings were enumerated for Escherichia coli, enterococci and Salmonella spp., and for the presence/absence of Cryptosporidium spp. Overall prevalence of E. coli and enterococci in samples was 95% and 94%, respectively. Cryptosporidium spp. was detected in 2% of the samples, whereas no Salmonella spp. were detected in the survey. Preliminary estimates of daily microbial outputs suggest that ducks will produce the highest loadings of E. coli and enterococci per bird, whereas Canada geese will produce the highest loadings of Campylobacter spp. per bird. This study provides the first set of indicator and pathogen counts for one of the largest sources of diffuse faecal contamination of natural waters in New Zealand.

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.

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.

Detection of the faecal pollution contribution from wildfowl is an important adjunct in determining the sources of faecal pollution in waterways. This is particularly true, where human waste and other animal faecal sources have been eliminated as the pollution source. A polymerase chain reaction (PCR) marker was developed as a duck-specific marker of faecal pollution. The semi-nested primer system targeted an unknown bacterium (E2) isolated from mallard ducks. E2 had the closest 16S rRNA sequence similarity to members of the Desulfovibrio genus, which was further confirmed by phenotypic characterisation of the bacterium. Testing of the prevalence of E2 identified the marker in 76% of duck faecal samples (n=42), 20% of swan faecal samples (n=10) and 15% of Canada geese faecal samples (n=20). It was also identified in the faeces of two out of 15 domestic goats (13%). The marker was not detected in any samples derived from human faeces or effluent, dairy cows or sheep. It is proposed that this PCR marker would be useful in conjunction with faecal taxation indicators in the determination of pollution derived from duck faecal inputs in waterways.

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.

This chapter describes the procedure of generating pulsed-field gel electrophoresis (PFGE) profiles (DNA fingerprints) of Shiga toxin-producing Escherichia coli O157:H7 (STEC O157) and non-O157 STEC strains within 48 h, based on the standardized laboratory protocol developed by the Centers for Disease Control and Prevention, USA. The protocol describes the preparation of agarose plugs containing STEC O157 and non-O157 STEC cells, the digestion of bacterial DNA in the plugs using restriction endonuclease enzymes, and the electrophoresis conditions to generate the characteristic PFGE profiles of STEC O157 and non-O157 STEC isolates.

Discrimination of the source of faecal pollution in water bodies is an important step in the assessment and mitigation of public health risk. One tool for faecal source tracking is the analysis of faecal sterols which are present in faeces of animals in a range of distinctive ratios. Published ratios are able to discriminate between human and herbivore mammal faecal inputs but are of less value for identifying pollution from wildfowl, which can be a common cause of elevated bacterial indicators in rivers and streams. In this study, the sterol profiles of 50 avian-derived faecal specimens (seagulls, ducks and chickens) were examined alongside those of 57 ruminant faeces and previously published sterol profiles of human wastewater, chicken effluent and animal meatwork effluent. Two novel sterol ratios were identified as specific to avian faecal scats, which, when incorporated into a decision tree with human and herbivore mammal indicative ratios, were able to identify sterols from avian-polluted waterways. For samples where the sterol profile was not consistent with herbivore mammal or human pollution, avian pollution is indicated when the ratio of 24-ethylcholestanol/(24-ethylcholestanol + 24-ethylcoprostanol + 24-ethylepicoprostanol) is ≥0.4 (avian ratio 1) and the ratio of cholestanol/(cholestanol + coprostanol + epicoprostanol) is ≥0.5 (avian ratio 2). When avian pollution is indicated, further confirmation by targeted PCR specific markers can be employed if greater confidence in the pollution source is required. A 66% concordance between sterol ratios and current avian PCR markers was achieved when 56 water samples from polluted waterways were analysed.

Quantitative real-time PCR is one of the newer methods for measurement of the amount of nucleic material in biological systems. However, reliable measurement requires an appropriate estimation of uncertainty and this paper has developed the uncertainty budget associated with this procedure using as an example, data from a quantitative real-time PCR method for the enumeration of Campylobacter jejuni. This uncertainty is relatively large and for instance, a measured result of 151 units of DNA would have a 95% confidence interval of +/-84 units of DNA with the main sources of uncertainty being the measurement of the threshold cycle (Ct) value, the predicted DNA content of the unknown sample from the calibration line and the molar absorbance value for DNA.

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.

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.

Identifying the source of faecal pollution is important to enable appropriate management of faecal pollution of water. Four independent assays for faecal source discrimination have been developed and implemented in our lab. These assays detect fluorescent whitening agents, faecal sterols, Bifidobacterium adolescentis and Rhodococcus coprophilus. The combination of these indicators is able to identify human derived faecal pollution in rivers containing inputs from septic tanks, municipal oxidation ponds, farmed animals and feral animals.

Both human and non‐human faecal contamination of environmental waters pose a risk to human health that can be reduced if the source can be identified and responsibility for mitigation accepted. Traditional microbiological indicators are rarely adequate for source identification but some chemical indicators offer promise. In this work, the similarities and differences of selected chemical profiles of different faecal effluent types have been modelled using classification and regression tree (CART) analysis. Human effluents can be distinguished from non‐human effluents by the presence of fluorescent whitening agents, and the concentrations or ratios of concentrations of the faecal stanols coprostanol and 24‐ethylcoprostanol can distinguish most of the non‐human effluent types considered. To allow for the effect of variable dilution when effluents enter environmental waters, ratios of concentrations will be required to establish a model, and a model more complex than CART will be required to apporti...

Campylobacteriosis is the most commonly reported form of human bacterial gastroenteritis in the world. Sound identification of infectious sources requires subtyping, but the most widely used methods have turnaround times measured in days and require specialist equipment and skills. A multiplex ligation-dependent probe amplification-binary typing (MBiT) assay was developed for subtyping Campylobacter jejuni and Campylobacter coli . It was tested on 245 isolates, including recent isolates from Belgium and New Zealand, and compared to multilocus sequence typing (MLST). When used in an outbreak setting, MBiT identified the predominant genotype and possible additional cases days before pulsed-field gel electrophoresis (PFGE) results were available. MBiT was more discriminatory than MLST and, being a single assay with results produced within 6 h, was more rapid and cost-effective than both MLST and PFGE. In addition, MBiT requires only basic molecular biology equipment and skills.

This chapter summarizes the various modern genotypic methods available for subtyping the various Campylobacter species, the use of genetic methods for investigating outbreaks of disease, the application and interpretation of large-scale techniques in broader epidemiological studies, and a perspective of future developments in the field. Discussions of three methods (pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and amplified fragment length polymorphism (AFLP)) used to compare relatively large numbers of strains follow in a section aimed at summarizing our knowledge of Campylobacter molecular epidemiology in a broader perspective. Limitations of MLST include cost and the degree of resolution that is possible with the quantity of data provided by seven highly conserved gene fragments. The molecular epidemiology of Campylobacter coli is less well studied, but more recent work offers some insights into host association. Current results suggest that the attribution of C. jejuni and C. coli isolates from human cases to their source will be best undertaken by means of a phylogenetic approach that uses analysis based on allele frequencies, or that follows robust definition of host associated sublineages within the main clonal groups. Molecular epidemiological studies of Campylobacter species have matured considerably since the early applications of plasmid profiling and restriction enzyme analysis in homogeneous electric fields.

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.

Tetranectin is a C-type lectin that occurs in the mammalian musculoskeletal system. In the present report we describe the first studies on an avian tetranectin. A full-length chicken tetranectin cDNA was isolated. Comparison of the deduced amino acid sequence of chicken tetranectin with mouse and human tetranectin showed an identity of 67 and 68%, respectively. Northern blot analysis demonstrated broad expression of chicken tetranectin mRNA, which was first detected on embryonic day 4. Tetranectin protein was detected in chicken serum and egg yolk. Since muscle is one of few tissues in which tetranectin protein is retained, we examined the distribution of tetranectin in various muscle types in chicken. Myofibers strongly positive for tetranectin were observed in several muscles including m. tibialis ant. and m. sartorius (from embryonic day 10 to adult). Using antibodies to fast and slow myosin heavy chains (MHC) and double immunostaining techniques, we found that tetranectin was restricted to slow (type I) muscle fibers. Similarly only slow intrafusal fibers accumulated tetranectin. The pattern of immunostaining in chickens differs markedly from that seen in mouse muscles, indicating that tetranectin performs a role in muscle that is not associated with a hitherto recognized muscle type or function.

The ADAMs (a disintegrin and metalloprotease) family of proteins is involved in a variety of cellular interactions, including cell adhesion and ecto- domain shedding. Here we show that ADAM 12 binds to cell surface syndecans. Three forms of recombinant ADAM 12 were used in these experiments: the cys-teine-rich domain made in Escherichia coli (rADAM 12-cys), the disintegrin-like and cysteine-rich domain made in insect cells (rADAM 12-DC), and full-length human ADAM 12-S tagged with green fluorescent protein made in mammalian cells (rADAM 12-GFP). Mesenchymal cells specifically and in a dose-dependent manner attach to ADAM 12 via members of the syndecan family. After binding to syndecans, mesenchymal cells spread and form focal adhesions and actin stress fibers. Integrin β1 was responsible for cell spreading because function-blocking monoclonal antibodies completely inhibited cell spreading, and chondroblasts lacking β1 integrin attached but did not spread. These data suggest that mesenchymal cells use syndecans as the initial receptor for the ADAM 12 cysteine-rich domain–mediated cell adhesion, and then the β1 integrin to induce cell spreading. Interestingly, carcinoma cells attached but did not spread on ADAM 12. However, spreading could be efficiently induced by the addition of either 1 mM Mn2+ or the β1 integrin–activating monoclonal antibody 12G10, suggesting that in these carcinoma cells, the ADAM 12–syndecan complex fails to modulate the function of β1 integrin.

Immature cotyledons from six cultivars and breeding lines of Pisum sativum were cocultivated with Agrobacterium tumefaciens and selected on a medium containing kanamycin. Transformed plants were recovered with a success rate of between 0.8 and 3.4% of explants treated. The plasmids used were based on pGA643 and have a nos promoter driving an nptII gene. Progeny from the primary transformants showed stable inheritance of the nos-nptII gene.