Steps towards operationalizing One Health approaches.

One Health recognizes the health of humans, agriculture, wildlife, and the environment are interrelated. The concept has been embraced by international health and environmental authorities such as WHO, WOAH, FAO, and UNEP, but One Health approaches have been more practiced by researchers than national or international authorities. To identify priorities for operationalizing One Health beyond research contexts, we conducted 41 semi-structured interviews with professionals across One Health sectors (public health, environment, agriculture, wildlife) and institutional contexts, who focus on national-scale and international applications. We identify important challenges, solutions, and priorities for delivering the One Health agenda through government action. Participants said One Health has made progress with motivating stakeholders to attempt One Health approaches, but achieving implementation needs more guidance (action plans for how to leverage or change current government infrastructure to accommodate cross-sector policy and strategic mission planning) and facilitation (behavioral change, dedicated personnel, new training model).

A cross-sectional investigation of Leptospira at the wildlife-livestock interface in New Zealand.

There has been a recent upsurge in human cases of leptospirosis in New Zealand, with wildlife a suspected emerging source, but up-to-date knowledge on this topic is lacking. We conducted a cross-sectional study in two farm environments to estimate Leptospira seroprevalence in wildlife and sympatric livestock, PCR/culture prevalence in wildlife, and compare seroprevalence and prevalence between species, sex, and age groups. Traps targeting house mice (Mus musculus), black rats (Rattus rattus), hedgehogs (Erinaceus europaeus) and brushtail possums (Trichosurus vulpecula) were set for 10 trap-nights in March-April 2017 on a dairy (A) and a beef and sheep (B) farm. Trapped wild animals and an age-stratified random sample of domestic animals, namely cattle, sheep and working dogs were blood sampled. Sera were tested by microagglutination test for five serogroups and titres compared using a Proportional Similarity Index (PSI). Wildlife kidneys were sampled for culture and qPCR targeting the lipL32 gene. True prevalence in mice was assessed using occupancy modelling by collating different laboratory results. Infection profiles varied by species, age group and farm. At the MAT cut-point of ≥ 48, up to 78% of wildlife species, and 16-99% of domestic animals were seropositive. Five of nine hedgehogs, 23/105 mice and 1/14 black rats reacted to L. borgpetersenii sv Ballum. The sera of 4/18 possums and 4/9 hedgehogs reacted to L. borgpetersenii sv Hardjobovis whilst 1/18 possums and 1/9 hedgehogs reacted to Tarassovi. In ruminants, seroprevalence for Hardjobovis and Pomona ranged 0-90% and 0-71% depending on the species and age group. Titres against Ballum, Tarassovi and Copenhageni were also observed in 4-20%, 0-25% and 0-21% of domestic species, respectively. The PSI indicated rodents and livestock had the most dissimilar serological responses. Three of nine hedgehogs, 31/105 mice and 2/14 rats were carrying leptospires (PCR and/or culture positive). True prevalence estimated by occupancy modelling in mice was 38% [95% Credible Interval 26, 51%] on Farm A and 22% [11, 40%] on Farm B. In the same environment, exposure to serovars found in wildlife species was commonly detected in livestock. Transmission pathways between and within species should be assessed to help in the development of efficient mitigation strategies against Leptospira.

"We don't really do doctors." messages from people diagnosed with occupational leptospirosis for medical professionals on infection, hospitalisation, and long-term effects.

Leptospirosis is largely an occupational disease for people working with livestock in Aotearoa New Zealand. Introduction of livestock vaccination and use of personal protective equipment has been associated with a reduction in the incidence. However, the incidence of occupational leptospirosis remains high, with significant burdens for affected families and healthcare system. For this article, a subset of thirteen participants from a nationwide leptospirosis case-control study (2019-2021) who were diagnosed with leptospirosis and worked with livestock at the time of illness were invited and agreed to a semi-structured interview. Interviewees reflected on their experiences as messages for medical professionals. The analysis of transcripts reveals widely shared experiences with infection, hospitalisation, and treatment, as well as long-term effects and recovery. Conclusions for medical professionals include that ill workers continue to have their diagnosis of leptospirosis delayed. This delay may contribute to more than half the people ill with leptospirosis hospitalised. Further, medical professionals' communication and relationship with ill people strongly colours the latter's experience, for good or for bad. Moreover, most interviewees experienced a recovery process that took several months of feeling tired, which undermined professional performance and emotional wellbeing.

The characterisation of antimicrobial resistant Escherichia coli from dairy calves.

Introduction. Dairy calves, particularly pre-weaned calves have been identified as a common source of multidrug resistant (MDR) Escherichia coli.Gap statement. E. coli strains isolated from dairy calves and the location of their resistance genes (plasmid or chromosomal) have not been well characterised.Aim. To characterise the phenotypic and genotypic features as well as the population structure of antimicrobial-resistant E. coli isolated from calves located on dairy farms that feed waste-milk to their replacement calves.Methodology. Recto-anal swab enrichments from 40 dairy calves (≤ 14 days old) located on four dairy farms were examined for tetracycline, streptomycin, ciprofloxacin, and third-generation cephalosporin resistant E. coli. Whole genome sequencing was performed using both short- and long-read technologies on selected antimicrobial resistant E. coli.Results. Fifty-eight percent (23/40) of calves harboured antimicrobial resistant E. coli: 43 % (17/40) harboured tetracycline resistant, and 23 % (9/40) harboured chromosomal mediated AmpC producing E. coli. Whole genome sequencing of 27 isolates revealed five sequence types, with ST88 being the dominant ST (17/27, 63 % of the sequenced isolates) followed by ST1308 (3/27, 11 %), along with the extraintestinal pathogenic E. coli lineages ST69 (3/27, 11 %), ST10 (2/27, 7 %), and ST58 (2/27, 7 %). Additionally, 16 isolates were MDR, harbouring additional resistance genes that were not tested phenotypically. Oxford Nanopore long-read sequencing technologies enabled the location of multiple resistant gene cassettes in IncF plasmids to be determined.Conclusion. Our study identified a high incidence of tetracycline and streptomycin-resistant E. coli in dairy calves, and highlighted the presence of multidrug-resistant strains, emphasising the need for further investigation into potential associations with farm management practices.

Leptospirosis in Aotearoa New Zealand: Protocol for a Nationwide Case-Control Study.

BACKGROUND: In Aotearoa New Zealand, 90% of patients with notified leptospirosis (a zoonotic bacterial disease) have been men working in agricultural industries. However, since 2008, the epidemiology of notified cases has been gradually changing, that is, more women are affected; there are more cases associated with occupations traditionally not considered high risk in New Zealand; infecting serovars have changed; and many patients experience symptoms long after infection. We hypothesized that there is a shift in leptospirosis transmission patterns with substantial burden on affected patients and their families. OBJECTIVE: In this paper, we aimed to describe the protocols used to conduct a nationwide case-control study to update leptospirosis risk factors and follow-up studies to assess the burden and sources of leptospirosis in New Zealand. METHODS: This study used a mixed methods approach, comprising a case-control study and 4 substudies that involved cases only. Cases were recruited nationwide, and controls were frequency matched by sex and rurality. All participants were administered a case-control questionnaire (study 1), with cases being interviewed again at least 6 months after the initial survey (study 2). A subset of cases from two high-risk populations, that is, farmers and abattoir workers, were further engaged in a semistructured interview (study 3). Some cases with regular animal exposure had their in-contact animals (livestock for blood and urine and wildlife for kidney) and environment (soil, mud, and water) sampled (study 4). Patients from selected health clinics suspected of leptospirosis also had blood and urine samples collected (study 5). In studies 4 and 5, blood samples were tested using the microscopic agglutination test to test for antibody titers against Leptospira serovars Hardjo type bovis, Ballum, Tarassovi, Pomona, and Copenhageni. Blood, urine, and environmental samples were also tested for pathogenic Leptospira DNA using polymerase chain reaction. RESULTS: Participants were recruited between July 22, 2019, and January 31, 2022, and data collection for the study has concluded. In total, 95 cases (July 25, 2019, to April 13, 2022) and 300 controls (October 19, 2019, to January 26, 2022) were interviewed for the case-control study; 91 cases participated in the follow-up interviews (July 9, 2020, to October 25, 2022); 13 cases participated in the semistructured interviews (January 26, 2021, to January 19, 2022); and 4 cases had their in-contact animals and environments sampled (October 28, 2020, and July 29, 2021). Data analysis for study 3 has concluded and 2 manuscripts have been drafted for review. Results of the other studies are being analyzed and the specific results of each study will be published as individual manuscripts.. CONCLUSIONS: The methods used in this study may provide a basis for future epidemiological studies of infectious diseases. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/47900.

An exploratory qualitative enquiry into workers' experiences of leptospirosis and post-leptospirosis in Aotearoa New Zealand.

AIMS: This pilot study describes the experiences of six people who reported post-leptospirosis symptoms. Our aim was to perform an exploratory qualitative study to document participants' experiences and to identify themes to gain understanding of the impact and burden experienced. METHODS: Participants self-recruited, meaning they had directly contacted the first author prior to the study commencing and had offered to tell their stories. Face-to-face semi-structured interviews were conducted in January 2016 and summative content analysis was used to distil themes. RESULTS: The participants were male, had been employed in livestock slaughter plants (n=2) or farming (n=4) when they first contracted leptospirosis and claimed they had been suffering from post-leptospirosis symptoms for 1-35 years. Symptoms included exhaustion, brain fog and mood swings, and participants' lifestyles and relationships were severely affected. Participants and their partners reported poor awareness and knowledge of leptospirosis when they sought help, and that employers and the Accident Compensation Corporation (ACC) were dismissive of post-leptospirosis symptoms. Participants also reported some positive experiences and had advice to share. CONCLUSION: Leptospirosis may have severe long-term consequences for patients, their families and their communities. We recommend that the aetiology, pathogenesis and burden of the persistence of leptospirosis symptoms become topics for future research.

Extended-spectrum ß-lactamase- and AmpC ß-lactamase-producing Enterobacterales associated with urinary tract infections in the New Zealand community: a case-control study.

OBJECTIVES: To assess whether having a pet in the home is a risk factor for community-acquired urinary tract infections associated with extended-spectrum ß-lactamase (ESBL)- or AmpC ß-lactamase (ACBL)- producing Enterobacterales. METHODS: An unmatched case-control study was conducted between August 2015 and September 2017. Cases (n = 141) were people with community-acquired urinary tract infection (UTI) caused by ESBL- or ACBL-producing Enterobacterales. Controls (n = 525) were recruited from the community. A telephone questionnaire on pet ownership and other factors was administered, and associations were assessed using logistic regression. RESULTS: Pet ownership was not associated with ESBL- or ACBL-producing Enterobacterales-related human UTIs. A positive association was observed for recent antimicrobial treatment, travel to Asia in the previous year, and a doctor's visit in the last 6 months. Among isolates with an ESBL-/ACBL-producing phenotype, 126/134 (94%) were Escherichia coli, with sequence type 131 being the most common (47/126). CONCLUSIONS: Companion animals in the home were not found to be associated with ESBL- or ACBL-producing Enterobacterales-related community-acquired UTIs in New Zealand. Risk factors included overseas travel, recent antibiotic use, and doctor visits.

Longitudinal Testing of Leptospira Antibodies in Horses Located near a Leptospirosis Outbreak in Alpacas.

The objectives of this study were to determine if horses located near an outbreak of leptospirosis in alpacas had Leptospira titres indicative of a previous or current infection and, if so, to determine the magnitude in change of titres over time. Further, the objective was to determine if horses with high titre results were shedding Leptospira in their urine. Blood samples were collected from twelve horses located on or next to the farm with the outbreak in alpacas, on day zero and at four subsequent time points (two, four, six and nine weeks). The microscopic agglutination test was used to test sera for five serovars endemic in New Zealand: Ballum, Copenhageni, Hardjo, Pomona and Tarassovi. A reciprocal MAT titre cut-off of ≥1:100 was used to determine positive horses. Seven out of twelve horses (58%) were positive to at least one serovar during one of the time points. Two horses recorded titres of ≥1600, one for both Pomona and Copenhageni and the other for Hardjo, and these two horses were both PCR positive for Leptospira in their urine samples. For five out of seven horses, the titres either remained the same or changed by one dilution across the sampling time points. The study confirmed endemic exposure to five endemic Leptospira serovars in New Zealand in a group of horses located near a confirmed leptospirosis outbreak in alpacas.

Using a Card Sort Technique to Determine the Perceptions of First-Year Veterinary Students on Veterinary Professionalism Attributes Important to Future Success in Clinical Practice.

First-year veterinary students' perceptions on the veterinary professionalism attributes important to future success in clinical practice were explored using a card sort technique. The key findings were that self-oriented attributes (overall mean: 3.20; 42% responses were essential) and people-oriented attributes (overall mean: 3.13; 39% essential) were rated more highly than task-oriented attributes (overall mean: 2.98; 31% essential) (1-4 scale: 1 = irrelevant, 4 = essential). Within these overall ratings, the establishment/maintenance of effective client relationships (people-oriented attribute; mean: 3.84) and the ability to be composed under pressure and recover quickly (self-oriented attribute; mean: 3.82) received the highest scores. The highest task-oriented score was the ability to work to a high standard and achieve results (mean: 3.57). There was no difference between ethnicities or between men and women, but respondents < 20 years of age gave higher scores to people-oriented attributes than did older respondents (≥ 20 years). The use of the card sort technique has not been widely reported in veterinary educational literature, and so this study represents a novel approach to garnering opinions from newly enrolled veterinary students-a group of stakeholders whose views on this subject are seldom sought. The results show that first-year veterinary students have well-developed opinions on the key attributes of veterinary professionalism and indicate that the early development of students' opinions needs to be taken into consideration in the design of professionalism curricula within veterinary programs.

Spread of Nontyphoidal Salmonella in the Beef Supply Chain in Northern Tanzania: Sensitivity in a Probabilistic Model Integrating Microbiological Data and Data from Stakeholder Interviews.

East Africa is a hotspot for foodborne diseases, including infection by nontyphoidal Salmonella (NTS), a zoonotic pathogen that may originate from livestock. Urbanization and increased demand for animal protein drive intensification of livestock production and food processing, creating risks and opportunities for food safety. We built a probabilistic mathematical model, informed by prior beliefs and dedicated stakeholder interviews and microbiological research, to describe sources and prevalence of NTS along the beef supply chain in Moshi, Tanzania. The supply chain was conceptualized using a bow tie model, with terminal livestock markets as pinch point, and a forked pathway postmarket to compare traditional and emerging supply chains. NTS was detected in 36 (7.7%) of 467 samples throughout the supply chain. After combining prior belief and observational data, marginal estimates of true NTS prevalence were 4% in feces of cattle entering the beef supply and 20% in raw meat at butcheries. Based on our model and sensitivity analyses, true NTS prevalence was not significantly different between supply chains. Environmental contamination, associated with butchers and vendors, was estimated to be the most likely source of NTS in meat for human consumption. The model provides a framework for assessing the origin and propagation of NTS along meat supply chains. It can be used to inform decision making when economic factors cause changes in beef production and consumption, such as where to target interventions to reduce risks to consumers. Through sensitivity and value of information analyses, the model also helps to prioritize investment in additional research.

Using the critical incident technique to determine veterinary professional competencies important for enhancing the veterinarian-client interaction.

BACKGROUND: While previous research has examined components of professionalism that veterinary employers and clients expect in veterinarians, little attention has been paid to practising clinical veterinarians' opinions on important professional competencies that help to enhance the veterinarian-client relationship. This study used a phenomenological approach and critical incident technique to gather the narratives of practising veterinarians in New Zealand about positive and negative veterinarian-client interactions. The intention was to identify the underlying principles of professionalism that resulted in the positive/negative outcomes of those interactions. METHODS: Twenty-two practising veterinarians were interviewed, and each respondent was asked to recollect and narrate a significant positive and a significant negative critical incident in their career that involved a veterinarian-client interaction within a clinical setting. The professional competencies that the veterinarian believed contributed to a successful or an unsuccessful outcome were elucidated using thematic analysis. RESULTS: Thematic analysis of the critical incident narratives revealed four major themes under the overarching theme of 'building a relationship between the veterinarian and the client': 'accountability and integrity', 'effective communication skills', 'personal wellbeing' and 'quality of care'. CONCLUSION: The description of the important professional competencies that helped to enhance the veterinarian-client relationship provides a reference for the practising profession to help improve the likelihood of veterinarians enjoying satisfied and successful careers.

Genomic adaptations of Campylobacter jejuni to long-term human colonization.

BACKGROUND: Campylobacter is a genus of bacteria that has been isolated from the gastrointestinal tract of humans and animals, and the environments they inhabit around the world. Campylobacter adapt to new environments by changes in their gene content and expression, but little is known about how they adapt to long-term human colonization. In this study, the genomes of 31 isolates from a New Zealand patient and 22 isolates from a United Kingdom patient belonging to Campylobacter jejuni sequence type 45 (ST45) were compared with 209 ST45 genomes from other sources to identify the mechanisms by which Campylobacter adapts to long-term human colonization. In addition, the New Zealand patient had their microbiota investigated using 16S rRNA metabarcoding, and their level of inflammation and immunosuppression analyzed using biochemical tests, to determine how Campylobacter adapts to a changing gastrointestinal tract. RESULTS: There was some evidence that long-term colonization led to genome degradation, but more evidence that Campylobacter adapted through the accumulation of non-synonymous single nucleotide polymorphisms (SNPs) and frameshifts in genes involved in cell motility, signal transduction and the major outer membrane protein (MOMP). The New Zealand patient also displayed considerable variation in their microbiome, inflammation and immunosuppression over five months, and the Campylobacter collected from this patient could be divided into two subpopulations, the proportion of which correlated with the amount of gastrointestinal inflammation. CONCLUSIONS: This study demonstrates how genomics, phylogenetics, 16S rRNA metabarcoding and biochemical markers can provide insight into how Campylobacter adapts to changing environments within human hosts. This study also demonstrates that long-term human colonization selects for changes in Campylobacter genes involved in cell motility, signal transduction and the MOMP; and that genetically distinct subpopulations of Campylobacter evolve to adapt to the changing gastrointestinal environment.

The epidemiology of AmpC-producing Escherichia coli isolated from dairy cattle faeces on pasture-fed farms.

Introduction. Antibiotic use, particularly amoxicillin-clavulanic acid in dairy farming, has been associated with an increased incidence of AmpC-hyperproducing Escherichia coli.Gap statement. There is limited information on the incidence of AmpC-hyperproducing E. coli from seasonal pasture-fed dairy farms.Aim. We undertook a New Zealand wide cross-sectional study to determine the prevalence of AmpC-producing E. coli carried by dairy cattle.Methodology. Paddock faeces were sampled from twenty-six dairy farms and were processed for the selective growth of both extended-spectrum beta-lactamase (ESBL)- and AmpC-producing E. coli. Whole genome sequence analysis was carried out on 35 AmpC-producing E. coli.Results. No ESBL- or plasmid mediated AmpC-producing E. coli were detected, but seven farms were positive for chromosomal mediated AmpC-hyperproducing E. coli. These seven farms were associated with a higher usage of injectable amoxicillin antibiotics. Whole genome sequence analysis of the AmpC-producing E. coli demonstrated that the same strain (<3 SNPs difference) of E. coli ST5729 was shared between cows on a single farm. Similarly, the same strain (≤15 SNPs difference) of E. coli ST8977 was shared across two farms (separated by approximately 425 km).Conclusion. These results infer that both cow-to-cow and farm-to-farm transmission of AmpC-producing E. coli has occurred.

Of Mice, Cattle, and Men: A Review of the Eco-Epidemiology of Leptospira borgpetersenii Serovar Ballum.

In New Zealand (NZ), leptospirosis is a mostly occupational zoonosis, with >66% of the recently notified cases being farm or abattoir workers. Livestock species independently maintain Leptospira borgpetersenii serovar Hardjo and L. interrogans serovar Pomona, and both are included in livestock vaccines. The increasing importance in human cases of Ballum, a serovar associated with wildlife, suggests that wildlife may be an overlooked source of infection. Livestock could also act as bridge hosts for humans. Drawing from disease ecology frameworks, we chose five barriers to include in this review based on the hypothesis that cattle act as bridge hosts for Ballum. Using a narrative methodology, we collated published studies pertaining to (a) the distribution and abundance of potential wild maintenance hosts of Ballum, (b) the infection dynamics (prevalence and pathogenesis) in those same hosts, (c) Ballum shedding and survival in the environment, (d) the exposure and competency of cattle as a potential bridge host, and (e) exposure for humans as a target host of Ballum. Mice (Mus musculus), rats (Rattus rattus, R. norvegicus) and hedgehogs (Erinaceus europaeus) were suspected as maintenance hosts of Ballum in NZ in studies conducted in the 1970s-1980s. These introduced species are distributed throughout NZ, and are present on pastures. The role of other wildlife in Ballum (and more broadly Leptospira) transmission remains poorly defined, and has not been thoroughly investigated in NZ. The experimental and natural Ballum infection of cattle suggest a low pathogenicity and the possibility of shedding. The seroprevalence in cattle appears higher in recent serosurveys (3 to 14%) compared with studies from the 1970s (0 to 3%). This review identifies gaps in the knowledge of Ballum, and highlights cattle as a potential spillover host. Further studies are required to ascertain the role that wild and domestic species may play in the eco-epidemiology of Ballum in order to understand its survival in the environment, and to inform control strategies.

Risk Factors for Hospitalisation amongst Leptospirosis Patients in New Zealand.

Leptospirosis is a neglected zoonotic disease that is widespread in tropical and subtropical regions such as Oceania, which includes New Zealand. The incidence rate of leptospirosis in New Zealand remains high in comparison to other high-income countries, with over half of the notified patients hospitalised, and the factors associated with hospitalisation are poorly understood. This study aimed to estimate the risk factors for hospitalisation amongst leptospirosis patients using passive surveillance data: notifications from 1 January 1999 to 31 December 2017 extracted from New Zealand's notifiable disease database. There were 771 hospitalised and 673 non-hospitalised patients. Multivariable logistic regression was used to identify risk factors. The year of notification was significantly and positively associated with hospitalisation, with adjusted (adj.) OR 1.03 (95% CI:1.01-1.05). Occupation was significantly associated with hospitalisation, with the adjusted odds of hospitalisation amongst dairy farmers notified with leptospirosis at adj. OR 1.44 (95% CI: 1.02-2.02) times the adjusted odds of hospitalisation amongst farmers that worked with other livestock. Seropositivity for Leptospira interrogans Copenhageni (adj. OR 5.96, 95% CI: 1.68-21.17) and Pomona (adj. OR 1.14, 95% CI: 0.74-1.74)) was more likely to result in hospitalisation when compared to Leptospira borgpetersenii Ballum (baseline). Seropositivity for Leptospira borgpetersenii Hardjo (adj. OR 0.71, 95% CI: 0.49-1.01) and Tarassovi (adj. OR 0.39, 95% CI: 0.23-0.66) was less likely to result in hospitalisation when compared to Ballum (baseline). All the estimates were additionally adjusted for the effect of sex, age, ethnicity, reported occupational exposure, geographical location, reported season, and deprivation status Although passive surveillance data has limitations we have been able to identify that the New Zealand dairy farming population may benefit from a targeted awareness campaign.

Genomic and phenotypic comparison of two Salmonella Typhimurium strains responsible for consecutive salmonellosis outbreaks in New Zealand.

Salmonella enterica serovar Typhimurium DT160 was the predominant cause of notified human salmonellosis cases in New Zealand from 2000 to 2010, before it was superseded by another S. Typhimurium strain, DT56 variant (DT56v). Whole genome sequencing and phenotypic testing were used to compare 109 DT160 isolates with eight DT56v isolates from New Zealand animal and human sources. Phylogenetic analysis provided evidence that DT160 and DT56v strains were distantly related with an estimated date of common ancestor between 1769 and 1821. The strains replicated at different rates but had similar antimicrobial susceptibility profiles. Both strains were resistant to the phage expressed from the chromosome of the other strain, which may have contributed to the emergence of DT56v. DT160 contained the pSLT virulence plasmid, and the sseJ and sseK2 genes that may have contributed to the higher reported prevalence compared to DT56v. A linear pBSSB1-family plasmid was also found in one of the DT56v isolates, but there was no evidence that this plasmid affected bacterial replication or antimicrobial susceptibility. One of the DT56v isolates was also sequenced using long-read technology and found to contain an uncommon chromosome arrangement for a Typhimurium isolate. This study demonstrates how comparative genomics and phenotypic testing can help identify strain-specific elements and factors that may have influenced the emergence and supersession of bacterial strains of public health importance.

Identification of pathogenic Leptospira species and serovars in New Zealand using metabarcoding.

Leptospirosis is a zoonotic disease of global importance. The breadth of Leptospira diversity associated with both human and animal disease poses major logistical challenges to the use of classical diagnostic techniques, and increasingly molecular diagnostic tools are used for their detection. In New Zealand, this has resulted in an increase in positive cases reported nationally that have not been attributed to the infecting serovar or genomospecies. In this study, we used data from all pathogenic Leptospira genomes to identify a partial region of the glmU gene as a suitable locus for the discrimination of the infecting species and serovars of New Zealand-endemic Leptospira. This method can be used in culture and culture-independent scenarios making it flexible for diagnostics in humans, animals, and environmental samples. We explored the use of this locus as a molecular barcoding tool via the Oxford Nanopore Technology (ONT) sequencing platform MinION. Sequences obtained by this method allowed specific identification of Leptospira species in mixed and enriched environmental cultures, however read error inherent in the MinION sequencing system reduced the accuracy of strain/variant identification. Using this approach to characterise Leptospira in enriched environmental cultures, we detected the likely presence of Leptospira genomospecies that have not been reported in New Zealand to date. This included a strain of L. borgpetersenii that has recently been identified in dairy cattle and sequences similar to those of L. mayottensis. L. tipperaryensis, L. dzianensis and L. alstonii.

An evaluation of diagnostic tests in a case series of suspected leptospirosis patients seen in primary care.

AIMS: This study describes 47 presentations of suspected leptospirosis in general practice in New Zealand. Our primary aim was to assess the laboratory diagnosis of leptospirosis in these patients, by comparing polymerase chain reaction (PCR) tests, microscopic agglutination test (MAT) and culture results. METHODS: Patients suspected of leptospirosis were recruited from general practices in the Waikato (n=17) and Wairoa (n=30) between August 2011 and June 2015. Blood and urine samples were tested for leptospirosis at two diagnostic laboratories and one research laboratory using PCR tests, MAT and culture. RESULTS: Forty-seven patients were recruited for this study: 37 during the acute phase of the illness (within 10 days of symptom onset) and 10 after the acute phase. Eleven of the acute phase patients (11/37, 30%) and two of the later phase patients (2/10, 20%) returned positive leptospirosis test results. The 11 acute phase leptospirosis positive patients had the following positive diagnostic tests: PCR and paired MAT (+/- blood culture) (n=3), PCR only (+/- blood culture) (n=4), paired MAT only (n=3) and blood culture only (n=1). Urine PCR (performed only on Wairoa patients) was the only positive test for two of these patients. CONCLUSION: About a quarter of farm workers and meat workers presenting to general practice with flu-like symptoms will have leptospirosis, but they will not be diagnosed unless appropriately tested, and then they may only test positive for some of the tests available. To increase the likelihood of making a diagnosis, clinicians should order multiple laboratory tests, including blood and urine PCR and a paired MAT.

Transmission Dynamics of Shiga Toxin-Producing Escherichia coli in New Zealand Cattle from Farm to Slaughter.

Cattle are asymptomatic carriers of Shiga toxin-producing Escherichiacoli (STEC) strains that can cause serious illness or death in humans. In New Zealand, contact with cattle feces and living near cattle populations are known risk factors for human STEC infection. Contamination of fresh meat with STEC strains also leads to the potential for rejection of consignments by importing countries. We used a combination of PCR/matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) and whole-genome sequencing (WGS) to evaluate the presence and transmission of STEC on farms and in processing plants to better understand the potential pathways for human exposure and thus mitigate risk. Animal and environmental samples (n = 2,580) were collected from six farms and three meat processing plants in New Zealand during multiple sampling sessions in spring of 2015 and 2016. PCR/MALDI-TOF analysis revealed that 6.2% were positive for "Top 7" STEC. Top 7 STEC strains were identified in all sample sources (n = 17) tested. A marked increase in Top 7 STEC prevalence was observed between calf hides on farm (6.3% prevalence) and calf hides at processing plants (25.1% prevalence). Whole-genome sequencing was performed on Top 7 STEC bacterial isolates (n = 40). Analysis of STEC O26 (n = 25 isolates) revealed relatively low genetic diversity on individual farms, consistent with the presence of a resident strain disseminated within the farm environment. Public health efforts should focus on minimizing human contact with fecal material on farms and during handling, transport, and slaughter of calves. Meat processing plants should focus on minimizing cross-contamination between the hides of calves in a cohort during transport, lairage, and slaughter.IMPORTANCE Cattle are asymptomatic carriers of Shiga toxin-producing E. coli (STEC) strains, which can cause serious illness or death in humans. Contact with cattle feces and living near cattle are known risk factors for human STEC infection. This study evaluated STEC carriage in young calves and the farm environment with an in-depth evaluation of six farms and three meat processing plants over 2 years. An advanced molecular detection method and whole-genome sequencing were used to provide a detailed evaluation of the transmission of STEC both within and between farms. The study revealed widespread STEC contamination within the farm environment, but no evidence of recent spread between farms. Contamination of young dairy calf hides increased following transport and holding at meat processing plants. The elimination of STEC in farm environments may be very difficult given the multiple transmission routes; interventions should be targeted at decreasing fecal contamination of calf hides during transport, lairage, and processing.