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To estimate animal-level seroprevalence of Leptospira interrogans serovar Copenhageni and L. borgpetersenii serovars Ballum and Tarassovi, in beef cattle, sheep and deer on New Zealand farms, and herd/flock-level seroprevalence of any serovar when existing same-sera data for serovars Hardjobovis and Pomona were included, and to determine associations between risk factors and animal-level seroprevalence. Banked sera from sheep (n = 82), beef (n = 54) and deer (n = 62) herds/flocks (n = 3,878 animals) from seven regions were analysed using the microscopic agglutination test. Titres of ≥48 were designated positive. Herds/flocks were considered positive if either ≥1, ≥2 or ≥3 animals were positive. Existing same-sera data for serovars Hardjobovis and Pomona were included to establish farm-level any-serovar seropositivity. Factors associated with serological status were analysed using generalised estimating equations. Animal-level seroprevalence for serovars Ballum, Copenhageni, and Tarassovi, respectively, was 13.7 (95% CI = 11.7–16.0)%, 12.6 (95% CI = 10.6–14.7)% and 18.0 (95% CI = 15.7–20.5)% for beef cattle, 10.5 (95% CI = 9.0–12.1)%, 16.7 (95% CI = 14.9–18.6)% and 14.0 (95% CI = 12.4–15.8)% for sheep and 6.6 (95% CI = 5.3–8.2)%, 15.5 (95% CI = 13.5–17.7)% and 3.6 (95% CI = 2.7–4.8)% for deer, respectively. Herd/flock-level seroprevalence for Ballum was 86.6, 52.4 and 39.0% for sheep, 85.2, 52.7 and 33.3% for beef cattle and 50.8, 27.9 and 21.3% for deer at definitions ≥1, ≥2 and ≥3 seropositive animals per species, respectively. For Copenhageni, corresponding data were 95.1, 73.2 and 56.1% for sheep, 68.5, 48.2 and 29.6% for beef cattle and 73.8, 57.4 and 41.0% for deer, and for Tarassovi, 80.5, 59.7 and 45.1% for sheep, 83.3, 68.5 and 61.1% for beef cattle, and 42.6, 16.4 and 4.9% for deer. Seropositivity to all serovars was observed from all regions, with some differences in seroprevalence observed between species and regions, but not between islands. Combining with Hardjobovis and Pomona data, herd/flock-level seropositivity for all animal species and all five Leptospira serovars was 100% at definition ≥1 animal positive, and 97.5 and 96.3% for sheep flocks, 87.8 and 97.8% for beef cattle herds, and 89.3 and 75% for deer herds at ≥2 and ≥3 animals positive, respectively. Seropositivity to serovars Ballum, Copenhageni and Tarassovi is common in sheep, beef cattle and deer New Zealand and most, or all farms have ≥1 livestock species seropositive to ≥1 serovar. Serovars Ballum, Tarassovi and Copenhageni should be considered when clinical or subclinical signs of leptospirosis are observed in sheep, beef cattle or deer. Livestock sector workers are potentially at risk of exposure. [ABSTRACT FROM AUTHOR]

AIMS: To determine within-farm prevalence, longitudinal pattern of exposure measured by serology, antibody titre longevity and point prevalence of shedding in urine ofLeptospira borgpeterseniiserovar Hardjo andL. interrogansserovar Pomona in naturally infected sheep on a sample of commercial farms in New Zealand. METHODS: On eight commercial sheep farms, between September 2011 and January 2014, blood samples were collected from 115–217 ewe lambs on each farm, at intervals of 2–11 months. They were analysed by microscopic agglutination test (MAT) for antibodies toL. borgpeterseniiserovar Hardjo andL. interrogansserovar Pomona, using a titre cut-point of 48. Urine from 98 animals was tested by quantitative PCR (qPCR). The half-life of antibodies was estimated in 185 sheep for serovar Hardjo and 21 for Pomona, and the seroprevalence and mean titre of animals lost to follow-up was compared with those remaining in the study. RESULTS: Within-flock seroprevalence for serovar Hardjo reached a maximum at 17–22 months of age, ranging from 79 to 100%. Seroprevalence for serovar Pomona rose above 10% on three farms and increased to 21–54% by 4–14 months. Seroconversions occurred mainly from late autumn to early summer at 7–15 months of age. Seroprevalences ranging from 3 to 76% for serovar Hardjo and 0.5 to 15% for serovar Pomona were observed up to 3 months of age, likely due to maternally derived immunity. The half-life of antibody in response to infection was estimated to be 6.7 (95% CI=5.8–7.9) months for serovar Hardjo and 6.3 (95% CI=4.8–9.0) months for Pomona. The prevalence of sheep with urine positive for leptospires on qPCR on each farm ranged from 11 to 88%. All but one of the qPCR-positive animals were seropositive for serovar Hardjo. On two farms where Pomona exposure was observed, animals that were lost to follow-up had a higher geometric mean titre for serovar Pomona than those remaining in the study. CONCLUSIONS: This study demonstrated seasonal exposure from autumn to early summer in young sheep, a wide range of within-flock serological and shedding prevalence, and gives an estimation of the half-life of MAT titres in sheep. More extensive data are needed to fully understand the epidemiology of leptospirosis in sheep flocks across New Zealand and, along with economic analysis, to justify and design cost-effective and efficient control measures to protect human and animal health. [ABSTRACT FROM AUTHOR]

AIMS: To determine the seroprevalence and quantify putative risk factors for exposure to leptospirosis both within and outside the veterinary curriculum among undergraduate veterinary students at Massey University, New Zealand. METHODS: A cross-sectional study was conducted from September 2010 to November 2011. In total, 302 students were blood sampled, with serum tested by microscopic agglutination test (MAT) for antibodies toLeptospira borgpeterseniiserovars Hardjobovis,Leptospira interrogansPomona andLeptospira borgpeterseniiBallum. Information on demographic characteristics, potential exposure within and outside the veterinary curriculum in the previous 18 months, and previous leptospirosis-like clinical history were recorded using an online questionnaire. RESULTS: All students were MAT negative for each serovar, using a cut-point of ≥1:48. Potential exposure to animal urine within and outside the veterinary curriculum was reported by 259/302 (85.8%) and 150/302 (49.7%) of the students, respectively. The median number of potential exposures to animal urine by each student within the veterinary curriculum in the previous 18 months was 63 (min 1, max 155). The other potential exposures among respondents included home slaughter (63/302; 20.9%), hunting (43/302; 14.2%) and outdoor activities involving exposure to fresh water (241/302; 79.8%). CONCLUSIONS: This study demonstrated that these veterinary students were at low risk of contracting leptospirosis, despite frequent exposure to potential sources of infection. The findings in this study contribute to a broader understanding of the occupational risk of leptospirosis. Data describe the level of animal exposure in veterinary students, which can support other zoonotic disease studies in this group. [ABSTRACT FROM PUBLISHER]

The article investigates the prevalence of Leptospira serovars bacterium among the deer, sheep and cattle populations in the North Island of New Zealand. The two main bacterium focused on are Hardjo-bovis and Pomona, and the researchers found that both of these strands were widely distributed in the deer population of the North Island, and assert that one possible cause is the co-grazing of deer with local sheep.

The article discusses a study of the growth response and shedding of Leptospira spp. in urine following vaccination against leptospirosis in young farmed red deer under pastoral conditions in New Zealand. The researchers enrolled 230 female and 205 male, three-month-old deer from five farms and treated them with streptomycin. They found an induced agglutinating antibody (AA) against Leptospira serovar (LS) Hardjo-bovis in 57 vaccinated deer and an induced AA against LS Pomona in 97 deer.

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., Competing Interests: Nil, (© PMA.)

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.

A cross-sectional survey was conducted to determine the seroprevalence of Leptospira in a cohort of horses and to evaluate potential risk factors for Leptospira seropositivity in horses in New Zealand. The convenience sample included 499 Thoroughbred racing and breeding horses from 25 commercial properties in North Island, New Zealand. A questionnaire was used to collect demographic data on horses and property-level information on grazing and management practices, pest (rodent) management, access to natural waterways, other livestock on the property, and possible contact with wildlife. The microscopic agglutination test was used to test sera for serovars Ballum, Copenhageni, Hardjo (bovis), Pomona, and Tarassovi. Logistic regression was used to investigate the risk factors for Leptospira seropositivity to at least one serovar and for each serovar individually. A total of 124 (25%, 95% confidence interval (CI) 21-29%) horses had positive titres to any one of the five serovars. The seroprevalence of Ballum, Copenhageni, Hardjo (bovis), Pomona, and Tarassovi was 5% (95% CI 3-7%), 9% (95% CI 7-12%), 6% (95% CI 4-8%), 6% (95% CI 4-8%), and 6% (95% CI 4-8%), respectively. Broodmares, compared to racehorses and alternately grazing horses with sheep, increased the odds of exposure to any one serovar, whilst grazing the same time as sheep and alternately grazing horses with cattle increased the odds of exposure to Ballum and Hardjo (bovis), respectively. Historical exposure to Leptospira in racing and breeding horses was identified, and risk factors were consistent with pasture-based exposure.

Background: Ross River virus (RRV) is a zoonotic alphavirus transmitted by several mosquito species. Until recently, endemic transmission was only considered possible in the presence of marsupial reservoirs., Methods: RRV seroprevalence was investigated in placental mammals (including horses, cows, goats, pigs, dogs, rats, and mice) in Fiji, where there are no marsupials. A total of 302 vertebrate serum samples were collected from 86 households from 10 communities in Western Fiji., Results: Neutralizing antibodies against RRV were detected in 28% to 100% of sera depending on the species, and neutralization was strong even at high dilutions., Conclusions: These results are unlikely to be due to cross-reactions. Chikungunya is the only other alphavirus known to be present in the Pacific Islands, but it rarely spills over into non-humans, even during epidemics. The study findings, together with a recent report of high RRV seroprevalence in humans, strongly suggest that RRV is circulating in Fiji in the absence of marsupial reservoirs. Considering that all non-human vertebrates present in Fiji are pan-global in distribution, RRV has the potential to further expand its geographic range. Further surveillance of RRV and access to RRV diagnostics will be critical for the early detection of emergence and outbreaks., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Human leptospirosis mainly affects people in close occupational contact with domestic livestock and their products in New Zealand. The disease has an unquantified impact on both human health and animal production in the country. This study aimed to estimate the burden of leptospirosis in terms of disability-adjusted life years (DALYs) and cost associated with loss due to absence from work, treatment of disease, animal production loss and cost of vaccination., Methods: Previously published studies of abattoir workers farmers, and veterinarians, reporting annual risks of influenza-like illness attributable to Leptospira infection, were used to estimate the expected number of cases in a year. The cost of lost animal production was based on results of observational studies in beef cattle, sheep and deer conducted in New Zealand., Results: Expected median annual number of severe and mild cases of human leptospirosis was 2,025 (95% probability interval [95% PI] 1,138-3,422). Median annual DALYs were 0.42 (95% PI: 0.06-2.40) per 100,000 people for the entire population, and 15.82 (95% PI: 2.09-90.80) per 100,000 people working in at-risk occupations (i.e. abattoir workers, farmers and veterinarians). Human infection resulted in a median cost of 4.42 (95% PI: 2.04-8.62) million US dollars (USD) due to absence from work and disease treatment. Median production loss cost in beef cattle, sheep and deer was USD 7.92 (95% PI: 3.75-15.48) million, while median vaccination cost in cattle, (including dairy), sheep and deer was USD 6.15 (95% PI: 5.30-7.03) million. Total annual cost of leptospirosis plus vaccination was USD 18.80 (95% PI: 13.47-27.15) million, equivalent to USD 440,000 (95% PI: 320,000-640,000) per 100,000 people., Conclusion: This study provides an estimate of the disease burden and cost of leptospirosis in New Zealand that could support occupational health authorities and livestock industries in assessing interventions for this disease., (© 2019 Blackwell Verlag GmbH.)

In 2006, New Zealand had the highest notification rate of campylobacteriosis in the world, and poultry was considered the leading source of campylobacteriosis. Implementation of food safety interventions by the poultry industry led to a decrease in the campylobacteriosis notification rate. The aim is to examine the impact of targeted food safety interventions implemented by the New Zealand poultry industry on the source attribution of Campylobacter jejuni infections in a sentinel region. Campylobacter jejuni isolates collected from the Manawatu region of New Zealand between 2005 and 2007 ("before intervention") and 2008 and 2015 ("after intervention") from human clinical cases, chicken meat, ruminant feces, environmental water, and wild bird sources were subtyped by multilocus sequence typing. Viable counts of Campylobacter spp. from carcasses were analyzed using a zero-inflated Poisson regression model. In the period before intervention, sequence type 474 (ST-474) was the most common sequence type (ST) recovered from human cases, accounting for 28.2% of the isolates. After intervention, the proportion of human cases positive for ST-474 reduced to 9.3%. Modeling indicated that chicken meat, primarily from one supplier, was the main source of C. jejuni infection in the Manawatu region before intervention. However, after intervention poultry collectively had a similar attribution to ruminants, but more human cases were attributed to ruminants than any single chicken supplier. Viable counts on carcasses were lower in all poultry suppliers after intervention. This study provides evidence of changes in the source attribution of campylobacteriosis following targeted food safety interventions in one sector of the food supply chain. IMPORTANCE This study provides a unique insight into the effects of food safety interventions implemented in one sector of the food industry on the transmission routes of a major foodborne agent. Following the implementation of food safety interventions by the poultry industry, shifts in the molecular epidemiology of Campylobacter jejuni infections in a sentinel region of New Zealand were observed. Targeted interventions to reduce disease incidence are effective but require continued surveillance and analysis to indicate where further interventions may be beneficial., (Copyright © 2020 American Society for Microbiology.)

Campylobacter jejuni , a leading cause of gastroenteritis worldwide, has been frequently isolated from recreational rivers and streams in New Zealand, yet the public health significance of this is unknown. This study uses molecular tools to improve our understanding of the epidemiology and sources of Campylobacter in recreational waterways, with a view to preventing human infection. Epidemiological and microbiological data were collected between 2005 and 2009 from six high-use recreational waterways in the Manawatu-Wanganui region of the North Island. Campylobacter spp. and C. jejuni were isolated from 33.2% and 20.4% of 509 samples, respectively. Isolation of Campylobacter was observed in both low and high river flows. After adjusting for the confounding effects of river flow, there was a significantly higher likelihood of isolating Campylobacter in the winter month of June compared to January. A high diversity of C. jejuni multilocus sequence types was seen, with the most commonly isolated being the water rail-associated ST-2381 (19/91 isolates [20.9%]), ST-1225 (8/91 isolates [8.8%]), and ST-45 (6/91 isolates [6.6%]). The ST-2381 was found in all rivers, while the most commonly isolated ST from human cases in New Zealand, the poultry-associated strain ST-474, was isolated only in one river. Although the majority of Campylobacter sequence types identified in river water were strains associated with wild birds that are rarely associated with human disease, poultry and ruminant-associated Campylobacter strains that are found in human infection were also identified and could present a public health risk. IMPORTANCE In 2016, there was a large-scale waterborne outbreak of campylobacteriosis in New Zealand, which was estimated to have affected over 5,000 people. This highlighted the need for a greater understanding of the sources of contamination of both surface and groundwater and risks associated with exposure to both drinking and recreational water. This study reports the prevalence and population structure of Campylobacter jejuni in six recreational waters of the Manawatu-Wanganui region of New Zealand and models the relationship between Campylobacter spp. and ruminant-associated Campylobacter and the parameters "sites," "months," and "river flow." Here, we demonstrate that both low and high river flows, month of the year, and recreational sites could influence the Campylobacter isolation from recreational waters. The presence of genotypes associated with human infection allowed us to describe potential risks associated with recreational waters., (Copyright © 2019 American Society for Microbiology.)

An epidemiological investigation was conducted in an unvaccinated dairy farming enterprise in which three workers on one of the milking herds (Herd 1) were diagnosed with leptospirosis due to serovars Hardjo (H) (n = 2) and Pomona (P) (n = 1) between January and March 2015. Blood and urine samples were collected from milking cows in Herd 1 (N = 230) and Herd 2 (N = 400), rising one- (R1, N = 125) and rising two-year-old (R2, N = 130) replacement heifers, and four pigs associated with Herd 1, in March 2015. Sera were tested using the MAT for serovars H, P, Copenhageni (C), Ballum (B) and Tarassovi (T), and urine samples were tested by qPCR. Seventy-five per cent of 109 cows in Herd 1 and 36% of 121 in Herd 2 were seropositive (≥48), predominantly to H and P, and 23% of 74 cows in Herd 1 and 1% of 90 cows in Herd 2 were qPCR positive. Fifty-five per cent of 42 R2 heifers were seropositive to T. No R1 and 17% of 42 R2 heifers were qPCR positive. Subsequently, all cattle were vaccinated for H and P, and Herds 1 and 2 were given amoxicillin. After the booster vaccination, 7% of 91 in Herd 1, 2% of 82 in Herd 2 and 11% of 38 R1 heifers (sampled as R2) were PCR positive. After the amoxicillin treatment, no cows in Herd 1 and 5% of 62 cows in Herd 2 were urine PCR positive. Calves and pigs were seropositive to H, P, C and B. Vaccination and antibiotic treatment appeared effective in reducing the risk of exposure of workers to vaccine serovars. However, evidence of non-vaccine serovars indicated that workers likely remain at risk of exposure to Leptospira., (© 2019 Blackwell Verlag GmbH.)

In New Zealand, up to 97% of NZ sheep flocks are seropositive to Leptospira borgpetersenii serovar Hardjo and/or Leptospira interrogans Pomona, yet vaccination is rare. This study evaluated the impact of exposure to these serovars and of vaccination on sheep growth. One third of 2260 ewe lambs on eight farms were randomly selected and vaccinated with a primary and booster bivalent Hardjo and Pomona vaccine starting at one month of age on seven farms and at around five months of age on one farm. Repeated blood samples were taken over one (n = 6 farms, bred as ewe lambs at 7-8 months of age) or two (n = 2 farms, bred as rising 2-year-old ewes) years and tested by microscopic agglutination test to assess exposure to Hardjo and Pomona. Individual weights were recorded at the same time and modelled using a multilevel linear model accounting for within-farm clustering and repeated measures. Predicted average weights were computed and compared based on the vaccination status and within the control group based on exposure status (positive for Hardjo only, Pomona only, Hardjo and Pomona and negative) for each combination of farm and weighing episode. Statistical significance of the comparison was evaluated after adjustment for multiple comparisons. There was no difference in average weight between vaccinated and control sheep before or after vaccination in any of the flocks. The comparison between sheep seropositive for either or both serovars and seronegative sheep was inconclusive, with variations of direction and magnitude of the difference between farms and weighing episodes. In the absence of an overall growth response to vaccination, widespread adoption of vaccination would unlikely yield an economic response at the industry level. However, the inconsistency observed when comparing animals based on their exposure status suggests that the actual effect of leptospirosis on growth is difficult to predict. A study of the effect on sheep reproduction is needed to fully assess the effect of vaccination on sheep production., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In New Zealand, the major risk factor for campylobacteriosis has been identified as poultry consumption. New Zealanders consume different types of chicken meat which undergo different processing before entering the retail chain. The manipulations and jointing of chicken carcasses into pieces and the subsequent processing and packaging have the potential to cross-contaminate and reshuffle bacterial pathogens among the different products sold. The aim of this study was to analyse: (a) the differences in the viable count and population genetic structure between Campylobacter isolated from chicken drumsticks and whole carcass meat for retail sale over a 1-year period; and (b) the genetic relatedness of human and chicken isolates collected concurrently. Enumeration of Campylobacter was performed using a spiral plater combined with manual spread plating. Campylobacter isolates were identified by polymerase chain reaction and typed by multilocus sequence typing (MLST). C. jejuni was the dominant species among both whole carcasses (63.5%) and drumsticks samples (73.8%), followed by C. coli (27% and 23.1%, respectively). After sample weight adjustment, whole carcasses showed significantly higher Campylobacter counts than drumsticks, with a significant difference in the counts between the commercial suppliers in both types of retail meat. MLST revealed 28 different sequence types among the two types of meat. Using permutational multivariate analysis of variance, statistically significant differences in the population genetic structures were observed between different suppliers but were not observed between the two types of chicken retail meat. In conclusion, we found differences in Campylobacter viable counts, suggesting consumption of whole carcasses may determine an exposure to a higher number of Campylobacter bacteria than consumption of chicken drumsticks. The Campylobacter population genetic structure did not differ between the two types of chicken retail meat. Therefore, source attribution studies based on MLST are unlikely to be biased by the selection of these types of retail meat during sampling., (© 2018 Blackwell Verlag GmbH.)

Most New Zealand sheep flocks are seropositive to Leptospira serovars Hardjo and/or Pomona, yet vaccination is rare. This study evaluated the impact of exposure to these serovars and of vaccination, on primiparous one- (P1) and two-year-old (P2) sheep reproduction outcomes. The study was designed as a split-flock vaccination trial, with a third of the animals vaccinated starting at one month of age. Reproduction outcomes were the proportion of bred P1 (7 months old) and as P2 (19 months old) scanned pregnant, the proportion of pregnant ewes rearing a lamb to tail docking and the proportion of docked lambs that were weaned. Odds ratios and their 95% confidence intervals were calculated to compare reproductive performance between vaccinated and control sheep, and within the control group, between seropositive and seronegative sheep. Odds ratios (OR) were also calculated to assess the relationship between vaccination and loss to follow-up. There was no difference in pregnancy and docking rates between vaccinated and control sheep, or between seropositive and seronegative sheep. P1 with a Hardjo titre ≥1536 were significantly less likely (OR = 0.41, 95%CI = 0.19-0.93) to keep a lamb between docking and weaning than P1 with both Hardjo and Pomona titres <1536, for an observed difference in weaning rate of up to 22.6% points on one farm. A reduction of weaning rates in 2-tooths seropositive for Pomona alone and both Hardjo and Pomona was observed but this was non-significant, possibly because of a lack of power. No difference in weaning rate was observed between vaccinated and control P1 or P2. On one farm vaccinated P1 were less likely to be lost to follow-up (OR = 0.27, 95% CI 0.08 to 0.95) between breeding and weaning. Comparing reproductive performance of vaccinated and control sheep revealed no significant difference. However, comparing exposed and non-exposed ewes revealed a possible adverse effect of Leptospira on weaning rates. This suggests that a full vaccination program may result in an improvement of reproductive outcomes, possibly by providing herd immunity., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Leptospirosis is a zoonosis often associated with occupational exposure from livestock that can be prevented by animal vaccination. Several trials have assessed vaccine efficacy in livestock but there have been no attempts to evaluate these trials jointly. This systematic review and meta-analysis aimed to estimate vaccine efficacy to prevent urinary shedding of Leptospira serovar Hardjo (Hardjo) in cattle. Three databases were used to search for relevant papers published from 1980 to 2015 evaluating commercial vaccines to prevent urinary shedding of leptospires after artificial conjunctival or natural challenge. A total of 1237 articles were initially identified. Eight articles containing information from nine trials that assessed vaccine efficacy to prevent Hardjo urinary shedding, as per bacteriological culture, were included in the meta-analysis. Fixed effects Mantel-Haenszel (MH) and a Bayesian random effects meta-analyses were used to estimate the efficacy of vaccination to prevent Hardjo shedding in urine. Vaccine efficacy against Hardjo challenge was 88.7% (95% CI 81.0%-93.2%) in the MH meta-analysis and 89.9% (95% probability interval 80.6%-94.9%) in the Bayesian random effects meta-analysis. There was no evidence of heterogeneity of study results (p = 0.17). The estimated vaccine efficacy to prevent urinary shedding of Hardjo in cattle may be sufficient to reduce disease incidence in animals and exposure risk of people working in close contact with cattle., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Leptospirosis is a global zoonosis that in New Zealand affects primarily people occupationally exposed to livestock. The objective of this study was to estimate the seroprevalence of five Leptospira serovars in farmers working on cattle, sheep and deer farms that had the serological status of animals previously assessed and to identify risk factors for farmer seropositivity. A total of 178 farmers from 127 properties participated in the study. Blood samples were tested using the microscopic agglutination test (MAT) for the presence of antibodies to Leptospira. Samples with a MAT titre ≥48 were considered seropositive. Using Bayesian statistical analysis, the median seroprevalence of Leptospira, all serovars combined, was estimated to be 6.6% (95% probability interval (PI) 3.6-10.9%). Risk factors associated with seropositivity were assisting deer or cattle calving, farming deer, having ≥25% of flat terrain and high abundance of wild deer on farm, while high possum abundance on farm was negatively associated with seropositivity. No association was observed between farmer serostatus and previously recorded livestock serology. Leptospira seropositivity was associated with influenza-like illness of farmers (RR = 1.7; 95% PI 1.0-2.5). Assuming a causal relationship, this suggested an annual risk of 1.3% (95% PI 0.0-3.0%) of influenza-like illnesses due to Leptospira infection in the population of farmers. The association between seropositivity and disease can be used to estimate the public health burden of leptospirosis in New Zealand. Identifying and understanding risk factors for Leptospira seropositivity can inform preventive measures, hence contributing to the reduction of leptospirosis incidence in farmers., (© 2016 Blackwell Verlag GmbH.)

L. borgpetersenii serovar Hardjo and L. interrogans serovar Pomona are endemic in New Zealand sheep. An effective vaccine and vaccination strategy would protect both humans and livestock. Four to 12 lambs were selected from each of eight farms (total=84, vaccinated group), while four to 16 lambs (total=98) served as unvaccinated controls. A commercial Hardjo/Pomona vaccine was given at 1-6 weeks of age, 5-11 weeks later and 33-67 weeks later on seven farms and at 18 weeks of age and 5 weeks later on the eighth farm. Vaccinates and controls were grazed together. Blood was regularly collected from the control group to assess flock exposure. Urine was collected from both groups 26-82 weeks after the second vaccination and tested by quantitative PCR. Seroprevalence in controls at the time of urine sampling ranged from 2.7 to 98.2% for Hardjo and from 0 to 54.1% for Pomona with seroconversion occurring 13 to 67 weeks after the second vaccination in all but one farm where exposure had happened by the time of vaccination. The shedding prevalence adjusted for clustering in farms was 45.1% [95% CI 17.6-72.7] (for an observed number of 50/98) in the control animals and 1.8% [95% CI 0.0-10.1] (for an observed number of 5/84) in the vaccinated animals. The vaccine was 100% effective on five farms where animals were vaccinated before 12 weeks of age and before natural exposure occurred, but the effectiveness was 80% [0-97] on one farm where the lambs were exposed before vaccination and 65% [9-87] to 80% [0-97] on one farm where the animals were fully vaccinated by 24 weeks of age. The overall vaccine effectiveness was 86.3% [63.6-94.8%] despite maternal antibodies in some flocks at first vaccination. Vaccination timing seemed to be crucial in achieving optimum reduction in shedding in urine in vaccinated sheep., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Unlabelled: Campylobacteriosis is one of the most important foodborne diseases worldwide and a significant health burden in New Zealand. Campylobacter jejuni is the predominant species worldwide, accounting for approximately 90% of human cases, followed by Campylobacter coli Most studies in New Zealand have focused on C. jejuni; hence, the impact of C. coli strains on human health is not well understood. The aim of this study was to genotype C. coli isolates collected in the Manawatu region of New Zealand from clinical cases, fresh poultry meat, ruminant feces, and environmental water sources, between 2005 and 2014, to study their population structure and estimate the contribution of each source to the burden of human disease. Campylobacter isolates were identified by PCR and typed by multilocus sequence typing. C. coli accounted for 2.9% (n = 47/1,601) of Campylobacter isolates from human clinical cases, 9.6% (n = 108/1,123) from poultry, 13.4% (n = 49/364) from ruminants, and 6.4% (n = 11/171) from water. Molecular subtyping revealed 27 different sequence types (STs), of which 18 belonged to clonal complex ST-828. ST-1581 was the most prevalent C. coli sequence type isolated from both human cases (n = 12/47) and poultry (n = 44/110). When classified using cladistics, all sequence types belonged to clade 1 except ST-7774, which belonged to clade 2. ST-854, ST-1590, and ST-4009 were isolated only from human cases and fresh poultry, while ST-3232 was isolated only from human cases and ruminant sources. Modeling indicated ruminants and poultry as the main sources of C. coli human infection., Importance: We performed a molecular epidemiological study of Campylobacter coli infection in New Zealand, one of few such studies globally. This study analyzed the population genetic structure of the bacterium and included a probabilistic source attribution model covering different animal and water sources. The results are discussed in a global context., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

This study assessed seroprevalence and risk factors for Leptospira (serovars Hardjo, Pomona, Ballum, Copenhageni, Tarassovi) exposure in New Zealand veterinarians. Veterinarians (n = 277) at one of two conferences were voluntarily enrolled and blood samples taken. Microscopic agglutination test (MAT) titres ⩾48 were considered seropositive. Fourteen veterinarians (5·1%, 95% confidence interval 2·8-8·3) were seropositive to Leptospira. Home slaughter of cattle or pigs were significant risk factors for Leptospira exposure. There were no clear relationships between the animal species handled at work and serostatus. However, veterinarians spending a 'mid to high' proportion of their time (>50% to ⩽75%) with pets had higher odds of being seropositive than those not working with pets. A borderline positive association (P = 0·09) was observed between seropositivity and clinical influenza-like illness (⩾3 days off work) in the 18 months before the study. Assuming causality, this suggests that 8·3% of these cases may be attributed to Leptospira exposure.

A cross-sectional study was carried out on sheep and cattle slaughtered at a New Zealand abattoir from September to November 2010 to investigate the supplier-specific shedding rate, renal carriage rate and seroprevalence of leptospires. In the 2008/2009 season, this abattoir experienced three human leptospirosis cases from 20 staff, of which two were hospitalized. Urine, kidney and blood samples were collected from carcasses of 399 sheep (six suppliers, 17 slaughter lines) and 146 cattle (three suppliers, 22 slaughter lines). The urine and kidney samples were tested by quantitative real-time PCR (qPCR), while serum samples (from coagulated blood samples) were tested by microscopic agglutination test (MAT). In total, 27% (73/274; 95% CI: 18-37) of urine samples tested positive by qPCR. Species-specific shedding rates (prevalence of positive urine qPCR) were 31% (95% CI: 17-48) for sheep and 21% (95% CI: 14-30) for cattle. For 545 kidney samples tested, 145 were qPCR positive (27%; 95% CI: 17-39). The average prevalence of kidney qPCR positivity was 29% (95% CI: 17-45) for sheep and 21% (95% CI: 15-28) for cattle. Three hundred and thirty of 542 sampled sheep and cattle had antibodies against Leptospira borgpetersenii serovar Hardjobovis (Hardjobovis) and/or Leptospira interrogans serovar Pomona (Pomona), based on reciprocal MAT titre ≥1 : 48 (overall seroprevalence of 61%; 95% CI: 48-73). Seroprevalence was 57% (95% CI: 40-72) for sheep and 73% (95% CI: 59-83) for cattle. Among the seropositive animals, 41% (70/170; 95% CI: 30-54) were shedding (tested positive by urine qPCR) and 42% (137/330; 95% CI: 30-54) had renal carriage (tested positive by kidney qPCR). Some risk management options for abattoirs or farms to prevent human leptospirosis infections include vaccination of maintenance hosts, the use of personal protective equipment, and the application of urine qPCR to detect shedding status of stock as surveillance and as an alert., (© 2014 Blackwell Verlag GmbH.)

A study was performed to investigate interlaboratory test agreement between a research and a commercial veterinary diagnostic laboratory on blood and urine samples, and to investigate test agreement between blood, urine, and kidney samples (research laboratory) for leptospirosis diagnosis. Samples were sourced from 399 sheep and 146 beef cattle from a local abattoir. Interlaboratory agreement for real-time quantitative polymerase chain reaction (qPCR) results on urine samples was almost perfect (kappa = 0.90), despite the use of different amplification targets (DNA gyrase subunit B gene vs. 16s ribosomal RNA gene), chemistries (SYTO9 vs. TaqMan probe), and pre-PCR processing. Interlaboratory agreement for microscopic agglutination test (MAT) positivity was almost perfect (kappa = 0.93) for Leptospira borgpetersenii serovar Hardjo subtype Hardjobovis (Hardjobovis) but moderate (kappa = 0.53) for Leptospira interrogans serovar Pomona (Pomona). Among animals that had different titers recorded, higher Hardjobovis and lower Pomona titers were reported by the commercial laboratory than by the research laboratory (P < 0.005). These interlaboratory comparisons can assist researchers and diagnosticians in interpreting the sometimes discrepant test results. Within the research laboratory, the comparison of qPCR results on urine and kidney showed almost perfect agreement (kappa = 0.84), suggesting that the qPCR on these 2 specimens can be used interchangeably. The agreement between MAT positivity and urine and kidney qPCR results was fair (kappa = 0.32 and kappa = 0.33, respectively). However, the prevalence ratio of urine and kidney qPCR positivity in Hardjobovis-seropositive versus Hardjobovis-seronegative sheep indicated that Hardjobovis seropositivity found in sheep may be able to predict shedding or renal carriage., (© 2014 The Author(s).)

Until recently New Zealand had one of the highest rates of human campylobacteriosis reported by industrialized countries. Since the introduction of a range of control measures in the poultry production chain a reduction in human cases of around 50% has been observed nationwide. To inform risk managers a combination of spatial, temporal and molecular tools - including minimum spanning trees, risk surfaces, rarefaction analysis and dynamic source attribution modelling - was used in this study to formally evaluate the reduction in disease risk that occurred after the implementation of control measures in the poultry industry. Utilizing data from a sentinel surveillance site in the Manawatu region of New Zealand, our analyses demonstrated a reduction in disease risk attributable to a reduction in the number of poultry-associated campylobacteriosis cases. Before the implementation of interventions poultry-associated cases were more prevalent in urban than rural areas, whereas for ruminant-associated cases the reverse was evident. In addition to the overall reduction in prevalence, this study also showed a stronger intervention effect in urban areas where poultry sources were more dominant. Overall a combination of molecular and spatial tools has provided evidence that the interventions aimed at reducing Campylobacter contamination of poultry were successful in reducing poultry-associated disease and this will inform the development of future control strategies., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A SYTO9 real-time polymerase chain reaction assay for detection of pathogenic Leptospira spp. based on amplification of DNA gyrase subunit B (gyrB) gene has been optimized and evaluated for sensitivity and specificity on kidney and urine samples of New Zealand farmed deer. The detection limit was 10(3) cells/ml (2-10 copies/reaction). Comparison of the assay on deer kidneys (n = 268) with culture as the gold standard revealed a sensitivity and specificity of 85% and 99.2%, respectively. For deer urine (n = 113), the assay was compared with known inoculated samples and revealed a sensitivity and specificity of 96.7% and 100%, respectively. The assay was applied for quantifying pathogenic leptospires shed naturally in deer urine and revealed a detectable concentration of 3.7 × 10(3) to 1.7 × 10(6) cells/ml. To assess the assay's capability for identifying pathogenic Leptospira spp., 14 field isolates of L. borgpetersenii serovar Hardjo-bovis and L. interrogans serovar Pomona were amplified for polymerase chain reaction (PCR) product, purified, and sequenced. When compared with the National Center for Biotechnology Information database, sequence data matched with L. borgpetersenii serovar Hardjo-bovis in 13 samples and L. interrogans serovar Pomona in 1 sample, which was consistent with the microscopic agglutination test (MAT). Sequence analysis of purified PCR product amplified directly from kidney and urine samples also yielded serovar-comparable MAT results. Results suggest that the assay is rapid, sensitive, and specific for detection of pathogenic leptospires in deer clinical samples. The developed assay can also be used for estimating the concentration of leptospires and identifying Leptospira spp. in combination with DNA sequencing.

This study estimated the frequency of exposure of meat workers to carcasses infected with Leptospira serovars Hardjobovis or Pomona in a sheep-only abattoir in New Zealand. A stochastic spreadsheet model was developed to assess the daily risk of exposure of eviscerators, meat inspectors and offal handlers to live leptospires in sheep carcasses from May to November 2004 (high-risk period), and from December 2004 to June 2005 (low-risk period). The average sheep processed per day were 225 for an eviscerator, 374 for a meat inspector, and 1123 for an offal handler. The median daily exposures during high- and low-risk periods were 11 [95% distribution interval (DI) 5-19] and three (95% DI 1-8) infected carcasses/day for eviscerators, 18 (95% DI 9-29) and six (95% DI 2-12) for meat inspectors, and 54 (95% DI 32-83) and 18 (95% DI 8-31) for offal handlers, respectively. Stochastic risk modelling provided evidence that processing of sheep carcasses exposed meat workers regularly to live leptospires with substantial seasonal variation.

Aim: To find evidence for localisation in the uterus, and fetal infection, of Leptospira spp. in farmed deer in the lower North Island of New Zealand during and shortly after the breeding season., Methods: Between February and July 2008, 116 blood samples, 120 kidneys, 120 uteri and 27 fetuses were collected from 120 mixed-age hinds from lines from nine farms, at a deer slaughter premises. Serum samples were tested for antibodies against Leptospira borgpetersenii serovar Hardjo-bovis and Leptospira interrogans serovar Pomona, using the microscopic agglutination test (MAT). For both serovars, a titre of ≥1:48 was considered positive. Samples from kidneys, uteri and fetal tissue were subjected to bacterial culture, using Ellinghausen-McCullough-Johnson-Harris (EMJH) medium, and real-time PCR, using DNA gyrase subunit B gene primers., Results: Thirty-four of 116 (29.3%) serum samples were positive for serovar Hardjo-bovis, and 13 (11.2%) for serovar Pomona. Seven of 120 kidneys were positive for serovar Hardjo-bovis by culture, and five of these, but no others, were positive by real-time PCR. Of 120 uteri, none was culture- or PCR-positive. None of 27 fetal samples was culture-positive but one was positive by real-time PCR. The dam of the PCR-positive fetus was culture-negative from the kidney, but had an MAT titre of 1:192 for Hardjo-bovis., Conclusions: Attempts to isolate Leptospira spp. from the genital tracts and early fetuses of farmed deer were unsuccessful. However, molecular evidence suggested fetal infection in one case. This finding justifies further study of the role of leptospires in the genital tract and fetus and its association with reproductive loss in farmed deer.

The epidemiology of human campylobacteriosis is complex but in recent years understanding of this disease has advanced considerably. Despite being a major public health concern in many countries, the presence of multiple hosts, genotypes and transmission pathways has made it difficult to identify and quantify the determinants of human infection and disease. This has delayed the development of successful intervention programmes for this disease in many countries including New Zealand, a country with a comparatively high, yet until recently poorly understood, rate of notified disease. This study investigated the epidemiology of Campylobacter jejuni at the genotype-level over a 3-year period between 2005 and 2008 using multilocus sequence typing. By combining epidemiological surveillance and population genetics, a dominant, internationally rare strain of C. jejuni (ST474) was identified, and most human cases (65.7%) were found to be caused by only seven different genotypes. Source association of genotypes was used to identify risk factors at the genotype-level through multivariable logistic regression and a spatial model. Poultry-associated cases were more likely to be found in urban areas compared to rural areas. In particular young children in rural areas had a higher risk of infection with ruminant strains than their urban counterparts. These findings provide important information for the implementation of pathway-specific control strategies.

In New Zealand the number of campylobacteriosis notifications increased markedly between 2000 and 2007. Notably, this country's poultry supply is different than that of many developed countries as the fresh and frozen poultry available at retail are exclusively of domestic origin. To examine the possible link between human cases and poultry, a sentinel surveillance site was established to study the molecular epidemiology of Campylobacter jejuni over a 3-year period from 2005 to 2008 using multilocus sequence typing. Studies showed that 60.1 to 81.4% of retail poultry carcasses from the major suppliers were contaminated with C. jejuni. Differences were detected in the probability and level of contamination and the relative frequency of genotypes for individual poultry suppliers and humans. Some carcasses were contaminated with isolates belonging to more than one sequence type (ST), and there was evidence of both ubiquitous and supplier-associated strains, an epidemiological pattern not recognized yet in other countries. The common poultry STs were also common in human clinical cases, providing evidence that poultry is a major contributor to human infection. Both internationally rare genotypes, such as ST-3069 and ST-474, and common genotypes, such as ST-45 and ST-48, were identified in this study. The dominant human sequence type in New Zealand, ST-474, was found almost exclusively in isolates from one poultry supplier, which provided evidence that C. jejuni has a distinctive molecular epidemiology in this country. These results may be due in part to New Zealand's geographical isolation and its uniquely structured poultry industry.

Integrated surveillance of infectious multi-source diseases using a combination of epidemiology, ecology, genetics and evolution can provide a valuable risk-based approach for the control of important human pathogens. This includes a better understanding of transmission routes and the impact of human activities on the emergence of zoonoses. Until recently New Zealand had extraordinarily high and increasing rates of notified human campylobacteriosis, and our limited understanding of the source of these infections was hindering efforts to control this disease. Genetic and epidemiological modeling of a 3-year dataset comprising multilocus sequence typed isolates from human clinical cases, coupled with concurrent data on food and environmental sources, enabled us to estimate the relative importance of different sources of human disease. Our studies provided evidence that poultry was the leading cause of human campylobacteriosis in New Zealand, causing an estimated 58-76% of cases with widely varying contributions by individual poultry suppliers. These findings influenced national policy and, after the implementation of poultry industry-specific interventions, a dramatic decline in human notified cases was observed in 2008. The comparative-modeling and molecular sentinel surveillance approach proposed in this study provides new opportunities for the management of zoonotic diseases.

Campylobacter jejuni is widely distributed in the environment, and river water has been shown to carry high levels of the organism. In this study, 244 C. jejuni isolates from three river catchment areas in New Zealand were characterized using multilocus sequence typing. Forty-nine of the 88 sequence types identified were new. The most common sequence types identified were ST-2381 (30 isolates), ST-45 (25 isolates), and ST-1225 (23 isolates). The majority of the sequence types identified in the river water could be attributed to wild bird fecal contamination. Two novel clonal complexes (CC) were identified, namely, CC ST-2381 (11 sequence types, 46 isolates) and CC ST-3640 (6 sequence types, 12 isolates), in which all of the sequence types were new. CC ST-2381 was the largest complex identified among the isolates and was present in two of the three rivers. None of the sequence types associated with the novel complexes has been identified among human isolates. The ST-2381 complex is not related to complexes associated with cattle, sheep, or poultry. The source of the novel complexes has yet to be identified.

Aim: To determine the prevalence of the two most commonly diagnosed pathogenic Leptospira spp. serovars, Hardjobovis and Pomona, in sheep in a sheep-only abattoir in New Zealand, and to determine the prevalence of kidneys which were leptospire culture-positive collected from sheep seropositive or seronegative to the microscopic agglutination test (MAT)., Methods: A repeated cross-sectional observational study was conducted of serological and kidney culture prevalences of Leptospira borgpetersenii serovar Hardjobovis and Leptospira interrogans serovar Pomona. Lines of sheep and individual sheep were systematically randomly selected at a sheep-only abattoir during 18 May 2004 to November 2004 and 06 December 2004 to 14 June 2005. Additionally, a cross-sectional study examined prevalences in a purposively selected line of sheep from a flock with clinical evidence of an outbreak of leptospirosis., Results: In the study population of 15,855 sheep of which 2,758 were sampled, 5.7 (95% CI=4.9-6.7)% were seropositive to one or both serovars; 44.2 (95% CI=34.6-54.2)% of 95 lines of sheep and 44.9 (95% CI=35.0-55.3)% of 89 farms showed serological evidence of infection. The serological prevalence of serovar Hardjobovis was significantly higher than that of serovar Pomona both at line (33% and 4%, respectively) and individual (5% and 1%, respectively) levels. A low but persistent seroprevalence of Hardjobovis throughout both years suggested low-level endemicity to this serovar, whereas Pomona infections appeared to be sporadic. Leptospires were isolated from kidneys of 8/37 (22%) Hardjobovis- and 1/6 (17%) Pomona-seropositive, and 5/499 (1%) seronegative animals. Of the animals purposively sampled from a farm with a clinical outbreak of leptospirosis, all kidneys from the 13 seropositive animals were culture-positive, indicating a high risk of exposure of meat workers in outbreak situations. Kidneys of MAT-seropositive sheep were 21.7 (95% CI=7.6-61.9) times more likely to test culture-positive than kidneys from animals with negative MAT titres. In general, the results indicated that 13/1,000 sheep slaughtered were potentially shedding leptospires., Conclusions: The study demonstrated the presence of a definite risk of occupational exposure of meat workers in a sheep-only slaughterhouse to the two most commonly diagnosed pathogenic Leptospira spp. serovars in New Zealand.

The size and distribution of histological lesions was studied in 14 cases of avian pseudotuberculosis using a combination of serotype-specific immunohistochemistry and image analysis. The material was derived from recent and archival cases in six canaries (Serinus canaria), two zebra finches (Poephila guttata), three psittaciformes (a kaka, Nestor meriondalis, one rainbow lorikeet, Trichoglossus mollucanus, and one budgerigar, Melopsittacus undulatus), and three New Zealand wood pigeons (Hemiphaga novaeseelandiae). The disease observed in the passerine species appeared to have an acute clinical course and the bacterial lesions were predominately enteric. In the pigeons and the psittaciformes examined, the clinical course of the disease was more chronic in nature and involved the liver and spleen. A correlation was found between the amount of stainable iron in the liver of affected birds and the area of bacterial lesions. All of the 11 strains of Yersinia pseudotuberculosis isolated from fresh necropsy material were serotype II, as determined using a standard serum agglutination test. Immunohistochemistry indicated the presence of antigen(s) common to serotype II in histological material from confirmed cases and in another three cases where the organism had been cultured but the serotype not specified. The in vitro virulence characteristics and plasmid profiles of Y. pseudotuberculosis isolates were also determined.

A mutational and structural analysis of Rhizobium loti nodulation genes in strains NZP2037 and NZP2213 was carried out. Unlike the case with other Rhizobium strains examined to date, nodB was found on an operon separate from nodACIJ. Sequence analysis of the nodACIJ and nodB operon regions confirm that R. loti common nod genes have a gene organization different from that of other Rhizobium spp. At least 4 copies of nodD-like sequences were identified in R. loti. The complete nucleotide sequence of one of these, nodD3, was determined. A new host-specific nod gene, nolL, was identified adjacent to nodD3. NolL shares homology with NodX and other O-acetyl transferases. Mutational analysis of the nod regions of strains NZP2037 and NZP2213 showed that nodD3, nodI, nodJ, and nolL were all essential for R. loti strains to effectively nodulate the extended host Lotus pedunculatus, but were not necessary for effective nodulation of the less restrictive host, Lotus corniculatus. Both nodD3 and nolL were essential for R. loti strains to nodulate Leucaena leucocephala.