Wastewater-based epidemiology in hazard forecasting and early-warning systems for global health risks.

With the advent of the SARS-CoV-2 pandemic, Wastewater-Based Epidemiology (WBE) has been applied to track community infection in cities worldwide and has proven succesful as an early warning system for identification of hotspots and changingprevalence of infections (both symptomatic and asymptomatic) at a city or sub-city level. Wastewater is only one of environmental compartments that requires consideration. In this manuscript, we have critically evaluated the knowledge-base and preparedness for building early warning systems in a rapidly urbanising world, with particular attention to Africa, which experiences rapid population growth and urbanisation. We have proposed a Digital Urban Environment Fingerprinting Platform (DUEF) - a new approach in hazard forecasting and early-warning systems for global health risks and an extension to the existing concept of smart cities. The urban environment (especially wastewater) contains a complex mixture of substances including toxic chemicals, infectious biological agents and human excretion products. DUEF assumes that these specific endo- and exogenous residues, anonymously pooled by communities' wastewater, are indicative of community-wide exposure and the resulting effects. DUEF postulates that the measurement of the substances continuously and anonymously pooled by the receiving environment (sewage, surface water, soils and air), can provide near real-time dynamic information about the quantity and type of physical, biological or chemical stressors to which the surveyed systems are exposed, and can create a risk profile on the potential effects of these exposures. Successful development and utilisation of a DUEF globally requires a tiered approach including: Stage I: network building, capacity building, stakeholder engagement as well as a conceptual model, followed by Stage II: DUEF development, Stage III: implementation, and Stage IV: management and utilization. We have identified four key pillars required for the establishment of a DUEF framework: (1) Environmental fingerprints, (2) Socioeconomic fingerprints, (3) Statistics and modelling and (4) Information systems. This manuscript critically evaluates the current knowledge base within each pillar and provides recommendations for further developments with an aim of laying grounds for successful development of global DUEF platforms.

Vaccination practices for Leptospira spp. on New Zealand dairy farms.

AIMS: To describe leptospiral vaccination practices in dairy herds in New Zealand and evaluate conformity with best practice guidelines issued by the New Zealand Veterinary Association using data from a questionnaire administered by participating veterinary practices. METHODS: A cross-sectional study of 200 randomly selected dairy farms stratified by herd size and region throughout New Zealand was conducted from January to April 2016 to investigate leptospiral vaccination practices in dairy herds in New Zealand. Using a pre-tested questionnaire administered during a face-to-face interview, vaccination practice details such as vaccine types, time, and age of vaccination and whether vaccines were administered by veterinary or farm staff, were collected. RESULTS: Leptospiral vaccination programmes had been implemented on 199/200 (99.5 (95% CI = 97.2-99.9)%) farms, and on 178 (89.4%) of those, programmes had been running for ≥5 years. Most farmers used bivalent vaccines containing antigens for leptospiral serovars Pomona and Hardjo (144/179 (80.4%) in calves, 112/167 (60.7%) in heifers, and 112/163 (68.7%) in cows), rather than trivalent vaccines which also include antigens for L. interrogans serovar Copenhageni. In total, 123/200 (61.5%) of farmers purchased only vaccinated animals but 51/199 (25.6%) were unsure of the vaccination status of purchased cattle. Sixty-one percent (105/172) of farmers had other livestock on their farms and of them, 78/186 (42%) vaccinated some or all for Leptospira spp. Leptospiral vaccines were administered always or sometimes with other animal remedies on 30/190 (15.8%) and 91/190 (47.9%) of farms, respectively. Most farmers had not made changes to their vaccination programme in the previous 5 years. Timing of first vaccination of calves ranged from 2 weeks to 10 months of age, with 112/189 (59.3%) vaccinating by 4 months of age. Approximately half of the farms followed the best practice guideline for the timing of vaccinations for calves (high-risk farms; 67/162; 41.4%) heifers (72/165, 43.6%), and cows (171/184; 92.9%). CONCLUSIONS: The results of this survey suggest that there is almost universal adoption of leptospiral vaccination for dairy cattle in New Zealand. However, there remain areas for improvement regarding the proportion of farmers following best practice guidelines and refinement of vaccination programmes, particularly with respect to timing of vaccination in calves.

Serological study of Leptospira interrogans serovar Copenhageni and L. borgpetersenii serovars Tarassovi and Ballum in beef cattle, sheep and deer in New Zealand.

AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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. CLINICAL RELEVANCE: 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.

A survey of gastrointestinal nematode species in red deer (Cervus elaphus) farms in New Zealand using PCR.

Gastrointestinal nematodes are recognised as an animal health issue for farmed red deer. The aim of this study was to explore the range of species infecting farmed deer herds and their farm-level prevalence in New Zealand. Faecal samples were collected from 12-24-month-old deer (n = 6-26; mean 19) on 59 farms located in the North (n = 25) and South (n = 34) Islands. Sub-samples of faeces were pooled by farm and cultured to recover third stage larvae. Twenty four larvae were randomly selected and identified to species using a multiplex PCR (total = 1217 larvae). At farm-level the most prevalent nematodes were Oesophagostomum venulosum 83% (n = 49) and the deer-specific nematodes in the subfamily Ostertagiinae (=Ostertagia-type) including, Spiculoptera asymmetrica 73% (n = 43), Ostertagia leptospicularis 47% (n = 28), Spiculoptera spiculoptera 47% (n = 28). The recently identified Trichostrongylus askivali was present on 32% (n = 19) of the farms and Oesophagostomum sikae on 17% (n = 10). In the analysis of the total number of larvae identified, the proportion was in similar order, 45% (n = 548) were O. venulosum, 14% (n = 173) S. asymmetrica, 10% (n = 124) S. spiculoptera, 9% (n = 114) O. leptospicularis, T. askivali, 3% (n = 40) and only 2% were O. sikae (n = 20). This study is the first to show the farm-level prevalence of nematode species in deer in New Zealand and the first to use PCR as a diagnostic tool. It provides data consistent with cross-infection from sheep/cattle to deer, and provided tentative insights into the proportions of the main GIN species across the deer population including O. sikae and T. askivali which have only recently been identified in New Zealand.

Investigation of association between bovine viral diarrhoea virus and cervid herpesvirus type-1, and abortion in New Zealand farmed deer.

This study tested for association between bovine viral diarrhoea virus (BVDv) and cervid herpesvirus type-1 (CvHV-1) exposure and abortion in New Zealand farmed red deer. Rising two-year-old (R2, n = 22,130) and mixed-age (MA, n = 36,223) hinds from 87 and 71 herds, respectively, throughout New Zealand were pregnancy tested using ultrasound early in gestation (Scan-1) and 55-89 days later (Scan-2) to detect mid-term abortion. Sera from aborted and non-aborted hinds at Scan-2 were tested for BVDv and CvHV-1 using virus neutralisation tests. Available uteri from aborted hinds and from hinds not rearing a calf to weaning were tested by PCR for herpesvirus DNA. In herds with aborted hinds, 10.3% of 639 R2 and 17.2% of 302 MA hinds were sero-positive for BVDv and 18.6% of 613 R2 and 68.5% of 232 MA hinds were sero-positive for CvHV-1. There was no association between BVDv sero-status and abortion at animal level (R2 p = 0.36, MA p = 0.76) whereas CvHV-1 sero-positivity was negatively associated with abortion in MA hinds (p = 0.01) but not in R2 hinds (p = 0.36), MA). Eleven of 108 uteri from aborted R2 hinds but no MA hinds were positive for herpesvirus DNA. Vaginal samples from four R2 and one MA aborted hinds tested were negative for herpesvirus DNA. A Cervid Rhadinovirus type-2 (CRhV-2) was identified in seven PCR positive uteri samples. Findings suggest that BVDv and CvHV-1 may not be associated with abortion in R2 hinds, but association needs to be tested further in MA hinds. The role of CRhV-2 requires clarification.

Meta-analysis of the efficacy of Leptospira serovar Hardjo vaccines to prevent urinary shedding in cattle.

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.

Lack of association between Leptospira spp. serovars Hardjobovis and Pomona and pregnancy and mid-term abortion in New Zealand farmed red deer.

This paper investigates Leptospira borgpeterseni serovar Hardjobovis and L. interrogans serovar Pomona as potential causes of sub-optimum pregnancy rates and mid-term abortion in farmed red deer. Rising two-year-old (R2, n = 22,130) and mixed-age (MA, n = 36,223) hinds from 87 and 71 herds, respectively, throughout New Zealand were ultrasound scanned early in gestation (Scan-1) and a sub-sample re-scanned (Scan-2) 55-89 days later and mid-term daily abortion rate calculated. A sub-sample of sera from pregnant and non-pregnant hinds at both scans, both with (case herds) and without aborted hinds was tested for Leptospira using the microscopic agglutination test with titre cut-point ≥1:48 as positive. At Scan-1, 44.3% of 661 and 4.6% of 647 hinds were sero-positive for Hardjobovis and Pomona, respectively. The geometric mean titre (GMT) for Pomona was higher in pregnant than non-pregnant MA hinds (p = 0.015) but not in R2 hinds. At Scan-2 in case herds, 40.3% of 2242 and 7.1% of 2243 hinds were sero-positive for Hardjobovis and Pomona, respectively. There was no association between Hardjobovis or Pomona serology and non-pregnancy (Scan-1) or mid-term abortion (Scan-2) at animal or herd level. In case herds, GMT was higher in non-aborted than aborted hinds for Hardjobovis (p = 0.018) in MA herds and for Pomona in R2 herds (p = 0.006). No uteri from hinds not pregnant or aborting at either scan, or not rearing a calf to weaning, and fetuses as available, were positive on PCR. Evidence is insufficient to confirm that Leptospira Hardjobovis and Pomona play a significant role in sup-optimum early pregnancy or mid-term abortion rates in deer.

Control of clinical paratuberculosis in New Zealand pastoral livestock.

This review summarises current control measures for clinical paratuberculosis (Johne's disease; JD) in New Zealand pastoral livestock. Most New Zealand sheep, deer, beef and dairy cattle herds and flocks are infected by Mycobacterium avium ssp. paratuberculosis (Map). Dairy cattle and deer are mostly infected with bovine (Type II), and sheep and beef cattle with ovine (Type I) strains. Control in all industries is voluntary. While control in sheep and beef cattle is ad hoc, the dairy and deer industries have developed resources to assist development of farm-specific programmes. The primary target for all livestock is reduction of the incidence rate of clinical disease rather than bacterial eradication per se. For dairy farms, a nationally instituted JD-specific programme provides guidelines for risk management, monitoring and testing clinically suspect animals. While there is no formal programme for sheep farms, for those with annual prevalences of clinical disease >2%, especially fine wool breeds, vaccination may be a cost effective control option. The deer industry proactively monitors infection by a national abattoir surveillance programme and farmers with an apparent high disease incidence are encouraged to engage with a national network of trained consultants for management and control advice. Evaluation of the biological and economic effectiveness of control in all industries remains to be undertaken. Nevertheless, opportunities exist for farmers, who perceive significant JD problems in their herds/flocks, to participate in systematic best-practice activities that are likely to reduce the number of clinical infections with Map on their farms, and therefore the overall prevalence of JD in New Zealand's farming industries.

Establishment rate of cattle gastrointestinal nematodes in farmed red deer (Cervus elaphus).

Red deer can be infected with some gastrointestinal nematodes (GIN) of cattle but it is unknown to what extent. An indoor study was conducted to determine the establishment rate of cattle GIN in young deer. Five young calves and 5 young red deer were used. They were effectively treated with anthelmintics when housed and then infected 2 weeks later. After four weeks they were killed for total worm counts. Establishment rates were assessed comparing worm counts to the infective dose which were identified morphologically, and to the relative establishment rate of different species. The establishment rates (%) in cattle and deer respectively were H. contortus (8.0, 18.7, p=0.18), Ostertagia ostertagi (30.8, 0.7, p<0.001), Cooperia spp. (72.3, 2.3, p<0.001) and Trichostrongylus spp. (19.0, 25.3, p=0.12). The majority (>98%) of Trichostrongylus spp. were Trichostrongylus axei in both hosts and there were no differences between hosts for this species (p=0.11). In cattle >98% of Cooperia were Cooperia oncophora and the mean burden was much higher than in deer (p<0.01) where there were similar proportions of Cooperia oncophora, Cooperia punctata and C. curticei. Small numbers of Oesophagostomum venulosum were also present with 3X as many found in deer as in cattle (p<0.05). This study has shown that some cattle-origin GIN can establish in red deer. In particular, the establishment of H. contortus and T. axei could allow sufficient burdens to build up to be clinically significant. Importantly, almost no cattle Ostertagia species or small intestinal species established in deer.

Seroprevalence and Risk Factors for Leptospira Seropositivity in Beef Cattle, Sheep and Deer Farmers in New Zealand.

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.

Serological patterns, antibody half-life and shedding in urine of Leptospira spp. in naturally exposed sheep.

AIMS: To determine within-farm prevalence, longitudinal pattern of exposure measured by serology, antibody titre longevity and point prevalence of shedding in urine of Leptospira borgpetersenii serovar Hardjo and L. interrogans serovar 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 to L. borgpetersenii serovar Hardjo and L. interrogans serovar 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.

Prevalence and risk factors for Leptospira exposure in New Zealand veterinarians.

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.

Establishment rate of sheep gastrointestinal nematodes in farmed red deer (Cervus elaphus).

To investigate the establishment of sheep gastrointestinal nematodes (GIN) in red deer, five red deer and five sheep aged 5-6 months were challenged with a mixed burden of sheep GIN at a rate of 327L3/kg bodyweight. The LSmean (SE) establishment rates (%) for Haemonchus contortus, Teladorsagia circumcincta, Cooperia curticei, Trichostrongylus spp. and Oesophagostomum+Chabertia spp. were 18.6 (0.03), 35.5 (0.04), 30.7 (0.04), 74.9 (0.05), 19.9 (0.06), respectively in sheep and 10.5 (0.03), 1.0 (0.04), 0.1 (0.04), 1.0 (0.05), 4.8 (0.06) respectively, in deer. Establishment rates were significantly different (p<0.05) between hosts for all genera. No Trichostrongylus colubriformis or Trichostrongylus vitrinus were seen in any deer but were present in all sheep. Trichostrongylus axei were seen in both hosts but there were relatively more which established in sheep than in deer (p<0.01). No Chabertia ovina were seen in any deer but were present in four of five sheep in low numbers. The only species of Oesophagostomum seen in either host was Oesophagostomum venulosum. These results suggest that the sheep GIN most likely to infect red deer grazing the same pastures are H. contortus, T. axei and O. venulosum.

Shedding and seroprevalence of pathogenic Leptospira spp. in sheep and cattle at a New Zealand Abattoir.

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.

Liver biopsy histopathology for diagnosis of Johne's disease in sheep.

Sheep with Johne's disease develop epithelioid macrophage microgranulomas, specific to Mycobacterium avium subsp. paratuberculosis (Map) infection, in the terminal ileum, mesenteric lymph nodes, and organs distant to the alimentary tract such as the liver. The objectives of this study were to determine whether liver pathology was present in ewes affected by Map and whether liver cores provide adequate tissue for this potential diagnostic marker. One hundred and twenty-six adult, low body condition ewes were euthanized, necropsied, and underwent simulated liver biopsy. Ileal lesions typical of Map were found in 60 ewes. Hepatic epithelioid microgranulomas were observed in all ewes with Type 3b (n = 40) and 82% (n = 11) with Type 3c ileal lesions. None were found in ewes unaffected by Map or with Type 1, 2, or 3a ileal lesions. Liver biopsy core samples provided adequate tissue for histopathology with a sensitivity and specificity of 96% (95% confidence interval [CI], 0.87-0.99) and 100% (95% CI, 0.95-1), respectively for detection of types 3b and 3c ileal lesions.

A randomised controlled trial of Silirum vaccine for control of paratuberculosis in farmed red deer.

A randomised controlled trial to assess the efficacy of Silirum vaccine in control of paratuberculosis in young farmed deer was carried out in 2008-2009 in six New Zealand herds with a history of clinical disease. Vaccination with Silirum was carried out in four-month-old deer, and vaccinates (n=1671) and controls (n=1664) were weighed at vaccination and at 8 and 12 months old, when faecal samples were collected from 125 vaccinates and 123 controls on five farms. Deer were slaughtered between 11 and 20 months of age, and the incidence of gross visceral lymph node (VLN) pathology typical of paratuberculosis in deer, that is, enlarged and/or granulomatous VLN, was recorded. Clinical disease was confirmed in 18 controls and seven vaccinates, representing a vaccine efficacy estimate of 60 per cent (95% CI 3 per cent to 83 per cent, P=0.04). Forty-seven percent (95% CI 38 per cent to 56 per cent) of faecal samples from vaccinates and 55 per cent (95% CI 46 per cent to 64 per cent) from controls were Mycobacterium avium subspecies paratuberculosis positive (P=0.5). Average daily liveweight gain did not differ between the cohorts. At slaughter, 1.4 per cent of vaccinates and 4.5 per cent of controls had VLN pathology, RR=0.32 (95% CI 0.19 to 0.54, P<0.001). These data indicate that vaccination with Silirum may be useful as an aid to control losses associated with clinical paratuberculosis in young deer.

Prevalence of Mycobacterium avium subsp. paratuberculosis in farmed red deer (Cervus elaphus) with grossly normal mesenteric lymph nodes.

AIM: To estimate the prevalence of Mycobacterium avium subsp. paratuberculosis (Map) in farmed deer with no gross post-mortem evidence of Map infection slaughtered in New Zealand, and to assess predictors of infection. METHODS: Mesenteric lymph node (MLN) samples (n = 251) were collected from 60 lines of deer presented at two slaughterhouses in the North and two in the South Island of New Zealand between October 2008 and January 2009 and cultured for Map. Estimates of individual animal prevalence for each island were adjusted to account for the clustering of individual observations within herds. The national herd prevalence estimate was calculated as a weighted mean, with weights being the proportion of herds from which deer were slaughtered at North and South Island slaughterhouses among all herds slaughtering deer throughout New Zealand. Age, gender, and the presence of other carcasses with enlarged and/or granulomatous MLN in the same line (line status) were assessed as predictors of infection using multivariable logistic regression. RESULTS: A national cluster-adjusted individual animal prevalence of 45 (95% CI = 30-60)% was estimated, with North and South Island prevalences of 29 (95% CI = 16-45)% and 51 (95% CI = 36-66)%, respectively. Line status was a strong predictor of infection in young deer (OR 7.1, 95% CI = 2.4-21.5), but not in older deer. Herd-level prevalence was 44 (95% CI = 24-64)% in the North Island and 67 (95% CI = 49-85)% in the South Island. Weighted adjustment resulted in a national herd-level prevalence estimate of 59 (95% CI = 41-78)%. CONCLUSIONS: This study has provided a national baseline prevalence estimate for Map infection at the individual and herd-level, showing a contrast between the North and South Islands. More research to investigate the factors contributing to the difference in infection prevalence seen between the islands may help to identify suitable control measures for Map in deer herds.

Association between Mycobacterium avium subspecies paratuberculosis and lymph node size in New Zealand farmed deer (Cervus elaphus).

AIMS: To develop and validate criteria for identification of abnormal lymph nodes (LN) at commercial slaughter, for the purpose of national surveillance for Mycobacterium avium subspecies paratuberculosis (Map) in New Zealand farmed deer. This included estimation of the predictive value of abnormal LN for Map infection; a standard circumference cut-point for measurement of abnormal LN; and identification of risk factors associated with increasing LN circumference. METHODS: In Study 1, official assessors sampled 129 LN with macroscopically visible abnormalities (abnormal LN) from 76 deer herds between May and November 2007. LN samples were cultured for Map, with culture-negative LN further examined for typical histopathological changes. The predictive value of abnormal LN for Map infection was calculated and significance of herd location (North or South Island) assessed. In Study 2, the circumferences of 412 mesenteric LN (MLN) from 79 herds were measured between October 2007 and January 2009, with samples cultured for Map and examined for eight histopathological features. The minimum circumference of an abnormal MLN was defined, based on an arbitrary >95% specificity of a culture-positive Map diagnosis. Associations between the predictor variables Map culture status, carcase weight, animal age and gender, and histopathological features, and increasing MLN circumference were assessed using a linear mixed-effects model. RESULTS: Based solely on culture, the predictive value of abnormal LN for Map infection was 92.2 (95% CI: 86.2-96.2)% with no difference between the North and South Islands (p = 0.09). Inclusion of three culture-negative LN with histopathological changes typical of Map infection increased the predictive value estimate to 94.6 (95% CI: 89.2-97.3)%. The minimum circumference of an abnormal MLN was defined as 55 mm, with a sensitivity of Map detection at this cut-point of approximately 12%. Increasing MLN circumference was positively associated with the presence of moderate follicular hyperplasia (p < 0.01), focal granulomas (p < 0.01) and a synergistic interaction between focal granulomas and Map status (p = 0.03). CONCLUSIONS: Deer MLN with macroscopically visible abnormalities and/or a circumference of >55 mm have >95% likelihood of Map infection. However, sensitivity of Map diagnosis in MLN with circumference of >55 mm was 12%, indicating use of abnormal LN as a sole criterion in national surveillance for Map in slaughtered deer will underestimate animal-level prevalence.

Sensitivity, specificity and level of agreement of meat inspector detection of abnormal lymph nodes of farmed deer (Cervus elaphus) in New Zealand.

AIMS: To assess the sensitivity (Se), specificity (Sp) and level of agreement in the detection of abnormal deer mesenteric lymph nodes (MLN) by official assessors (meat inspectors) for the purpose of slaughter premise surveillance for Johne's disease in New Zealand farmed deer. METHODS: To determine Se and Sp, four meat inspectors in two commercial deer slaughter premises (DSP) each examined between 153 and 925 MLN under normal visual examination, palpation and/or incision inspection procedures. Abnormal MLN were independently defined as those with a circumference measurement of  ≥ 55 mm and/or grossly visible pathological changes such as caseation, necrosis and/or mineralisation, while those with a circumference of <55 mm and without gross lesions were defined as normal. The inspectors' diagnosis of normal or abnormal was retrieved from the national DSP-based surveillance database for Johne's disease in deer. To determine between-inspector level of agreement, 54 meat inspectors visually examined two photographic images each of 19 normal and 10 abnormal MLN, and recorded their diagnosis. Between-inspector agreement was calculated based on a modification of Cohen's kappa statistic for multiple raters. The influence of covariates such as inspector age and experience on the between-inspector level of agreement were assessed using the Breslow-Day statistic and test for equal kappa coefficients. RESULTS: The weighted average Se and Sp of inspector detection of abnormal MLN was 13.3 (minimum 4.8, maximum 41.2)% and 99.9 (minimum 99.5, maximum 100.0)%, respectively. The level of between-inspector agreement in the diagnosis of abnormal and normal MLN was fair (κ = 0.32). Employment location, inspector age, experience inspecting deer or other species, and the number of shifts inspecting deer within the previous four weeks had no influence on between-inspector agreement (p > 0.10). CONCLUSIONS: Inspectors diagnosed abnormal deer MLN with a high specificity, but low sensitivity. These data supported that visual assessment of MLN characteristics was suitable for national surveillance for paratuberculosis, while highlighting the need for further emphasis on training of inspectors in abnormal lymph node detection.

The prevalence of disseminated Mycobacterium avium subsp. paratuberculosis infection in tissues of healthy ewes from a New Zealand farm with Johne's disease present.

AIM: To determine the prevalence of disseminated Mycobacterium avium subsp. paratuberculosis (Map) infection in healthy ewes in a flock with a history of clinical Johne's disease. METHODS: Twenty-four healthy ewes, from a large sheep and cattle farm with a history of clinical Johne's disease in the ewe flock, were randomly selected, euthanased, blood sampled, and examined at necropsy. BACTEC™ radiometric culture for Map was performed on samples of faeces, ileum, mesenteric lymph node, biceps femoris muscle and mononuclear cells in peripheral blood. Serum antibody ELISA tests were performed. Histological sections and Ziehl Neelsen (ZN) stains of impression smears of ileum and mesenteric lymph node were examined for pathological lesions characteristic of Johne's disease and acid fast organisms (AFO). Indirect quantification of Map was performed, using BACTEC radiometric growth indices measuring the time taken for the production of (14)CO(2.) RESULTS: No histological evidence of Johne's disease or AFO was found in the ileum and mesenteric lymph nodes. Twelve of the 24 ewes (50%) had Map cultured from the ileum (n=6) and/or mesenteric lymph nodes (n=8) while none had Map cultured from the faeces, biceps femoris muscle or blood mononuclear cells. One of the 12 Map culture positive ewes was serum ELISA positive. The culture growth rates in liquid medium suggest low numbers of Map were present in the tissues of the culture positive ewes. CONCLUSION: Fifty per cent of clinically healthy ewes exposed to Map within a Johne's infected flock were Map culture positive in the ileum and/or mesenteric lymph node(s), while the ELISA was positive in 8% of those animals (n=1). There was no faecal shedding of Map and no Map was cultured from skeletal muscle or from blood mononuclear cells suggesting that systemic Map infection, defined as positive culture of Map from skeletal muscle and/or blood, may be uncommon in healthy mixed age ewes without clinical Johne's disease. CLINICAL RELEVANCE: ELISA serology detected 1 of 12 ewes infected with Map whilst none were detected from faecal BACTEC radiometric culture, suggesting biosecurity measures used to control the spread of Map may be of limited use. Map was not cultured from blood mononuclear cells or skeletal muscle, indicating that meat from healthy ewes, from farms where Johne's disease is present, is an unlikely source of Map exposure for humans. Further research is warranted to establish the prevalence and dissemination of Map in tissues outside the alimentary tract of healthy ewes from farms throughout New Zealand where Map is present.