Anne C. Midwinter

Assigning the source of human campylobacteriosis in New Zealand: a comparative genetic and epidemiological approach.

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.

Molecular Epidemiology of Campylobacter jejuni Isolates from Wild-Bird Fecal Material in Children's Playgrounds

ABSTRACT In many countries relatively high notification rates of campylobacteriosis are observed in children under 5 years of age. Few studies have considered the role that environmental exposure plays in the epidemiology of these cases. Wild birds inhabit parks and playgrounds and are recognized carriers of Campylobacter, and young children are at greater risk of ingesting infective material due to their frequent hand-mouth contact. We investigated wild-bird fecal contamination in playgrounds in parks in a New Zealand city. A total of 192 samples of fresh and dried fecal material were cultured to determine the presence of Campylobacter spp. Campylobacter jejuni isolates were also characterized by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST), and the profiles obtained were compared with those of human isolates. C. jejuni was isolated from 12.5% of the samples. MLST identified members of clonal complexes ST-45, ST-682, and ST-177; all of these complexes have been recovered from wild birds in Europe. PFGE of ST-45 isolates resulted in profiles indistinguishable from those of isolated obtained from human cases in New Zealand. Members of the ST-177 and ST-682 complexes have been found in starlings (Sturnus vulgaris) in the United Kingdom, and these birds were common in playgrounds investigated in New Zealand in this study. We suggest that feces from wild birds in playgrounds could contribute to the occurrence of campylobacteriosis in preschool children. Further, the C. jejuni isolates obtained in this study belonged to clonal complexes associated with wild-bird populations in the northern hemisphere and could have been introduced into New Zealand in imported wild garden birds in the 19th century.

Molecular Epidemiology of Campylobacter jejuni in a Geographically Isolated Country with a Uniquely Structured Poultry Industry

ABSTRACT In New Zealand the number of campylobacteriosis notifications increased markedly between 2000 and 2007. Notably, this countrys 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 Zealands geographical isolation and its uniquely structured poultry industry.

Molecular and spatial epidemiology of human campylobacteriosis: source association and genotype-related risk factors.

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.

Prevalence of pathogenic Leptospira spp. in sheep in a sheep-only abattoir in New Zealand

Abstract 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 14June 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 besporadic. 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.

Leptospirosis in farmed deer in New Zealand: A review

Abstract Current knowledge of leptospirosis in farmed deer in New Zealand is reviewed. Over the past 25 years, leptospirosis has been reported to occur in individual cases as well as in herd outbreaks in farmed deer and in human cases linked to farmed deer. Serological studies and evidence from bacterial culture suggest infection is widespread. Mixing of young stock from several sources appears to be a significant risk factor for outbreaks. The culture of Leptospira interrogans Hardjobovis, Pomona and Copenhageni has been reported. Infection with serovar Hardjobovis had the highest prevalence, either individually or mixed with serovar Pomona. Infection with serovar Copenhageni appears uncommon and its pathogenicity in deer is unproven. Titres to serovars Australis, Ballum, Balcanica and Tarassovi have been reported. Deer appear to be maintenance hosts for serovar Hardjobovis, incidental or accidental hosts and probably a maintenance population for serovar Pomona, since some infections persist for several months, and accidental hosts for serovar Copenhageni. Serovar Pomona appears to produce clinical and probably subclinical disease, whereas serovar Hardjobovis appears to cause only subclinical disease, although the relative risk of disease causation has not been determined. Clinical disease is usually manifested by haemolysis, jaundice, renal lesions, haemoglobinuria and often by sudden death. Renal lesions are commonly observed at slaughter and many are associated with leptospiral infections. Occupationally, slaughterhouse workers appear to beat greatest risk of contracting the disease from deer. Vaccination produces serological responses, but its effectiveness in protecting against disease, and prevention or reduction of shedding in urine, has not yet been confirmed in deer. More robust knowledge of the epidemiology of leptospiral infections in deer, and the effectiveness of vaccines and vaccination regimes, is needed to assist the deer industry to develop a strategy to manage this disease.

VACCINATION OF MICE WITH LIPOPOLYSACCHARIDE (LPS) AND LPS-DERIVED IMMUNO-CONJUGATES FROM LEPTOSPIRA INTERROGANS

Mice were vaccinated with lipopolysaccharide (LPS) from Leptospira interrogans serovar pomona or hardjo, or with the polysaccharide (PS) fraction of the LPS, or with an immunoconjugate of PS and diphtheria toxoid (DT). Maximum agglutinin titres were found 6-10 weeks after vaccination with LPS or PS-DT conjugate; the latter elicited antibody titres at least 10 times higher than those produced in response to LPS. Animals failed to react significantly to PS. Titres elicited by antigens of serovar pomona were higher than those elicited by serovar hardjo.

Assessment of occupational exposure to leptospirosis in a sheep-only abattoir.

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.

Novel Clonal Complexes with an Unknown Animal Reservoir Dominate Campylobacter jejuni Isolates from River Water in New Zealand

ABSTRACT 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.

Regional seroprevalence of leptospirosis on deer farms in New Zealand

Abstract AIM: To undertake a seroprevalence survey to estimate the distribution of infection by Leptospira spp. serovars Hardjobovis, Pomona and Copenhageni in farmed deer herds regionally throughout New Zealand. METHODS: Between March 2003 and February 2005, 111 red and red x wapiti deer herds not using a leptospiral vaccine were selected from nine regions from Northland to Southland. Eighty-three herds were selected for on-farm collection of 12– 20 blood samples from hinds predominantly 9–30 months of age, by jugular venepuncture, while similar numbers, predominantly males9–30 months of age, per farm were collected from 28 herds at sticking at deer slaughter premises (DSP). Serum was stored frozen until analysed using the microscopic agglutination test (MAT). Titre cut-points of 1:24 for serovar Hardjobovis and 1:48 for serovars Pomona and Copenhageni were considered positive at the individual animal level. Herds were considered seropositive if there were at least three animals per herd seropositive to any serovar. RESULTS: Eighty-one percent of herds were seropositive for Leptospira spp. Hardjobovis was the predominant serovar at the herd (78%) and individual animal (60.8%) levels. The seroprevalence of Pomona was 20% and 8.4% at the herd and animal levels, respectively. Dual seroreactivity to both Hardjobovis and Pomona was observed in 16% of herds and 6.6% of individuals; within-herd seroprevalence for both serovars ranged from 0 to 100%.No herds and 1.2% of animals were seropositive for serovar Copenhageni. There was a regional difference in the prevalence of herds seropositive to both Hardjobovis and Pomona (p=0.02), but no difference between regions in the prevalence of herds seropositive to either Hardjobovis or Pomona alone. CONCLUSION: The seroprevalence of Leptospira spp. serovars is high, and distributed in farmed deer herds throughout New Zealand. CLINICAL SIGNIFICANCE: Results confirm the risk of disease and potential loss of production in deer, and exposure of infection to humans and other livestock from deer is widespread, demonstrating that control of the disease needs to be considered at a national rather than regional level.