Lynn Rogers

Infectivity and persistence of an outbreak strain of Salmonella enterica serotype Typhimurium DT160 for house sparrows (Passer domesticus) in New Zealand

Abstract AIM: To examine the infective dose, incubation period and disease progression of an isolate of Salmonella enterica serotype Typhimurium definitive type 160 (DT160) originating from a naturally-infected house sparrow (Passer domesticus) during an outbreak of the disease in New Zealand. METHODS: Thirty-six house sparrows captured from the wild and free of Salmonella spp were divided into six groups of six birds, housed individually, and inoculated orally with phosphate buffered saline (PBS) or 101, 102, 103, 105, 2 × 108 colony forming units (cfu) of the outbreak strain of S. Typhimurium DT160. The birds were observed for 10 days for clinical signs and/or mortality, and faecal samples were collected to determine excretion of S. Typhimurium. The birds were eutha- nised 11 days post-inoculation (p.i.) and a wide range of tissue samples were collected for histopathological examination, and culture and typing of Salmonella spp. Macro-restriction profiling by pulsed-field gel electrophoresis (PFGE) using XbaI was performed for the epidemiological typing of S. Typhimurium DT160 isolates. RESULTS: Mortality in house sparrows inoculated with S. Typhimurium DT160 was dose-dependent, and 2/6 birds inoculated with 105 cfu and all six birds inoculated with 2 × 108 cfu died during the study. Infected sparrows displayed few clinical signs, apart from diarrhoea and/or polyuria, fluffed plumage, and sitting on the floor of the cage. Faecal excretion of DT160 occurred briefly in two birds inoculated with 102 cfu and four birds inoculated with 103 cfu, on most days in five birds inoculated with 105 cfu, and continuously in six birds inoculated with 2 × 108 cfu. DT160 was isolated from the livers of three birds which received 103 cfu, five birds dosed with 105 cfu, and all six birds given 2 × 108 cfu. Following necropsy, histopathological lesions similar to those seen in the natural disease were observed in the liver or spleen of three birds which received 103 cfu, and all birds dosed with ≥105 cfu. CONCLUSION: The results indicate that an isolate of S. Typhimurium DT60 originating from house sparrows in New Zealand is pathogenic to these birds and that the response is dose- dependent. The persistence and excretion of the pathogen may last for at least 10 days. This confirms that sparrows infected with DT160 could be a source of infection to humans and other in-contact animals.

Septicaemia and meningitis caused by infection of New Zealand sea lion pups with a hypermucoviscous strain of Klebsiella pneumoniae

This study describes a syndrome of neonatal septicemia and meningitis in New Zealand sea lions, caused by a strain of Klebsiella pneumoniae that is phenotypically similar to strains causing environmentally-acquired septicemia and neuro-invasive disease in humans. Between late 2006 and early 2010, 123 pups from the Enderby Island breeding colony died of K. pneumoniae infection, with lesions including fibrinous to fibrinosuppurative meningitis, subdural hemorrhage, septic arthritis, herniation and hemorrhage of the cerebellar vermis, lymphadenitis and cellulitis. This infection was responsible for 58% of observed pup mortality over this time period, with most deaths occurring in the latter part of the breeding season (mid February onwards). The results of this study suggest that the pattern of this disease has changed since it was first described in 2002, when most deaths occurred early in the season (early to mid-January), and that it is an important and consistent cause of pup mortality in this population. In addition, a similar disease syndrome and bacterial strain was diagnosed in a single pup in a fragile recolonizing New Zealand sea lion population on mainland New Zealand, and the potential effect on this population is unknown but could have a negative impact on recolonisation at this site.

Epidemiological and molecular evidence of a monophyletic infection with Staphylococcus aureus causing a purulent dermatitis in a dairy farmer and multiple cases of mastitis in his cows.

An epidemiological and molecular investigation of a cutaneous suppurative infection with Staphylococcus aureus in a dairy farmer, occurring concurrently with an outbreak of clinical mastitis in his herd, was carried out. A common aetiology for the diseases in the farmer and his cows was established by combining clinical evidence with a molecular genomic analysis of the bacterial isolates using pulsed field gel electrophoresis of DNA macro-restriction fragments. This case indicates the possibility of the emergence and circulation of anthropozoonotic clones of S. aureus in dairy herds. It also provides further evidence of the severe impact of infection with highly virulent clones on dairy lactating cattle.

Evidence for the clustering of antibacterial resistance phenotypes of enterococci within integrated poultry companies.

From July to December 2006, a panel of 401 enterococci was isolated from carcass rinse samples collected in five poultry processing plants in New Zealand. Agar diffusion assays for nine antibacterial drugs were used to obtain a resistance phenotype for each isolate. Hierarchical clustering techniques and diversity indices showed a high diversity of resistance phenotypes within each plant, with populations of Enterococcus faecalis showing greater heterogeneity than Enterococcus faecium. Bayesian modelling identified three clusters of phenotype patterns within the panel: the E. faecium isolates showed a high probability of containing two distinct clusters, whilst the E. faecalis isolates all grouped together to form the third cluster. The validity of these three clusters was examined using pairwise fixation indices and analysis of variance. Comparing the three clusters to the structure of the participating companies showed that resistance phenotypes for E. faecium isolated from processing plants that were geographically separated but were operated by the same integrated poultry company were more similar than E. faecium isolated from unconnected companies. Company-level management factors, such as the routine use of antibacterial drugs and the genetic line of birds reared, mirrored the structure of these clusters, thus indicating that company-level factors were the dominant selective pressures upon resistance phenotypes across all operating units within these integrated poultry companies.

Serologic Evaluation of New Zealand Sea Lions for Exposure to Brucella and Leptospira spp.

A serologic survey of anti-Brucella and antileptospiral antibodies was conducted on 147 adult, female New Zealand sea lions (Phocarctos hookeri). Most sea lions (n=138) were sampled at Sandy Bay, Enderby Island, Auckland Islands (50°30′S, 166°17′E), January 2000–March 2005. Nine were sampled at Otago, New Zealand (46°0′S, 170°40′E); four in April 2008 and five in March 2009. Serum from one of the Enderby Island females was weakly positive for antibodies to Brucella abortus using the competitive enzyme-linked immunosorbent assay, and one female had a low titer for Leptospira interrogans serovar pomona using the microscope agglutination test. All serum samples from Otago animals were negative. Brucellosis and leptospirosis are therefore considered unlikely to play a major role in population dynamics of these populations, and the low antibody prevalence of these agents suggests that they are an unlikely source of infection for humans, wildlife, or domestic species on mainland New Zealand.

Isolation of Campylobacter spp. from Client‐Owned Dogs and Cats, and Retail Raw Meat Pet Food in the Manawatu, New Zealand

Campylobacter causes acute gastroenteritis in people worldwide and is frequently isolated from food, animals and the environment. The disease is predominately food‐borne but many routes of transmission and sources of infection have been described, including contact with pets. The prevalence of Campylobacter spp. in dogs and cats varies widely, and data on New Zealand pets are limited. This study aimed to investigate the prevalence of Campylobacter spp. in dogs, cats and retail raw meat pet food products in New Zealand and to characterize Campylobacter jejuni isolates using multilocus sequence typing (MLST). Ninety dogs and 110 cats examined at the Massey University Veterinary Teaching Hospital for elective procedures, and fifty locally purchased retail raw meat pet diets were sampled. Two culture protocols combining Bolton broth enrichment and mCCDA and CAT agars in a microaerobic atmosphere at 42°C and 37°C with species identification using PCR were performed. The prevalence of Campylobacter spp., C. jejuni, Campylobacter upsaliensis and Campylobacter helveticus was 36%, 13%, 23% and 1% in dogs and 16%, 5%, 5% and 7% in cats, respectively. One dog had Campylobacter lari confirmed, and three dogs and one cat had multiple Campylobacter spp. detected. Significantly more animals tested positive using CAT than mCCDA agar (P < 0.001). Being neutered, vaccinated for Bordetella bronchiseptica, fed dry diets and brought in for neutering were protective factors for dogs, whereas attendance for dental treatment was a risk factor for cats. Campylobacter spp. were isolated from 28%, C. jejuni 22%, C. lari 6% and Campylobacter coli 6% of food samples. Six isolates positive by Campylobacter genus PCR were identified as Arcobacter butzleri. Poultry meat was more likely to be positive than non‐poultry meat (P = 0.006). Of the 13 C. jejuni pet isolates with full MLST profiles, eight were of different sequence types (ST) and all nine food isolates were of different STs.

Variation in the limit-of-detection of the ProSpecT Campylobacter microplate enzyme immunoassay in stools spiked with emerging Campylobacter species

Campylobacter enteritis in humans is primarily associated with C. jejuni/coli infection. The impact of other Campylobacter spp. is likely to be underestimated due to the bias of culture methods towards Campylobacter jejuni/coli diagnosis. Stool antigen tests are becoming increasingly popular and appear generally less species-specific. A review of independent studies of the ProSpecT® Campylobacter Microplate enzyme immunoassay (EIA) developed for C. jejuni/coli showed comparable diagnostic results to culture methods but the examination of non-jejuni/coli Campylobacter spp. was limited and the limit-of-detection (LOD), where reported, varied between studies. This study investigated LOD of EIA for Campylobacter upsaliensis, Campylobacter hyointestinalis and Campylobacter helveticus spiked in human stools. Multiple stools and Campylobacter isolates were used in three different concentrations (10(4)-10(9)CFU/ml) to reflect sample heterogeneity. All Campylobacter species evaluated were detectable by EIA. Multivariate analysis showed LOD varied between Campylobacter spp. and faecal consistency as fixed effects and individual faecal samples as random effects. EIA showed excellent performance in replicate testing for both within and between batches of reagents, in agreement between visual and spectrophotometric reading of results, and returned no discordance between the bacterial concentrations within independent dilution test runs (positive results with lower but not higher concentrations). This study shows how limitations in experimental procedures lead to an overestimation of consistency and uniformity of LOD for EIA that may not hold under routine use in diagnostic laboratories. Benefits and limitations for clinical practice and the influence on estimates of performance characteristics from detection of multiple Campylobacter spp. by EIA are discussed.

Low levels of antibacterial drug resistance expressed by Gram-negative bacteria isolated from poultry carcasses in New Zealand

Abstract AIM: To provide baseline data on the levels and patterns of antibacterial drug resistance expressed by Gram-negative bacteria isolated from poultry carcasses in New Zealand. METHODS: Between July and December 2006, isolates of Escherichia coli (n=407) and Salmonella spp. (n=3) originating from carcass-rinse samples were submitted by testing laboratories affiliated to five major poultry processing plants. Isolates of Campylobacter jejuni (n=193) originating from retail poultry carcasses in 2005–2006 were retrieved from the Massey University archives. All isolates underwent disc diffusion susceptibility testing against panels of 12 (Enterobacteriaceae) and six (Campylobacter spp.) antibacterial drugs. Cephalothin-resistance in isolates of E. coli was confirmed using ETest strips, and confirmation of the resistance phenotypes for a subset of C. jejuni isolates used microbroth dilution assays. Patterns within the resistance phenotypes of the isolates were investigated using hierarchical clustering, and logistic regression modelling. RESULTS: The majority of isolates (71.5% E. coli, 99% C. jejuni, and all three Salmonella spp. isolates) were fully susceptible to the drugs that were tested. Four (1%) E. coli isolates showed resistance to three or more drugs. The proportions of susceptible E. coli differed between the five processing plants. Resistances were detected in E. coli isolates, using disc diffusion to cephalothin (18.2%), ampicillin (4.4%), tetracycline (4.4%) and gentamicin (1.5%). There was an association between cephalothin-resistant isolates of E. coli and decreased susceptibility to gentamicin. Using ETests to ascertain the minimum inhibitory concentrations (MIC) of E. coli for cephalothin gave inconsistent results. One of 193 C. jejuni isolates was resistant to erythromycin, and microbroth dilution assays confirmed that this panel of C. jejuni was generally susceptible to antibacterial drugs. CONCLUSIONS: The levels of resistance shown by Gram-negative bacteria isolated from chicken carcasses in New Zealand are among the lowest reported around the world. No resistance to extended-spectrum cephalosporin drugs was detected in E. coli, suggesting that CTX-M and AmpC beta-lactamases are rare or absent. Salmonella spp. are rarely isolated from poultry carcasses during routine testing in New Zealand, and the isolates identified during this study were fully susceptible to the drugs tested. A panel of C. jejuni isolates originating from retail poultry carcasses were susceptible to first-line and second-line antibacterial drugs. The use of cephalothin as a marker of resistance to first-generation cephalosporins may not be appropriate for non-type-specific E. coli of animal origin.

Updating the genomic taxonomy and epidemiology of Campylobacter hyointestinalis

Campylobacter hyointestinalis is a member of an emerging group of zoonotic Campylobacter spp. that are increasingly identified in both gastric and non-gastric disease in humans. Here, we discovered C. hyointestinalis in three separate classes of New Zealand ruminant livestock; cattle, sheep and deer. To investigate the relevance of these findings we performed a systematic literature review on global C. hyointestinalis epidemiology and used comparative genomics to better understand and classify members of the species. We found that C. hyointestinalis subspecies hyointestinalis has an open pangenome, with accessory gene contents involved in many essential processes such as metabolism, virulence and defence. We observed that horizontal gene transfer is likely to have played an overwhelming role in species diversification, favouring a public-goods-like mechanism of gene ‘acquisition and resampling’ over a tree-of-life-like vertical inheritance model of evolution. As a result, simplistic gene-based inferences of taxonomy by similarity are likely to be misleading. Such genomic plasticity will also mean that local evolutionary histories likely influence key species characteristics, such as host-association and virulence. This may help explain geographical differences in reported C. hyointestinalis epidemiology and limits what characteristics may be generalised, requiring further genomic studies of C. hyointestinalis in areas where it causes disease.

Genome-by-genome approach for fast bacterial genealogical relationship evaluation

Motivation Large-scale whole-genome sequencing dataset-based studies are becoming increasingly common in pathogen surveillance and outbreak investigations. A highly discriminative and time-efficient bioinformatics tool is needed to transform large amounts of sequencing data into usable biological information. To replace the intuitive, yet inefficient, way of gene-by-gene allele calling algorithm, a new algorithm using genome-by-genome approach was developed. Results Tests showed that the program equipped with the new algorithm achieved significant improvements in allele calling efficiency compared to a conventional gene-by-gene approach. The new program, Fast-GeP, rendered a fast and easy way to infer high-resolution genealogical relationships between bacterial isolates using whole-genome sequencing data. Availability and implementation FAST-GeP is freely available from: https://github.com/jizhang-nz/fast-GeP. Supplementary information Supplementary data are available at Bioinformatics online.