A comparison of methods for extracting DNA from Coxiella burnetii as measured by a duplex qPCR assay.

AIMS: To determine the optimal DNA extraction method for the detection of Coxiella burnetii including the small-cell variant (SCV) by real-time PCR (qPCR) in clinical samples. METHODS AND RESULTS: A duplex qPCR detecting two Coxiella burnetii gene targets (com1 and IS1111a genes) was developed. Each target in this PCR had a sensitivity of one copy number per reaction. DNA extraction methods were compared on spiked negative samples and included a silica column kit, a chloroform separation prior to a silica column method and a chloroform/phenol separation and DNA precipitation method. CONCLUSIONS: The silica column extraction method was more efficient at recovering C. burnetii DNA, from large-cell and small-cell variants, than a chloroform or chloroform/phenol method. The silica column method was useful on spiked human samples including serum, buffy coat and bone marrow samples. SIGNIFICANCE AND IMPACT OF STUDY: This study demonstrated that a simple column kit method is efficient to use for the detection of C. burnetii in clinical samples including the SCV.

Comparative susceptibility of planktonic and 3-day-old Salmonella Typhimurium biofilms to disinfectants.

AIMS: To compare the susceptibility of a 3-day-old biofilm and planktonic Salmonella to disinfectants at different exposure times. We hypothesize that Salmonella biofilms are more resilient to disinfectants compared to planktonic Salmonella. METHODS AND RESULTS: The susceptibility of planktonic cells to disinfectants was tested by a modified version of the Council of Europe suspension test EN 1276. Salmonella biofilms were formed using the Calgary Biofilm Device. Results show that 3-day-old Salmonella biofilms are less susceptible to the disinfectants benzalkonium chloride, chlorhexidine gluconate, citric acid, quaternary ammonium compounds, sodium hypochlorite (SH) and ethanol, compared to planktonic Salmonella. Surprisingly, the results also demonstrate that low concentrations of SH were more effective against a 3-day-old biofilm compared to high concentrations of SH. CONCLUSIONS: While all the disinfectants evaluated were able to reduce biofilm-associated cells at concentrations and contact times sufficient to eliminate planktonic cells, there were still sufficient viable cells remaining in the biofilm to cause further contamination and potential infection. SIGNIFICANCE AND IMPACT OF THE STUDY: Protocols for the use of chemical disinfectants need to include biofilm susceptibility testing. There is a requirement for an effective and standardized tool for determining the susceptibility of biofilms to disinfectants.

Essential veterinary education in the virology of domestic animals, wild animals and birds: diagnosis and pathogenesis of viral infections.

An education in veterinary virology should establish a basis for life-long learning and enable veterinary graduates to address professionally the control and eradication of viral diseases, both locally and globally. It is therefore more important that the curriculum focuses on a sound understanding of the nature and behaviour of viruses and their interactions with animal hosts, rather than imparting detailed information on an ever-increasing number of individual viral diseases in a widening range of animal species. Graduate veterinarians should be prepared with a comprehensive knowledge of the nature of viruses and their close dependence on the hosts thatthey infect, as well as a good understanding of pathogenesis, immunology, epidemiology, diagnostic approaches and control options. All these are necessary if the profession is successfully to meet familiar and new challenges in viral diseases in a wide range of host species, under different management conditions, in various geographic areas of the world.

Integrating the issues of global and veterinary public health into the veterinary education curriculum: an Australian perspective.

This article discusses the integration of global and veterinary public health issues into the Australian veterinary curriculum. Formal veterinary education in Australia has a history of over 100 years and veterinarians have played a major role in the control of zoonotic and transboundary diseases for an even longer period. Australia is the largest exporter of red meat and live animals in the world. Therefore, educating veterinarians to promote and ensure food safety and animal welfare is prominent in Australian veterinary curricula. Veterinary degrees are accredited to allow Australian graduates to work professionally overseas, including in the United Kingdom and United States of America, and, in recent years, globalisation of the student body at Australian veterinary schools has occurred. For this reason, an appropriately broad curriculum is required to produce graduates who are able to address challenges in veterinary public health throughout the world. A Public Health University Network has been established to harmonise the veterinary public health curricula at the various veterinary schools and to develop the 'Australian veterinary public health philosophy', with its links to global issues and the 'One World, One Health' concept. Finally, conclusions are drawn on the implications of veterinary public health teaching in Australia and the preparation of Australian graduates for the global profession.

Identification of uncultured bacteria from abscesses of exotic pet animals using broad-range nested 16S rRNA polymerase chain reaction and Sanger sequencing.

BACKGROUND: The Sanger sequencing technique has been questioned and challenged by advanced high-throughput sequencing approaches. Sanger sequencing seems to be an obsolete technology. However, there are still research problems that could be answered using the Sanger sequencing technology. Fastidious obligate anaerobic bacteria are mostly associated with abscesses in animals. These bacteria are difficult to isolate from abscesses and are frequently excluded due to the bias of conventional bacterial culturing. AIM: This study demonstrated the usefulness of a broad-range polymerase chain reaction (PCR) with Sanger sequencing to identify the majority population of bacteria in abscesses from exotic pet animals. MATERIALS AND METHODS: This study performed a pilot investigation of abscesses from 20 clinical cases (17 rabbits, 2 hedgehogs, and 1 sugar glider) using standard culture methods for both aerobes and anaerobes and broad-range nested PCR targeting the 16S rRNA gene followed by the Sanger sequencing technique. RESULTS: The standard culture and PCR techniques detected bacteria in 9 and 17 of 20 samples, respectively. From the 17 sequencings of the 16S rRNA, 10 PCR products were found to be closely related with obligate anaerobes including Bacteroides spp., Fusobacterium spp., Prevotella spp. Phylogenetic analysis using the rpoB gene revealed that the species for the Bacteroides was thetaiotaomicron and for the Fusobacterium was varium and nucleatum. However, the amplification of the rpoB gene for the Prevotella spp. was unsuccessful. Correlations between the standard culture and PCR techniques were found in 9 (6 positive and 3 negative samples) of 20 samples. Eleven samples were discordant between the standard culture and PCR techniques which were composed of eight samples negative by culture but positive by PCR and three samples had different bacteria by the culture and PCR techniques. CONCLUSION: According to this study, broad-range PCR combined with Sanger sequencing might be useful for the detection of dominant anaerobic bacteria in abscesses that were overlooked based on conventional bacterial culture.

Pulsed-field gel electrophoresis of Campylobacter jejuni sheep abortion isolates.

Campylobacter species are a significant cause of sheep abortion in most sheep-raising countries. In New Zealand, Campylobacter fetus subsp. fetus is the leading cause of diagnosed sheep abortion and the species C. jejuni and C. coli have also been implicated. To date, strain typing information of C. jejuni sheep abortion isolates is limited. The objective of the present study was to genotype C. jejuni isolates cultured from sheep abortions submitted to diagnostic laboratories in New Zealand during the 2000 breeding season, using pulsed-field gel electrophoresis (PFGE). In this study, C. jejuni isolates were cultured from approximately 10% of farms from which Campylobacter species were isolated from sheep abortions in the year 2000. This equated to 25 C. jejuni isolates from 21 farms. These isolates were obtained from the veterinary diagnostic laboratories and strain typed using the molecular typing technique PFGE. Ten distinct PFGE types were identified amongst the isolates. No particular PFGE type was found most frequently amongst these C. jejuni sheep abortion isolates. However, indistinguishable or similar C. jejuni PFGE types were identified from different aborted foetuses from the same flock, consistent with the role of C. jejuni as an infectious cause of abortion in sheep. These strain types were similar or indistinguishable from C. jejuni sheep abortion isolates obtained in 1999 in a smaller study (Mannering, S.A., Marchant, R.M., Middelberg, A., Perkins, N.R., West, D.M., Fenwick, S.G., 2003. Pulsed-field gel electrophoresis typing of C. fetus subsp. fetus from sheep abortions in the Hawke's Bay region of New Zealand. NZ Vet. J. 51, 33-37).

The application of One Health concept to an outdoor problem-based learning activity for veterinary students.

BACKGROUND: The One Health (OH) approach, which seeks to bring together human and animal health, is particularly suited to the effective management of zoonotic diseases across both sectors. To overcome professional silos, OH needs to be taught at the undergraduate level. Here, we describe a problem-based learning activity using the OH approach that was conducted outdoors for 3rd-year veterinary students in Malaysia. MATERIALS AND METHODS: A total of 118 students, divided into two groups, completed the activity which spanned 1½ days at a deer park adjacent to a wilderness area. Students were asked to evaluate the activity using an online survey that had quantitative and qualitative components. RESULTS: Response rate was 69.5%. The activity was rated excellent by 69.5% and good by 30.4%. Levels of satisfaction were high on a range of criteria. 97.5% of students intended to take action in their studies as a result of what they had learned. CONCLUSIONS: Delivery of an outdoor problem-based learning activity using OH approach was very successful in terms of participation, knowledge delivery and understanding, and the willingness of students to integrate OH into their future practice. For the improvement of future programs, the involvement of other disciplines (such as Medical, Biology, Biotechnology, Biomedical, and Public Health) is being considered.

Demonstration of polymorphism among Brucella ovis field isolates by pulsed-field gel electrophoresis.

Brucella ovis is recognized worldwide as an important pathogen of sheep, and has also been identified in farmed deer in New Zealand. Previously, only one strain type of B. ovis has been identified. The objective of this paper was to perform pulsed-field gel electrophoresis (PFGE) on field isolates of B. ovis to determine whether strain variations exist, whether sheep and deer are affected by the same strains, and to compare the performance of the rare-cutting restriction enzymes XbaI and SwaI. Ten B. ovis isolates from sheep and two from deer in New Zealand, as well as the type strain, were subjected to PFGE analysis using both XbaI and SwaI. PFGE of XbaI restriction fragments produced two banding patterns consisting of 27-28 bands, which were found to be 98% similar by cluster analysis, and were named X1 and X1a. PFGE of SwaI restriction fragments resulted in three banding patterns consisting of 13-15 bands each. Ten of the isolates had identical banding patterns and were named S1. One isolate differed by one band, representing a subtype named S1a. Two isolates differed by six bands, representing a different strain type of B. ovis and this was named S2. Cluster analysis showed S2 to be 78% similar to the S1/S1a cluster. Both strain types were isolated from both sheep and deer. Thus, two distinct strain types of B. ovis were identified in New Zealand, which is the first report of more than one strain type being identified worldwide. Neither strain was species-specific for sheep or deer. The restriction endonuclease SwaI was found to be more discriminatory than the enzyme XbaI, which has been used in previous studies.

Prevalence of Yersinia species in goat flocks.

AIMS: To investigate the epidemiology of Yersinia species in healthy goats in New Zealand, in particular to determine the prevalence of farms with infected goats, the prevalence of infected goats on those farms, the serotypes involved, and potential risk factors for carriage. METHODS: A cross-sectional study of the prevalence of Yersinia infection in infected flocks in a study population of thirty commercial goat farms in the Manawatu region of New Zealand. RESULTS: Infection was detected on 60% of farms in an initial study. In a prevalence study on 18 infected farms, the study population comprised 6770 animals (mean of 376, median of 175 and range of 36 to 1295 goats/farm). Of 902 goats (296 < 1 year, 178 1 to 2 years, and 428 > 2 years) sampled from the study population, 135 (73 < 1 year, 21 1 to 2 years, and 41 > 2 years) were excreting Yersinia spp, giving an overall prevalence of 14.97% (95% confidence interval [CI]; 12.8 to 17.4) with individual farm prevalences ranging from 0.0 (+ 7.9) to 58.14% (95% Cl, 43.3 to 71.6). Goats < 1 year were more likely to be infected than 1-2 year and > 2 year old animals (relative risk [RR] = 2.1; 95% Cl, 1.3 to 3.3) and 2.6 (95% Cl, 1.8 to 3.6) respectively), but there was no significant difference between risks for 1 to 2 year and > 2 year goats (RR = 1.2; 95% CI, 0.7 to 2.0). Yersinia enterocolitica was the most common species isolated in the youngest age group, with prevalence declining with increasing age, while other species were more common in the older age groups. CONCLUSION: Yersinia infections were common in goats in the study region, with younger animals apparently more susceptible to infection and in particular to infection with Y enterocolitica. The prevalence on infected farms appeared to decrease as flock size increased and to increase as stocking rates and the number of paddocks grazed increased.

A cohort study of Yersinia infection in goats.

OBJECTIVE: To determine the temporal pattern of Yersinia infections in three goat flocks and examine the influence of management and seasonal factors on the incidence of those infections over a 1-year period. METHODS: A longitudinal study involving monthly culture of faeces for Yersinia spp. from age groups of randomly selected goats on three farms in the Manawatu region of New Zealand. RESULTS: The incidence of excretion of potentially pathogenic Yersinia (Yersinia pseudotuberculosis and Y enterocolitica biotypes 2, 3 and 5) peaked in winter and fell in summer. In contrast, environmental Yersinia (Y enterocolitica biotype 1A, Y frederiksenii, Y intermedia and Y rohdei) showed no clear pattern of seasonal variation. Pathogenic Yersinia were more prevalent in young animals than in adults, while environmental Yersinia were more prevalent in adults. The same type was isolated from the same animal in two or more successive months in about 20 to 25% of cases, and in the remaining cases there was a gap of at least one month between successive isolations, with many animals yielding a particular type on only a single occasion. A notable difference was that with the potentially pathogenic types, no animal had more than one period of time when it was found to be excreting a particular type, suggesting that immunity develops following exposure. In contrast, it was common for environmental types to be isolated from the same animal throughout the study period. Two goats were suspected to have developed clinical yersiniosis but all remaining infected animals showed no clinical signs of infection. CONCLUSIONS: Asymptomatic Yersinia carriage was common in goats in New Zealand, with a clear seasonal and age group pattern of infection with potentially pathogenic types. There was evidence that immunity developed to potentially pathogenic types. This is the first time that Y rohdei has been isolated from goats.

Unusual Yersinia enterocolitica strains recovered from domestic animals and people in New Zealand.

Twenty five isolates of Yersinia recovered from the faeces of cattle, sheep, goats and people in New Zealand were characterised biochemically and serologically and were found to be unrelated to any known Yersinia species. Two phenotypes were identified biochemically. Testing against all known O-factors of Yersinia showed that the isolates were serologically unique and two new serotypes were proposed, O:77 and O:78. Deoxyribonucleic acid relatedness studies indicated that the strains formed a homogeneous group, closely related to Yersinia enterocolitica. The definitive status of the isolates has not yet been decided.

A comparison of bacterial adherence to bare hands and gloves following simulated contamination from a beef carcass.

One of the risks for contamination of edible product in the pre-inspection area of processing lines in meat plants is cross contamination. This can occur directly as a result of carcass-to-carcass contact or indirectly via knives or the hands of butchers. Standard procedures require that operators rinse their hands and knives to remove any visible contamination. In New Zealand, protective gloves are not allowed in the pre-inspection area because they are considered a potential risk for cross contamination until the carcasses have passed the final meat inspection. However, the risk of injury to the bare hands is as high in this area as in other parts of the plant, where such gloves are permitted. There is therefore a need to evaluate the risk of bacterial cross contamination via bare hands and via protective gloves. The present study compared bacterial adherence to bare hands and to gloves after rinsing for 5 s in a shower of water at 40 degrees C and after rinsing gloves in hotter water (60 degrees C) following simulated contact with the hide of a recently slaughtered animal. Under laboratory conditions there were no statistically significant differences between bacterial adherence to bare hands or to gloves rinsed in water at 40 degrees C or 60 degrees C.

Genomic typing of Streptococcus uberis isolates from cases of mastitis, in New Zealand dairy cows, using pulsed-field gel electrophoresis.

Three hundred and forty-two Streptococcus uberis isolates were cultured from milk samples from subclinical and clinical cases of dairy cattle mastitis. The samples were collected from 15 different New Zealand farming regions, including eight specific farms, during field research trials and veterinary diagnostic investigations. Pulsed-field gel electrophoresis was used to determine and compare the degree of genetic dissimilarity between the restriction endonuclease fragment pattern of the 342 New Zealand and a single United States S. uberis isolate. The 343 isolates exhibited 330 different restriction endonuclease fragment patterns. The United States isolate had a pattern unlike any of the New Zealand isolates. Most of the isolates were genetically different strains (pattern deferred by at least 33%), but identical patterns were noted within the same or different quarters of an individual cow, different cows within the same farm, and from different cows from the same or different districts, farming regions or islands. Seven of the eight selected farms had at most only one pair of isolates with banding patterns, which differed by less than 33%. A high degree of dissimilarity was noted in individual herds in which all the samples were collected on the same day or over a 2-year period. The high degree of dissimilar isolates is an indication that S. uberis infections in New Zealand dairy cattle are largely due to the opportunistic nature of the organism in the cows' environment. Prevention and treatment of S. uberis mastitis will therefore need to be directed at a multitude of different strains present throughout the country as well as in individual herds.

Free available chlorine in commercial broiler chicken drinking water in New Zealand.

Free available chlorine (FAC) concentrations in drinking water supplied to broiler chickens grown commercially in New Zealand were monitored for 11 farms in two companies. Different sites within a growout house were examined at different times of the day to determine spatial and temporal differences in FAC concentrations. Taps provided water with significantly higher FAC concentrations than did drinkers. There were no significant differences between the concentrations of FAC taken from various drinkers around the growout house. There were differences in the variations of measurements taken from the same drinker within a growout house at different times of the day, with variations increasing in the afternoon. No growout houses provided an average FAC content of 2 ppm, the suggested standard in one company. Three growout houses consistently met the chlorine concentration of 0.2 ppm suggested by the New Zealand Drinking Water Standards.

An on-farm study of the epidemiology of Actinobacillus pleuropneumoniae infection in pigs as part of a vaccine efficacy trial.

Thirty cohort pigs were followed from birth to slaughter to study epidemiological patterns of porcine pleuropneumonia caused by Actinobacillus pleuropneumoniae. The study was conducted within a larger 380-animal study of vaccination against Mycoplasma hyopneumoniae and A. pleuropneumoniae in a 340-sow farrow-to-finish piggery with 4-month weaning, operating a continuous system of intensive production in the North Island of New Zealand. The cohort pigs were randomly allocated into two equal groups: vaccinated and control. Pigs in the first group were vaccinated at 2 and 4 weeks of age with both M. hyopneumoniae vaccine and A. pleuropneumoniae vaccine at separate vaccination sites. A series of nasal swabs was taken at 4, 8, 10, 11, 12, 14, 16 and 18 weeks of age. Each swab was streaked onto the surface of a selective medium on the farm and the plates were immediately transported to a laboratory and incubated at 37 degrees C for 5 days. After the trial, pigs were slaughtered at an average of 132 days of age, lungs were examined and samples taken for bacteriological culture and isolation. Thirty-five out of 256 samples produced haemolytic colonies which were Gram-negative, V-factor-dependent and positive to the CAMP test. A. pleuropneumoniae was first isolated at 4 weeks of age from one vaccinated pig. This finding suggests that piglets became infected in the farrowing pen and the source of infection might be a carrier sow. The interval-specific cumulative incidence of A. pleuropneumoniae infection reached a maximum of 54% and 40% at 11 weeks of age in the vaccinated and control groups, respectively. Infection status of the litter is considered to be a factor influencing morbidity in infected herds during weaner and grower periods. Our results suggest that simultaneous vaccination with M. hyopneumoniae and A. pleuropneumoniae vaccines at 2 and 4 weeks of age might lessen the prevalence but cannot absolutely prevent A. pleuropneumoniae infection during the weaner or grower-finisher periods.

A comparison of Salmonella serotypes isolated from New Zealand sea lions and feral pigs on the Auckland Islands by pulsed-field gel electrophoresis.

The Salmonella serotypes S. Cerro and S. Newport were isolated from New Zealand sea lions (Phocarctos hookeri) and feral pigs on the Auckland Islands in the New Zealand subantarctic region. The isolates were typed by pulsed-field gel electrophoresis using Xba1 as the restriction enzyme. The isolates were indistinguishable, which suggests that Salmonella infection cycles between sea lions and pigs in this environment. Apart from a previous isolation from a single New Zealand fur seal (Arctocephalus forsteri), S. Newport has not been recorded in any animals from New Zealand, but it is associated with gastroenteritis in humans. Contamination of the marine environment by human waste is a possible source of infection for marine mammals and warrants further investigation.

Yersinia enterocolitica is a common cause of gastroenteritis in Auckland.

AIMS: Infections with Yersinia enterocolitica are a significant cause of gastroenteritis in many countries, However, little information is available on the incidence of human disease in New Zealand. A study was performed between January 1988 and December 1993 to investigate aspects of the epidemiology of yersiniosis in the Auckland region including prevalence, age and sex distribution, strain definition and seasonal occurrence. METHODS: Culture of 231 128 faecal samples for enteric pathogens was performed at Diagnostic Laboratory, a community laboratory in Auckland, between January 1988 and December 1993. All yersinia isolates recovered were sent to the department of veterinary pathology and public health, Massey University for further typing. Relevant data were entered into a database and reviewed at the end of the study period. RESULTS: 1469 of the samples, representing 941 cases, were positive for strains of yersinia during the study period, a crude isolation rate of 0.6%. A single strain from each of the cases was further typed and yersinia enterocolitica was found to constitute the majority of the isolates (918), making it the third most common enteric pathogen isolated after campylobacter and salmonella. Of the strains isolated, 98% were recognised human pathogens, with biotype 4, serotype 0:3 being the most frequently recovered, as it is worldwide. No clear seasonal pattern emerged although most isolations were made in the autumn-winter period. The age distribution showed two peaks, 0-4 (17.6%) and 25-29 year olds (13%). Infections in males outnumbered those in females (56:44). CONCLUSION: The results of this study show that Y enterocolitica is an important enteric pathogen in the Auckland region, particularly in children and young adults and it has identified a need for a case-control study to assess the relevant risk factors for acquisition of Y enterocolitica infections in New Zealand.

The effects of parenteral iron dextran and/or desferrioxamine on the development of experimental pseudotuberculosis in the domestic chicken (Gallus domesticus).

Abstract The development of disease following oral challenge with Yersinia pseudotuberculosis (serotype 11) was compared in four groups of five birds treated with a parenteral dose of 10 mg iron dextran (Imferon), 10 mg of iron dextran plus 10 mg of the chelating agent desferrioxamine (Desferal), 10 mg of desferrioxamine or 10 mg of dextran 2 days before the experiment. Four groups of two birds received the above treatment regimens but no bacterial challenge. In iron dextran treated birds, oral challenge resulted in faecal shedding for the 10 day duration of the experiment, whereas in those birds which received dextran or desferrioxamine alone, the duration of faecal shedding was significantly less. Serological titres to the lipopolysaccharide antigen of the challenge bacteria were also lower in the groups not pretreated with iron dextran. The birds pretreated with iron dextran had diarrhoea and were clinically unwell 2 days following the initial oral challenge. Birds not given iron dextran showed no clinical signs of disease. Histological examination of five selected areas in the liver, spleen and intestine of each bird indicated that birds in the groups treated with iron dextran prior to bacterial challenge had significantly more intestinal lesions than birds in the groups not treated with iron. In contrast, there were significantly more lesions in the spleens of birds not pretreated with iron dextran. There was no evidence of stainable iron in the livers of birds challenged with Y pseudotuberculosis 10 days after an injection of 10 mg of iron dextran. This is in contrast to birds given iron dextran and no bacteria. It was concluded that pretreatment of birds with iron dextran resulted in more severe clinical disease, prolonged faecal shedding with associated intestinal lesions and higher serological titres to bacterial antigen. The number of lesions in the spleen and liver was not necessarily correlated with the severity of clinical disease, and in all infected birds the hepatic iron levels were significantly lower than in the non-infected control birds 10 days after oral challenge. It seems probable that the chicken has a high requirement for iron during infection with Y pseudotuberculosis and mobilises stored and exogenously supplied iron for tissue repair and immunological function.

Meningitis in a dog caused by Prevotella oralis.

Prevotella oralis, an obligate Gram-negative anaerobe, was shown to be the cause of meningitis in a two-year-old crossbreed dog. The dog had a six-week history of lethargy and neck pain. On presentation, the animal was reluctant to walk, had a stiff gait and neck pain on flexion. Analysis of cerebrospinal fluid documented a neutrophilic pleocytosis (7488 cells/microl) and the presence of occasional intracellular bacterial coccobacilli. The aetiological diagnosis was confirmed by routine aerobic and anaerobic culture, and identification of the isolates. This report also reviews the literature of the documented cases of bacterial meningitis in dogs.