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.

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.

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.

Use of a tetanus toxoid marker to allow differentiation of infected from vaccinated poultry without affecting the efficacy of a H5N1 avian influenza virus vaccine.

Tetanus toxoid (TT) was assessed as a positive marker for avian influenza (AI) virus vaccination in chickens, in a vaccination and challenge study. Chickens were vaccinated twice with inactivated AI H5N2 virus vaccine, and then challenged three weeks later with highly pathogenic AI H5N1 virus. Vaccinated chickens were compared with other groups that were either sham-vaccinated or vaccinated with virus with the TT marker. All sham-vaccinated chickens died by 36 hours postinfection, whereas all vaccinated chickens, with or without the TT marker, were protected from morbidity and mortality following exposure to the challenge virus. Serological testing for H5-specific antibodies identified anamnestic responses to H5 in some of the vaccinated birds, indicating active virus infection.

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.

Seroprevalence of Toxoplasma gondii in wild kangaroos using an ELISA.

Infection with Toxoplasma gondii is a significant problem in Australian marsupials, and can lead to devastating disease and predispose animals to predation. T. gondii infection in kangaroos is also of public health significance due to the kangaroo meat trade. A moderate seroprevalence of T. gondii was observed in a study of western grey kangaroos located in the Perth metropolitan area in Western Australia. Of 219 kangaroos tested, 15.5% (95%CI: 10.7-20.3) were positive for T. gondii antibodies using an ELISA developed to detect T. gondii IgG in macropod marsupials. When compared with the commercially available MAT (modified agglutination test), the ELISA developed was in absolute agreement and yielded a kappa coefficient of 1.00. Of 18 kangaroos tested for the presence of T. gondii DNA by PCR, the 9 ELISA positive kangaroos tested PCR positive and the 9 ELISA negative kangaroos tested PCR negative indicating the ELISA protocol was both highly specific and sensitive and correlated 100% with the more labour intensive PCR assay.

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.

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).

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.

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.

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.

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.

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.

Duration of infection and strain variation in Streptococcus uberis isolated from cows' milk.

The duration of infection and pulsed-field gel electrophoresis (PFGE) types of bovine intramammary Streptococcus uberis isolates were examined. Milk samples were collected in duplicate from all 4 glands of 503 cows from 5 herds within 1 to 3 d of parturition and from 113 cows with clinical mastitis in the same herds throughout lactation. Glands from which S. uberis was isolated were resampled at 28-d intervals.The prevalence of S. uberis was 12% for cows around parturition, and the median duration of infection was 16 d. Cows >2 yr old had a longer duration of infection than 2 yr old cows, and duration varied among herds. A total of 173 different PFGE types were identified from a total of 234 S. uberis isolates. Each farm had a unique set of PFGE types. Only 3 PFGE types were common to each of 3 pairs of cows, and these occurred on the same farm. Where S. uberis was isolated on more than one occasion from a gland, only 55% of the PFGE types were the same across time. For cows with multiple glands infected, only one-half (9 of 18) had the same PFGE type in more than one gland. No predominant PFGE type was identified in any herd. It is concluded that there was wide heterogeneity of PFGE types, that the environment rather than other cows was the likely source of S. uberis infections, and that glands may be infected with multiple S. uberis PFGE types over a lactation.

Some potential sources for transmission of Campylobacter jejuni to broiler chickens.

AIMS: The aim of the study was to determine Campylobacter jejuni contamination and prevalence on fomites moving between broiler farms and the processing plant in the period after cleaning and before departure to harvest chickens. In addition, changes in the proportion of contaminated fomites in the course of a day were assessed. METHODS AND RESULTS: Pooled swab samples were obtained from pallets, crates, wheels of trucks, tractors and forklifts, truck beds, and from drivers' and catchers' boots. After enrichment in Bolton's broth Campylobacter were recovered on modified blood-free Campylobacter selective agar (mCCDA). Isolates were identified using tests for phenotypic and biochemical characteristics. Of the 209 samples collected, 53% were positive for C. jejuni, with all fomites positive except tractor wheels. Pallets had the highest contamination rate at 75%. More than 50% of catchers' boots, drivers' boots, crates and truck wheels were positive. Forty-seven per cent and 31% of truck beds and forklift wheels, respectively, were contaminated. The proportion of contaminated fomites did not change significantly during the day. CONCLUSIONS: This study has identified trucks, forklifts, pallets, crates, drivers' and catchers' boots as potential sources of C. jejuni for broilers. SIGNIFICANCE AND IMPACT OF THE STUDY: Campylobacter jejuni contamination of broiler processing plant fomites was found to be extensive ranging from 31% for truck beds to 75% for pallets. The proportion of contaminated fomites was observed to be similar throughout the day. The impact of contaminated fomites as sources of colonization of broilers with C. jejuni is discussed.

Salmonella Brandenburg - emergence of a new strain affecting stock and humans in the South Island of New Zealand.

AIMS: To report information on the spread of a new strain of Salmonella Brandenburg, which affected livestock and humans in the South Island of New Zealand, and a series of small case studies designed to investigate potential transmission of infection. METHODS: Information on the occurrence and spread of S. Brandenburg in livestock was gathered from laboratory diagnostic submissions, from case studies on the faecal excretion rate in ewes, carrier status of black-backed gulls (Larus dominicanus), spread of S. Brandenburg organisms in sheep yards, infection in lambs going to meat plants, and from post-abortion pathological changes in the reproductive tract of ewes. RESULTS: A newly recognised strain of S. Brandenburg was first diagnosed in aborting sheep from a flock in mid Canterbury in the South Island in 1996. Subsequently, the disease spread to other farms in mid and south Canterbury in 1997 and to Southland and Otago in the lower half of the South Island in 1998-2003. In 1999, the same strain was responsible for abortions in cattle and gastroenteritis in calves and adult cattle. The same strain of bacterium also caused disease in horses, goats, deer, pigs and humans. Spread of the disease on farms was strongly associated with aborting ewes, which resulted in considerable environmental contamination. During the abortion season, black-backed gulls appeared to spread the disease to other farms. Other potential sources of infection were carrier sheep, contaminated water sources and contaminated sheepyard dust. Damage to the reproductive tract may affect the ability of surviving ewes to conceive. CONCLUSION: Important features of this disease are its high morbidity and mortality within a flock or herd, rapid local spread and its role as an occupational, health and safety risk to farm workers and their families.

Pulsed-field gel electrophoresis typing of Campylobacter fetus subsp. fetus isolated from sheep abortions in New Zealand.

AIMS: To genotype Campylobacter fetus subsp. fetus isolates cultured from sheep abortions submitted to diagnostic laboratories in New Zealand during the year 2000 breeding season. To compare the types found nationally with those found in the Hawke's Bay region in 1999, and strains held in the New Zealand Reference Culture Collection, Medical Section (NZRM) from a study published in 1987. METHODS: Campylobacter fetus subsp. fetus isolates cultured by veterinary diagnostic laboratories in the year 2000 breeding season, from sheep abortions from throughout New Zealand, were typed using pulsed-field gel electrophoresis (PFGE). In addition, seven freeze-dried C. fetus subsp. fetus isolates (strain numbers 2939-2945) from the NZRM, representing restriction types a-g found amongst sheep abortion isolates in a study published in 1987, were typed using PFGE. RESULTS: In total, 293 C. fetus subsp. fetus isolates from 200 farms were obtained from veterinary diagnostic laboratories. Twenty-two distinct PFGE profiles were identified amongst the isolates. PFGE type B1 was predominant in each region of New Zealand and was identified from 66% of farms overall. Of the C. fetus subsp. fetus restriction types a-g lodged with the NZRM, 3/7 had PFGE profiles indistinguishable from profiles found in the current study. The other four restriction types had PFGE profiles that were unique but similar to those found in the current study. CONCLUSIONS: PFGE type B1 was predominant amongst the C. fetus subsp. fetus isolates cultured from sheep abortions in each region of New Zealand in the year 2000, as was found in Hawke's Bay in 1999. The similarity between PFGE profiles of C. fetus subsp. fetus sheep abortion isolates from 1987 and 2000, and the relative prevalence of the PFGE groups, suggests that there has been no major genotypic shift in the population of C. fetus subsp. fetus implicated in sheep abortion in New Zealand during this time.

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.