Identification of bla(CMY-7) and associated plasmid-mediated resistance genes in multidrug-resistant Escherichia coli isolated from dogs at a veterinary teaching hospital in Australia

Objectives: To determine clonality and identify plasmid-mediated resistance genes in 11 multidrug-resistant Escherichia coli (MDREC) isolates associated with opportunistic infections in hospitalized dogs in Australia. Methods: Phenotypic (MIC determinations, modified double-disc diffusion and isoelectric focusing) and genotypic methods (PFGE, plasmid analysis, PCR, sequencing, Southern hybridization, bacterial conjugation and transformation) were used to characterize, investigate the genetic relatedness of, and identify selected plasmid-mediated antimicrobial resistance genes, in the canine MDREC. Results: Canine MDRECs were divided into two clonal groups (CG 1 and 2) with distinct restriction endonuclease digestion and plasmid profiles. All isolates possessed bla(CMY-7) on an similar to 93 kb plasmid. In CG 1 isolates, bla(TEM), catA1 and class 1 integron-associated dfrA17-aadA5 genes were located on an similar to 170 kb plasmid. In CG 2 isolates, a second similar to 93 kb plasmid contained bla(TEM) and unidentified class 1 integron genes, although a single CG 2 strain carried dfrA5. Antimicrobial susceptibility profiling of E. coli K12 transformed with CG 2 large plasmids confirmed that the bla(CMY-7)-carrying plasmid did not carry any other antimicrobial resistance genes, whereas the bla(TEM)/class 1 integron-carrying plasmid carried genes conferring resistance to tetracycline and streptomycin also. Conclusions: This is the first report on the detection of plasmid-mediated bla(CMY-7) in animal isolates in Australia. MDREC isolated from extraintestinal infections in dogs may be an important reservoir of plasmid-mediated resistance genes.

Real-time PCR in the microbiology laboratory

Use of PCR in the field of molecular diagnostics has increased to the point where it is now accepted as the standard method for detecting nucleic acids from a number of sample and microbial types. However, conventional PCR was already an essential tool in the research laboratory. Real-time PCR has catalysed wider acceptance of PCR because it is more rapid, sensitive and reproducible, while the risk of carryover contamination is minimised. There is an increasing number of chemistries which are used to detect PCR products as they accumulate within a closed reaction vessel during real-time PCR. These include the non-specific DNA-binding fluorophores and the specific, fluorophore-labelled oligonucleotide probes, some of which will be discussed in detail. It is not only the technology that has changed with the introduction of real-time PCR. Accompanying changes have occurred in the traditional terminology of PCR, and these changes will be highlighted as they occur. Factors that have restricted the development of multiplex real-time PCR, as well as the role of real-time PCR in the quantitation and genotyping of the microbial causes of infectious disease, will also be discussed. Because the amplification hardware and the fluorogenic detection chemistries have evolved rapidly, this review aims to update the scientist on the current state of the art. Additionally, the advantages, limitations and general background of real-time PCR technology will be reviewed in the context of the microbiology laboratory.

A comparison of the responses of first and second year veterinary science students to group project work

A change in curriculum permitted a direct and simultaneous comparison between first and second year responses to group project work while assuming similar prior experience with this method of learning. Responses were obtained by a survey form and by meetings with individual groups. Overall, there were no differences between first and second year responses, although analyses of gender responses suggested trends whereby males indicated they had developed greater creativity and felt they had contributed more to the group. The majority of students responded that group project work was a positive experience and a useful learning experience.

Survey for papillomatous digital dermatitis in Australian dairy cattle

Objective To determine whether Treponema-associated papillomatous digital dermatitis (PDD) occurs in Australian dairy cattle. Design Mail-out questionnaire and histological and bacteriological examination of biopsy tissue from suspect PDD lesions. Procedure The questionnaire was mailed to 375 veterinarians to evaluate their knowledge of PDD, determine if they had observed the disease in Australian dairy cattle, and to request biopsy material from suspicious cases. Biopsies were examined for histological and bacteriological evidence of PDD, including for the presence of spirochaetes. Results Eighty-eight replies to the questionnaire were received (23.5%). Of 52 respondents who were aware of PDD as a possible cause of lameness, 26 reported observing the condition in Australian cattle. Of 32 respondents who were unaware of the condition, 6 reported observing lesions that might have been PDD. The majority of reports of PDD-like lesions came from the southern Australian states, the condition occurring during periods of high rainfall and proving responsive to topical or parenteral application of antimicrobials. Biopsies from five erosive lesions showed histological similarity to PDD whereas biopsies from five proliferative lesions were consistent with chronic inflammation, fibroma or cutaneous papilloma. The presence of spirochaetes was not demonstrated in any of the lesions by histological or bacteriological methods. Conclusion Anecdotal reports and analysis of biopsy material confirm that a condition similar to PDD does occur sporadically in dairy cattle in southern Australia. However, this condition has so far not been shown to be associated with the presence of spirochaetes in the lesions.

Validation of assembled nucleic acid-based tests in diagnostic microbiology laboratories

Medical microbiology and virology laboratories use nucleic acid tests (NAT) to detect genomic material of infectious organisms in clinical samples. Laboratories choose to perform assembled (or in-house) NAT if commercial assays are not available or if assembled NAT are more economical or accurate. One reason commercial assays are more expensive is because extensive validation is necessary before the kit is marketed, as manufacturers must accept liability for the performance of their assays, assuming their instructions are followed. On the other hand, it is a particular laboratory's responsibility to validate an assembled NAT prior to using it for testing and reporting results on human samples. There are few published guidelines for the validation of assembled NAT. One procedure that laboratories can use to establish a validation process for an assay is detailed in this document. Before validating a method, laboratories must optimise it and then document the protocol. All instruments must be calibrated and maintained throughout the testing process. The validation process involves a series of steps including: (i) testing of dilution series of positive samples to determine the limits of detection of the assay and their linearity over concentrations to be measured in quantitative NAT; (ii) establishing the day-to-day variation of the assay's performance; (iii) evaluating the sensitivity and specificity of the assay as far as practicable, along with the extent of cross-reactivity with other genomic material; and (iv) assuring the quality of assembled assays using quality control procedures that monitor the performance of reagent batches before introducing new lots of reagent for testing.

West Nile virus vaccines

West Nile virus (WNV) is a mosquito-borne flavivirus that is emerging as a global pathogen. In the last decade, virulent strains of the virus have been associated with significant outbreaks of human and animal disease in Europe, the Middle East and North America. Efforts to develop human and veterinary vaccines have taken both traditional and novel approaches. A formalin-inactivated whole virus vaccine has been approved for use in horses. DNA vaccines coding for the structural WNV proteins have also been assessed for veterinary use and have been found to be protective in mice, horses and birds. Live attenuated yellow fever WNV chimeric vaccines have also been successful in animals and are currently undergoing human trials. Additional studies have shown that immunisation with a relatively benign Australian variant of WNV, the Kunjin virus, also provides protective immunity against the virulent North American strain. Levels of efficacy and safety, as well as logistical, economic and environmental issues, must all be carefully considered before vaccine candidates are approved and selected for large-scale manufacture and distribution.

Application of nox-restriction fragment length polymorphism for the differentiation of Brachyspira intestinal spirochetes isolated from pigs and poultry in Australia

Sixty-nine intestinal spirochetes isolated from pigs and poultry in eastern Australia were selected to evaluate the effectiveness of a species-specific PCR-based restriction fragment length polymorphism (RFLP) analysis of the Brachyspira nox gene. For comparative purposes, all isolates were subjected to species-specific PCRs for the pathogenic species Brachyspira hyodysenteriae and Brachyspira pilosicoli, and selected isolates were examined further by sequence analysis of the nox and 16S ribosomal RNA genes. Modifications to the original nox-RFLP method included direct inoculation of bacterial cells into the amplification mixture and purification of the PCR product, which further optimized the nox-RFLP for use in a veterinary diagnostic laboratory, producing sufficient product for both species identification and future comparisons. Although some novel profiles that prevented definitive identification were observed, the nox-RFLP method successfully classified 45 of 51 (88%) porcine and 15 of 18 (83%) avian isolates into 5 of the 6 recognized species of Brachyspira. This protocol represents a significant improvement over conventional methods currently used in veterinary diagnostic laboratories for rapid specific identification of Brachyspira spp. isolated from both pigs and poultry.

Stress in veterinary science students: A study at the University of Queensland

This paper reports on the results of a survey of selected University of Queensland (UQ) veterinary students aimed at elucidating factors causing stress during the five undergraduate years of the program. Students from each of the five years were asked to form six- or seven-member focus groups. Each focus group was then interviewed and their opinions sought on causes of ongoing stress and the ranking of those causes into predetermined categories. They were also asked to give their opinions on counseling services available within the university and what, if any, services they would like to see in place to help students with stress-related problems. Students in the first, third, and fourth years of the program rated academic issues as the most likely causes of ongoing stress, while students in the second and fifth years of the program ranked lifestyle and financial issues as more likely to cause ongoing stress. in most cases, students coped well with these causes of stress and tended not to use counseling services available to all UQ students. When faced with stressful issues, students looked to their classmates or family members for help and not to university counseling services. Students were also happy to approach staff members in the Veterinary School when faced with a problem. The authors nevertheless conclude that mechanisms set in place at the undergraduate level to help veterinary students cope with stress should particularly benefit those students when they become new graduates and are faced with the stresses of veterinary practice.

A comparison of responses to group learning between first-year Asian and first-year Australian veterinary science students

Introduction - Group learning has been used to enhance deep (long-term) learning and promote life skills, such as decision making, communication, and interpersonal skills. However, with increasing multiculturalism in higher education, there is little information available as to the acceptance of this form of learning by Asian students or as to its value to them. Methodology - Group-learning projects, incorporating a seminar presentation, were used in first-year veterinary anatomical science classes over two consecutive years (2003 and 2004) at the School of Veterinary Science, University of Queensland. Responses of Australian and Asian students to survey forms evaluating the learning experience were analyzed and compared. Results - All students responded positively to the group learning, indicating that it was a useful learning experience and a great method for meeting colleagues. There were no significant differences between Asian and Australian students in overall responses to the survey evaluating the learning experience, except where Asian students responded significantly higher than Australian students in identifying specific skills that needed improving. Conclusions - Group learning can be successfully used in multicultural teaching to enhance deep learning. This form of learning helps to remove cultural barriers and establish a platform for continued successful group learning throughout the program.