Differential and defective transcription of koala retrovirus indicates the complexity of host and virus evolution.

Koala retrovirus (KoRV) is unique amongst endogenous (inherited) retroviruses in that its incorporation to the host genome is still active, providing an opportunity to study what drives this fundamental process in vertebrate genome evolution. Animals in the southern part of the natural range of koalas were previously thought to be either virus-free or to have only exogenous variants of KoRV with low rates of KoRV-induced disease. In contrast, animals in the northern part of their range universally have both endogenous and exogenous KoRV with very high rates of KoRV-induced disease such as lymphoma. In this study we use a combination of sequencing technologies, Illumina RNA sequencing of 'southern' (south Australian) and 'northern' (SE QLD) koalas and CRISPR enrichment and nanopore sequencing of DNA of 'southern' (South Australian and Victorian animals) to retrieve full-length loci and intregration sites of KoRV variants. We demonstrate that koalas that tested negative to the KoRV pol gene qPCR, used to detect replication-competent KoRV, are not in fact KoRV-free but harbour defective, presumably endogenous, 'RecKoRV' variants that are not fixed between animals. This indicates that these populations have historically been exposed to KoRV and raises questions as to whether these variants have arisen by chance or whether they provide a protective effect from the infectious forms of KoRV. This latter explanation would offer the intriguing prospect of being able to monitor and selectively breed for disease resistance to protect the wild koala population from KoRV-induced disease.

Antimicrobial susceptibility, plasmid replicon typing, phylogenetic grouping, and virulence potential of avian pathogenic and faecal Escherichia coli isolated from meat chickens in Australia.

Globally, avian colibacillosis is a leading cause of morbidity and mortality in poultry, associated with economic losses and welfare problems. Here, clinical avian pathogenic E. coli isolates (CEC; n = 50) and faecal E. coli isolates from healthy (FEC; n = 187) Australian meat chickens collected between 2006 and 2014 were subjected to antimicrobial susceptibility testing, phylogenetic grouping, plasmid replicon (PR) typing, multilocus sequence typing, and virulence gene (VG) profiling. Extended-spectrum cephalosporin (ESC)- and fluoroquinolone (FQ)-resistant E. coli isolates underwent further genetic characterization. Significant proportions of CEC and FEC were, respectively, susceptible (13/50; 48/187) or MDR (9/50; 26/187) to 20 tested antimicrobials. Phylogenetic groups A and C, and PR types IncFIB and IncFrep were most represented. Five tested CEC-associated VGs were more prevalent in CEC (≥ 90%) than FEC (≤ 58%). Some isolates (CEC n = 3; FEC n = 7) were resistant to ESCs and/or FQs and possessed signature mutations in chromosomal FQ target genes and plasmid-mediated qnrS, blaCMY-2, and blaDHA-1 genes. Sequence type 354 (n = 4), associated with extraintestinal infections in a broad range of hosts, was prevalent among ESC- and/or FQ-resistant FEC. This study confirmed existence of a small reservoir of ESC- and FQ-resistant E. coli in Australian commercial meat chickens despite absence of use in the industry of these drugs. Otherwise, diversity of VGs and PR types in both FEC and CEC populations was identified. We hypothesize that the source of ESC- and FQ-resistant E. coli is external to poultry production facilities.RESEARCH HIGHLIGHTSLow-level resistance to older and newer generation antimicrobial drugs detected.The most common sequence type (ST) associated with FQ resistance was ST354 (4/10).A small proportion of CEC (n = 3) and FEC (n = 7) were resistant to ESCs and/or FQs.

Antimicrobial effects of cinnamon essential oil and cinnamaldehyde combined with EDTA against canine otitis externa pathogens.

AIMS: The antimicrobial activity of cinnamon essential oil and cinnamaldehyde against bacterial and fungal pathogens associated with canine otitis externa, as well as the effect of their combination with EDTA were investigated. METHODS AND RESULTS: Antimicrobial susceptibility testing was performed using the broth microdilution method while spot-plating technique was used to determine their bactericidal activity. Time-kill kinetics and checkerboard assays were performed to confirm the bactericidal activity and combination effects of the compounds. Cinnamon oil and cinnamaldehyde exhibited antimicrobial activity against Gram-positive and Gram-negative pathogens, as well as Malassezia pachydermatis. Synergistic interaction was shown when EDTA (672 µg ml-1 ) was combined with cinnamon oil (41 µg ml-1 ) and cinnamaldehyde (22 µg ml-1 ) against Pseudomonas aeruginosa. Cinnamaldehyde exhibited significantly stronger antimicrobial activity than cinnamon bark oil. CONCLUSIONS: Cinnamon essential oil and cinnamaldehyde, either used alone or in combination with EDTA, were effective against the causative micro-organisms of canine otitis externa. The data suggest that cinnamaldehyde could be a promising antimicrobial agent against canine otitis externa. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that cinnamon essential oil and cinnamaldehyde, especially the latter, could be used in combination with EDTA as novel treatment for sensitive and resistant bacterial and fungal pathogens involved in canine otitis externa.

A novel quantitative polymerase chain reaction to monitor urinary tract mycoplasma infection in a dog.

The aim of the study was to develop a quantitative real-time PCR assay for diagnosis and monitoring of mycoplasma urinary tract infections (UTI) in a dog. An English Cocker Spaniel dog with the history of urinary tract infection was physically examined and laboratory findings identified chronic renal insufficiency and urinary tract infection. Attempts to culture organisms from pyuric urine failed, and empirical antibiotic therapy did not resolve the pyuria. A mycoplasma species most closely resembling Ureaplasma canigenitalium was identified in urine samples by conventional PCR and sequencing. A quantitative PCR method was developed to monitor and finally verify successful treatment. This novel approach to monitoring mycoplasma urinary tract infections is conceptually simple, and provides rapid results. It may have wider application in monitoring treatment efficacy for infections with other Mycoplasma spp. as well as additional organisms that are difficult to culture. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, we highlight two different findings, detection of Ureaplasma canigenitalium in a dog with chronic urinary tract infection and development of a quantitative real-time PCR test to track treatment results in an infected dog. This report is the first report of detection of U. canigenitalium in one dog in Australia. This novel qPCR method for monitoring mycoplasma urinary tract infections is conceptually simple and provides results fast. It will have wider applications in monitoring treatment efficacy for infections with mycoplasmas and mycoplasma-like organisms that are difficult to culture, and provides a sensitive guide to treatment progress.

Sporadic bovine encephalopathy caused by Chlamydia pecorum secondary to bovine viral diarrhoea virus infection in calves in South Australia.

BACKGROUND: Despite bovine viral diarrhoea virus and Chlamydia pecorum being important endemic diseases of cattle, there are limited reports of theirco-occurrence. CASE REPORT: Several 12-18-week-old, weaned Hereford calves presented with ill-thriftiness and neurological signs on a mixed cattle and sheep farm in South Australia in July 2021. Immune suppression resulting from transient infection with bovine viral diarrhoea virus (BVDV) is implicated in predisposing to infection with Chlamydia pecorum, the causative agent of sporadic bovine encephalopathy (SBE). Chlamydia spp. are difficult to culture in vitro or definitively identify based on current standard molecular based tests. In this case, diagnosis was confirmed by immunohistochemistry. CONCLUSION: To the authors' knowledge, this case report is the first to document BVDV transient infection occurring in conjunction with SBE. Given the current high prevalence of BVDV on Australian farms, such co-infections may have significant future clinical relevance. This case also highlights the need for appropriate tests, such as immunohistochemistry to demonstrate the causative organism in histological lesions and thus reduce the occurrence of false negative diagnosis.

Assessing natural isothiocyanate air emissions after field incorporation of mustard cover crop.

A regional air assessment was performed to characterize volatile natural isothiocyanate (NITC) compounds in air during soil incorporation of mustard cover crops in Washington State. Field air sampling and analytical methods were developed specific to three NITCs known to be present in air at appreciable concentrations during/after field incorporation. The maximum observed concentrations in air for the allyl, benzyl, and phenethyl isothiocyanates were respectively 188, 6.1, and 0.7 µg m(-3) during mustard incorporation. Based on limited inhalation toxicity information, airborne NITC concentrations did not appear to pose an acute human inhalation exposure concern to field operators and bystanders.

Risk factors for multidrug-resistant Escherichia coli rectal colonization of dogs on admission to a veterinary hospital.

This study aimed to identify risk factors for intestinal colonization with multidrug-resistant (MDR) E. coli in dogs on admission to a veterinary teaching hospital. Exposures to potential risk factors, including prior treatments, hospitalizations and interventions during the 42 days prior to admission were assessed for 82 case admissions and 82 time-matched controls in a retrospective prevalence-based case-control study of 20 months duration. On multivariable analyses, risk of MDR E. coli colonization on admission was increased with prior hospitalization for 4-7 days and >7 days relative to shorter periods, and in dogs that had prior diagnostic imaging techniques. Univariable analyses indicated that risk was increased following prior treatment with several antimicrobial agents. However, on multivariable analysis, administration of fluoroquinolones was associated with increased risk but risk did not appear to increase following administration of other antimicrobials. These results can inform management of canine patients and infection control procedures to mitigate the risk of clinical disease due to MDR bacteria in hospitalized dogs.

Risk factors for dogs becoming rectal carriers of multidrug-resistant Escherichia coli during hospitalization.

This study aimed to identify risk factors for dogs becoming rectal carriers of multidrug-resistant (MDR) Escherichia coli while hospitalized in a veterinary teaching hospital. Exposures to potential risk factors, including treatments, hospitalization, and interventions during a 42-day pre-admission period and hospitalization variables, were assessed for 90 cases and 93 controls in a retrospective, risk-based, case-control study. On multivariable analyses, hospitalization for >6 days [odds ratio (OR) 2·91-8·00], treatment with cephalosporins prior to admission (OR 5·04, 95% CI 1·25-20·27), treatment with cephalosporins for >1 day (OR 5·18, 95% CI 1·86-14·41), and treatment with metronidazole (OR 7·17, 95% CI 1·01-50·79) while hospitalized were associated with increased risk of rectal carriage of MDR E. coli during hospitalization. The majority of rectal isolates obtained during the study period conformed to MDR E. coli clonal groups previously obtained from extraintestinal infections. These results can assist the development of improved infection control guidelines for the management of dogs in veterinary hospitals to prevent the occurrence of nosocomial clinical infections.

Haematological reference intervals of wild southern Australian koalas (Phascolarctos cinereus).

OBJECTIVE: Current haematology reference intervals (RIs) for koalas were developed in northern Australian koalas, using low numbers and/or individuals of unknown Chlamydia pecorum and koala retrovirus (KoRV) status. This study developed haematological RIs for wild, clinically healthy southern Australian koalas of known C. pecorum and KoRV infection status and investigated the effects of population, age and sex. METHODS: Haematological RIs were determined for 138 clinically healthy South Australian koalas (Mount Lofty Ranges [MLR], n = 68; Kangaroo Island, n = 70) examined in April 2016 and February 2017, respectively. C. pecorum and KoRV status were determined by PCR. RESULTS: RIs for southern koala haematological parameters were established for all koalas based on the finding that there were limited differences in haematological values in koalas with subclinical C. pecorum or KoRV infections (P > 0.05), except KoRV-infected koalas had a lower haematocrit than noninfected koalas. MLR koalas had significantly lower erythrocyte mass and leucocyte counts than Kangaroo Island koalas. Young koalas had significantly lower haemoglobin, haematocrit and higher mean cellular haemoglobin concentration and lymphocyte counts than adult koalas. MLR male koalas had elevated erythrocyte, leucocyte and neutrophil counts compared with MLR females. CONCLUSION: The haematological RIs developed in this study are based on a large number of clinically healthy koalas, where subclinical C. pecorum and KoRV infections had no effect on haematological values and will be a valuable tool during clinical examination and disease investigation by veterinarians and researchers Australia-wide.

Antimicrobial use and stewardship practices on Australian beef feedlots.

OBJECTIVE: Improving antimicrobial stewardship in the livestock sector requires an understanding of the motivations for antimicrobial use and the quantities consumed. However,detailed information on antimicrobial use in livestock sectors is lacking. This cross-sectional study aimed to better understand antimicrobial use in the beef feedlot sector in Australia. DESIGN: A self-administered questionnaire asking about antimicrobial use and reasons for use was designed and mailed to beef feedlot operators in Australia. Respondents were asked to report the percentage of animals treated, purpose of use, and disease conditions targeted for 26antimicrobial agents. RESULTS: In total, 83 of 517 (16.1%) beef feedlot operators completed the survey. Monensin (61.0%of respondents) and virginiamycin (19.5%of respondents) were the most commonly reported in-feed antimicrobials. In-feed antimicrobial agents were most frequently used by respondents for treatment of gastrointestinal diseases (52.8%). Antimicrobials were used for growth promotion by 42.1% of respondents, with most (85.7%) reporting the use of ionophores(a group of compounds not used in human medicine). Short-acting penicillin(69.1%), short-acting oxytetracycline, and tulathromycin (both 57.3%) werethe most common injectable antimicrobial agents used. Injectable antimicrobials were most frequently used to treat respiratory (72.3%) and musculoskeletal (67.5%) conditions. CONCLUSION: Overall,the use of antimicrobials was appropriate for the purpose indicated, and there was a strong preference for drugs of low-importance in human medicine. The data described here stand to be a strong influence on the implementation of an antimicrobial stewardship program in the sector.

Pathological Findings in Koala Retrovirus-positive Koalas (Phascolarctos cinereus) from Northern and Southern Australia.

Koala retrovirus (KoRV) infection shows differences in prevalence and load between northern and southern Australian koala populations; however, the effect of this on diseases such as lymphoma and chlamydial disease is unclear. This study compared clinicopathological findings, haematology and splenic lymphoid area of KoRV-positive koalas from northern (Queensland [Qld], n = 67) and southern (South Australia [SA], n = 92) populations in order to provide further insight into KoRV pathogenesis. Blood was collected for routine haematology and for measurement of KoRV proviral load by quantitative polymerase chain reaction (qPCR). Plasma samples were assessed for KoRV viral load by reverse transcriptase qPCR and conjunctival and cloacal swabs were collected for measurement of the load of Chlamydia pecorum (qPCR). During necropsy examination, spleen was collected for lymphoid area analysis. Lymphoma was morphologically similar between the populations and occurred in koalas with the highest KoRV proviral and viral loads. Severe ocular chlamydial disease was observed in both populations, but urinary tract disease was more severe in Qld, despite similar C. pecorum loads. No associations between KoRV and chlamydial disease severity or load were observed, except in SA where viral load correlated positively with chlamydial disease severity. In both populations, proviral and viral loads correlated positively with lymphocyte and metarubricyte counts and correlated negatively with erythrocyte and neutrophil counts. Splenic lymphoid area was correlated positively with viral load. This study has shown further evidence for KoRV-induced oncogenesis and highlighted that lymphocytes and splenic lymphoid tissue may be key sites for KoRV replication. However, KoRV infection appears to be highly complex and continued investigation is required to fully understand its pathogenesis.

Periodontal disease in free-ranging koalas (Phascolarctos cinereus) from the Mount Lofty Ranges, South Australia, and its association with koala retrovirus infection.

BACKGROUND: In northern Australian koala populations (Queensland and New South Wales), periodontal disease (gingivitis and periodontitis) is common while koala retrovirus subtype A is endogenous, with other subtypes transmitted exogenously. Koala retrovirus has been hypothesised to cause immune suppression and may predispose koalas to diseases caused by concurrent infections. In southern Australia populations (Victoria and South Australia) periodontal disease has not been investigated, and koala retrovirus is presumably exogenously transmitted. This study described oral health in South Australian koalas and investigated if an association between periodontal disease and koala retrovirus exists. METHODS: Oral health was examined for wild-caught koalas from the Mount Lofty Ranges (n = 75). Koala retrovirus provirus was detected in whole blood using nested PCR and proviral load determined with qPCR. Periodontal disease severity was recorded and used to calculate the Final Oral Health Index (0-normal, 24-severe).Results Periodontal disease was observed in 84% (63/75) of koalas; 77% had gingivitis (58/75) and 65% (49/75) had periodontitis. The average Final Oral Health Index was 5.47 (s.d 3.13). Most cases of periodontal disease were associated with the incisors. Koala retrovirus-infected koalas were more likely to present with periodontitis (p = 0.042) and the Final Oral Health Index was negatively correlated with proviral load (ρ = -0.353, p = 0.017). CONCLUSION: South Australian koalas had a high prevalence of gingivitis and periodontitis. Periodontal disease was more prevalent in the incisors. Exogenous koala retrovirus infection may also facilitate the development of periodontitis by modulation of the immune response to concurrent oral bacterial infections.

Egg yolk antibodies for detection and neutralization of Clostridium botulinum type A neurotoxin.

The objective of this research project was to determine the usefulness of an egg antibody platform for producing materials for the detection and neutralization of botulinum type A neurotoxin. Yield estimates for detection and neutralizing antibodies produced using methods described were calculated. Antibody specific to botulinum toxoid A (aToxoid) and toxin A (aBoNT/A) was produced by immunizing hens with botulinum toxoid A (toxoid) followed by increasing amounts of botulinum neurotoxin A (BoNT/A) in Freund incomplete adjuvant. Egg yolks were extracted with polyethylene glycol (PEG) for antibody detection and neutralization experiments. A model aToxoid/toxoid immunoassay using only egg yolk antibody was developed and had a detection limit of 1 pg/ml of toxoid. In an indirect enzyme-linked immunosorbent assay of BoNT/A-specific antibody, the aBoNT/A contained more BoNT/A-specific antibody than did the aToxoid, and aBoNT/A was as effective as commercial rabbit antibody. The aToxoid provided no protection against BoNT/A in a standard mouse neutralization assay; however, 1 mg of PEG-extracted aBoNT/A neutralized 4,000 lethal doses of BoNT/A injected intraperitoneally. Based on these results, we calculated that in 1 month one hen could produce more than 100 liters of antibody detection reagents or enough antibody to neutralize approximately 11.6 million mouse lethal doses of botulinum toxin. Utilization of an egg antibody platform is potentially rapid (28 to 70 days) and scalable to kilogram quantities using current egg production facilities with as few as 1,000 hens.

Antimicrobial use in the Australian pig industry: results of a national survey.

OBJECTIVE: To describe how various antimicrobials are used in commercial pig herds in Australia and for what disease conditions. PROCEDURE: Managers of large pig herds (> 200 sows) across Australia and their veterinarians participated in an internet-based survey in 2006. Questions were asked about herd management, the occurrence of bacterial diseases and the type and frequency of antimicrobial use. An antimicrobial usage index for each herd was derived as a summary of the risk of selection for antimicrobial resistance. Relationships between responses were explored with univariate and multivariate analysis. RESULTS: Responses were received for 197 herds estimated to represent at least 51% of all large pig herds in Australia. Most piggeries relied on drugs of low importance in human medicine (e.g. tetracyclines, penicillins and sulfonamides). For the two drugs of high importance in human medicine that can be legally prescribed to pigs in Australia, ceftiofur use was reported in 25% of herds and virginiamycin in none. Infections attributed to Lawsonia, Mycoplasma and Escherichia coli motivated the most use of antimicrobials. No useful association was found between management factors and the antimicrobial use index. CONCLUSION: Most antimicrobial use in the Australian pig industry is based on drugs of low importance to public health. Enhanced control of E. coli infections without reliance on antimicrobials would further reduce the risk of selecting for antimicrobial resistance relevant to public health. The amount of variation in the usage index between herds suggests that antimicrobial use should be constantly reviewed on a herd by herd basis.

Oxalate-degrading bacteria, including Oxalobacter formigenes, colonise the gastrointestinal tract of healthy koalas (Phascolarctos cinereus) and those with oxalate nephrosis.

BACKGROUND: Koalas in the Mount Lofty Ranges, South Australia, have a high prevalence of oxalate nephrosis, or calcium oxalate kidney crystals. Gastrointestinal tract oxalate-degrading bacteria, particularly Oxalobacter formigenes, have been identified in other animal species and humans, and their absence or low abundance is postulated to increase the risk of renal oxalate diseases. This study aimed to identify oxalate-degrading bacteria in the gastrointestinal tract of koalas and determine their association with oxalate nephrosis. METHODS: Caecal and faecal samples were collected at necropsy from 22 Mount Lofty Ranges koalas that had been euthanased on welfare grounds, with 8 koalas found to have oxalate nephrosis by renal histopathology. Samples were analysed by PCR for the oxc gene, which encodes oxalyl-CoA decarboxylase, and also by Illumina sequencing of the V3-V4 region of the bacterial 16S rRNA gene. RESULTS: The oxc gene was detected in 100% of koala samples, regardless of oxalate nephrosis status. Oxalobacter formigenes was detected in all but one faecal sample, with no difference in abundance between koalas affected and unaffected by oxalate nephrosis. Other species of known oxalate-degrading bacteria were infrequently detected. CONCLUSION: This is the first study to identify Oxalobacter and other oxalate-degrading bacterial species in koalas, but an association with oxalate nephrosis and absence or low abundance of Oxalobacter was not found. This suggests other mechanisms underlie the risk of oxalate nephrosis in koalas.

Malocclusions in the koala (Phascolarctos cinereus).

Malocclusions are a misalignment or incorrect positioning of the teeth when the upper and lower jaws close. These are poorly described in the koala and can result in irregular mastication which can have lifelong effects on body condition and oral health. A total of 370 koalas from two populations in Queensland (295) and one in South Australia (75) were examined for malocclusions. The prevalence of malocclusions in South Australian free-ranging koalas, captive Queensland koalas and Queensland free-ranging koalas was 39% (44), 30% (29) and 22% (29) respectively. Four types of malocclusion were identified based on severity of misalignment of the incisor/canine region, types 1, 2, 3 and 4. Maxillary overbite measurements of the molariform teeth were determined and these anisognathic values were then used to describe malocclusions within familial relationships in captive colonies. Captive koalas with a malocclusion had narrower mandibular width that ranged between 0.5 and 1% less than the normal measurements. The specific malocclusions reported in this study affected individuals by leading to tooth rotation, mobility and erosion with inefficient mastication of food and vegetation compaction. These changes increased the oral cavity pathology, by placing animals at risk of periodontal disease. There was evidence of familial links to malocclusion types in captive animals. Therefore captive breeding recommendations should consider known koala malocclusion traits to minimise their effect on future generations.

Multidrug-resistant E. coli and enterobacter extraintestinal infection in 37 dogs.

BACKGROUND: Extraintestinal infections caused by multidrug-resistant (MDR) Escherichia coli and Enterobacter are becoming more common in veterinary medicine. OBJECTIVE: To generate hypotheses for risk factors for dogs acquiring extraintestinal infection caused by MDR E. coli and Enterobacter, describe antimicrobial resistance profiles and analyze treatment and clinical outcomes. ANIMALS: Thirty-seven dogs diagnosed with extraintestinal infection caused by MDR E. coli and Enterobacter spp. between October 1999 and June 2006. METHODS: Retrospective case series assembled from hospital records data, including clinical history before 1st MDR isolation and treatment outcome. Identity and antimicrobial susceptibility profiles were confirmed by standard microbiological techniques for 57 isolates. RESULTS: Most dogs had an underlying disease condition (97%), received prior antimicrobial treatment (87%), were hospitalized for >or =3 days (82%), and had a surgical intervention (57%). The urinary tract was the most common infection site (62%), and urinary catheterization, bladder stasis, or both were common among dogs (24%). Some dogs were treated with high doses of co-amoxyclavulanate (n = 14) and subsequently recovered even though the isolates showed in vitro resistance to this antimicrobial. Other dogs were successfully treated with chloramphenicol (n = 11) and imipenem (n = 2). CONCLUSION AND CLINICAL IMPORTANCE: Predisposing disease condition, any prior antimicrobial use rather than a specific class of antimicrobial, duration of hospitalization, and type of surgical procedure might be risk factors for acquiring MDR extraintestinal infections. Whereas culture and sensitivity results can indicate use of last-resort antimicrobials such as imipenem for MDR infections, some affected dogs can recover after administration of high doses of co-amoxyclavulanate.

Additions of killed whole cell bacteria preparations to Freund complete adjuvant alter laying hen antibody response to soluble protein antigen.

Passive transfer of antibodies from hen to egg has value to both the producer of commercial polyclonal egg antibody and the producer of hatching eggs. Water-in-oil emulsions are commonly amended with immune stimulants such as Mycobacteria (e.g., Freund complete adjuvant; FCA) to increase antibody production to soluble protein antigens (SPA). Recent discoveries of the mechanisms by which microbial products act as adjuvants led us to hypothesize that additions of killed whole cell bacteria (bacterins) to FCA could improve antibody responses to SPA. All injections used in each experiment were water-in-oil emulsions (50:50) containing 3 mg/mL of phospholipase A(2) (PLA(2)) immunogen. Additionally, all primary control and treatment injections contained heat-killed Mycobacterium butyricum immunogens from FCA. In addition to PLA(2) and FCA, primary treatment injections contained various microbial bacterin immunogens. Hence, the experimental treatment of all experiments was addition of a commercial source of microbial bacterin to FCA for the primary injection only. Booster injections were the same as the primary control injections except Freund incomplete adjuvant replaced FCA. Anti-body titers to PLA(2) in yolk were determined by ELISA. Bacterins tested as additives to FCA were Escherichia coli, Staphylococcus aureus, Streptococcus suis, and Corynebacterium pseudotuberculosis. Escherichia coli bacterin added to FCA decreased egg yolk antibody titer to SPA by 23% in hens of different ages and strains (P < 0.0001). In a second experiment, a 51% decrease in antibody production associated with E. coli bacterin was sustained for several weeks after the primary immunization (P = 0.003). Staphylococcus aureus or Streptococcus suis combined with FCA increased egg yolk antibody 62 and 51%, respectively (P < 0.05), and Corynebacterium pseudotuberculosis had no effect. In conclusion, the addition of bacterin to FCA can influence hen antibody response to SPA as measured in egg yolks. It is hypothesized that the difference in antibody production may be related to the composition of various pathogen associated molecular patterns in the primary injection.

Present and Future Surveillance of Antimicrobial Resistance in Animals: Principles and Practices.

There is broad consensus internationally that surveillance of the levels of antimicrobial resistance (AMR) occurring in various systems underpins strategies to address the issue. The key reasons for surveillance of resistance are to determine (i) the size of the problem, (ii) whether resistance is increasing, (iii) whether previously unknown types of resistance are emerging, (iv) whether a particular type of resistance is spreading, and (v) whether a particular type of resistance is associated with a particular outbreak. The implications of acquiring and utilizing this information need to be considered in the design of a surveillance system. AMR surveillance provides a foundation for assessing the burden of AMR and for providing the necessary evidence for developing efficient and effective control and prevention strategies. The codevelopment of AMR surveillance programs in humans and animals is essential, but there remain several key elements that make data comparisons between AMR monitoring programs, and between regions, difficult. Currently, AMR surveillance relies on uncomplicated in vitro antimicrobial susceptibility methods. However, the lack of harmonization across programs and the limitation of genetic information of AMR remain the major drawbacks of these phenotypic methods. The future of AMR surveillance is moving toward genotypic detection, and molecular analysis methods are expected to yield a wealth of information. However, the expectation that these molecular techniques will surpass phenotypic susceptibility testing in routine diagnosis and monitoring of AMR remains a distant reality, and phenotypic testing remains necessary in the detection of emerging resistant bacteria, new resistance mechanisms, and trends of AMR.

Identification of stable reference genes for quantitative PCR in koalas.

To better understand host and immune response to diseases, gene expression studies require identification of reference genes with stable expression for accurate normalisation. This study describes the identification and testing of reference genes with stable expression profiles in koala lymph node tissues across two genetically distinct koala populations. From the 25 most stable genes identified in transcriptome analysis, 11 genes were selected for verification using reverse transcription quantitative PCR, in addition to the commonly used ACTB and GAPDH genes. The expression data were analysed using stable genes statistical software - geNorm, BestKeeper, NormFinder, the comparative ΔCt method and RefFinder. All 13 genes showed relative stability in expression in koala lymph node tissues, however Tmem97 and Hmg20a were identified as the most stable genes across the two koala populations.