Embryo mortality in a captive-bred, Critically Endangered amphibian.

The Critically Endangered southern corroboree frog Pseudophryne corroboree is dependent upon captive assurance colonies for its continued survival. Although the captive breeding programme for this species has largely been successful, embryonic mortality remains high (40-90% per year). This study aimed to investigate the causes of mortality in P. corroboree embryos in the captive collection at Melbourne Zoo. During the 2021 breeding season, we investigated 108 abnormal embryos to determine the impact of infections and anatomical deformities on survival and used culture and molecular methods to identify microbes. Overall, 100% of abnormal embryos had fungal infections, and of these, 41.6% also had anatomical deformities. The mortality rate in abnormal embryos was 89.8%; however, we detected no difference in survival in any of the 3 observed fungal growth patterns or between deformed and non-deformed embryos. Sanger sequencing of the ITS region identified fungal isolates belonging to the genus Ilyonectria, the first record in a vertebrate host, and another as a Plectosphaerella sp., which is the first record of infection in an embryo. Dominant bacteria identified were of the genera Herbaspirillum and Flavobacterium; however, their role in the mortality is unknown. Fungal infection and deformities have a significant impact on embryo survival in captive-bred P. corroboree. In a species which relies on captive breeding, identifying and reducing the impacts of embryonic mortality can inform conservation efforts and improve reintroduction outcomes.

Assessment of the efficacy of an autogenous vaccine against Yersinia pseudotuberculosis in young Merino sheep.

AIMS To assess the efficacy of an autogenous vaccine against Yersinia pseudotuberculosis III in preventing clinical disease and deaths due to yersiniosis in young Merino sheep, and to determine the effect of vaccination on the prevalence of faecal shedding of pathogenic Yersinia spp., daily liveweight gain, and development of antibodies to Yersinia spp. following vaccination and natural exposure. METHODS In six groups (three groups each from two farms) of young Merino sheep, 148-150 animals were systematically allocated to be vaccinated twice with an autogenous, formalin- killed bacterin vaccine containing Y. pseudotuberculosis serotype III or to remain non-vaccinated. All vaccinated and non-vaccinated sheep were run together in their original groups throughout the trial. Faecal and blood samples were collected, and liveweight measured, at the time of vaccination and subsequently over a 6-month period to determine faecal shedding of Y. enterocolitica and Y. pseudotuberculosis, seroprevalence of antibodies to Yersinia outer membrane proteins (YOP) and changes in liveweight. RESULTS None of the six trial groups experienced an outbreak of clinical yersiniosis during the study period. On Farm A, the prevalence of shedding of either or both Yersinia spp. was <40% on all but one sampling occasions. On Farm B the prevalence of shedding of both Yersinia spp. peaked at 98%, 96 days after vaccination. Mean liveweight and daily liveweight gain at the end of the study were similar in vaccinated and non-vaccinated groups on both farms (p>0.1), as was the prevalence of faecal shedding of Yersinia spp. (p>0.2), and the proportion of animals that became seropositive for antibodies to YOP following vaccination (p>0.1). CONCLUSIONS AND CLINICAL RELEVANCE This vaccine had, at most, limited effects on seroconversion and, under the conditions of this study, had no demonstrable impact on liveweight, mean daily liveweight gain or faecal shedding of Yersinia spp. Further studies are needed to determine the efficacy of this vaccine during outbreaks of yersiniosis or following experimental challenge with pathogenic Yersinia spp..

Antimicrobial susceptibility testing by Australian veterinary diagnostic laboratories.

BACKGROUND: The national strategy for tackling antimicrobial resistance highlights the need for antimicrobial stewardship in veterinary practice and for surveillance of antimicrobial susceptibility in veterinary pathogens. Diagnostic laboratories have an important role in facilitating both of these processes, but it is unclear whether data from veterinary diagnostic laboratories are similar enough to allow for compilation and if there is consistent promotion of appropriate antimicrobial use embedded in the approaches of different laboratories to susceptibility testing. METHODS: A cross-sectional study of antimicrobial susceptibility testing and reporting procedures by Australian veterinary diagnostic laboratories was conducted in 2017 using an online questionnaire. All 18 veterinary diagnostic laboratories in Australia completed the questionnaire. RESULTS: Kirby-Bauer disc diffusion was the method predominantly used for antimicrobial susceptibility testing and was used to evaluate 86% of all isolates, although two different protocols were used across the 18 laboratories (CLSI 15/18, CDS 3/18). Minimum inhibitory concentrations were never reported by 61% of laboratories. Common isolates were consistently reported on across all species, except for gram-negative isolates in pigs, for which there was some variation in the approach to reporting. There was considerable diversity in the panels of antimicrobials used for susceptibility testing on common isolates and no consistency was apparent between laboratories for any bacterial species. CONCLUSION: We recommend that nationally agreed and consistent antimicrobial panels for routine susceptibility testing should be developed and a uniform set of guidelines should be adopted by veterinary diagnostic laboratories in Australia.

A longitudinal study of faecal shedding of Yersinia enterocolitica and Yersinia pseudotuberculosis by Merino lambs in south-eastern Australia.

A prospective longitudinal study was conducted to investigate potential risk factors for faecal shedding of Yersinia enterocolitica and Y. pseudotuberculosis by Merino lambs in four flocks in south-eastern Australia. The primary aims of the study were to determine the seasonal patterns of shedding of pathogenic Y. enterocolitica and Y. pseudotuberculosis, and to evaluate putative risk factors for faecal shedding of these organisms, including worm egg count, live-weight and growth rate. The risk of shedding varied markedly between Yersinia spp., farms, seasons and years. Shedding of Y. pseudotuberculosis occurred predominately in winter, whereas Y. enterocolitica was commonly isolated from faeces throughout the year. Moderate to high prevalences of shedding of each organism occurred in the absence of outbreaks of yersiniosis. In general, for shedding of Y. pseudotuberculosis, animals with moderate or high worm egg counts were at increased risk of shedding compared with animals with low worm egg counts. Sheep with higher average daily weight gains were at decreased risk of shedding Y. enterocolitica but at increased risk of shedding Y. pseudotuberculosis. Live-weight was not significantly associated with risk of shedding either species. This study highlighted that exposure to determinants of shedding Y. enterocolitica and Y. pseudotuberculosis differ between farms and over time within farms. Shedding is likely influenced by environmental, animal and management factors. Our results indicate that different or additional risk factors are required for yersiniosis over those that cause faecal shedding of Yersinia spp., because moderate to high prevalences of shedding were not always associated with outbreaks of clinical disease.

Investigation of the Role of Campylobacter Infection in Suspected Acute Polyradiculoneuritis in Dogs.

BACKGROUND: Acute polyradiculoneuritis (APN) is an immune-mediated peripheral nerve disorder in dogs that shares many similarities with Guillain-Barré syndrome (GBS) in humans, in which the bacterial pathogen Campylobacter spp. now is considered to be a major triggering agent. Little information is available concerning the relationship between APN and Campylobacter spp. in dogs. HYPOTHESIS/OBJECTIVES: To estimate the association between Campylobacter spp. infection and APN. Associations with additional potential risk factors also were investigated, particularly consumption of raw chicken. ANIMALS: Twenty-seven client-owned dogs suffering from suspected APN and 47 healthy dogs, client-owned or owned by staff members. METHODS: Case-control study with incidence density-based sampling. Fecal samples were collected from each enrolled animal to perform direct culture, DNA extraction, and polymerase chain reaction (PCR) for detection of Campylobacter spp. In some cases, species identification was performed by sequence analysis of the amplicon. Data were obtained from the medical records and owner questionnaires in both groups. RESULTS: In cases in which the fecal sample was collected within 7 days from onset of clinical signs, APN cases were 9.4 times more likely to be positive for Campylobacter spp compared to control dogs (P < 0.001). In addition, a significant association was detected between dogs affected by APN and the consumption of raw chicken (96% of APN cases; 26% of control dogs). The most common Campylobacter spp. identified was Campylobacter upsaliensis. CONCLUSIONS AND CLINICAL IMPORTANCE: Raw chicken consumption is a risk factor in dogs for the development of APN, which potentially is mediated by infection with Campylobacter spp.

Survey of Victorian small ruminant herds for mycoplasmas associated with contagious agalactia and molecular characterisation of Mycoplasma mycoides subspecies capri isolates from one herd.

OBJECTIVE: Regarded as one of the most expensive production diseases of dairy sheep and goats, contagious agalactia (CA) is caused by any of four agents: Mycoplasma agalactiae, M. mycoides subspecies capri (Mmc), M. capricolum subspecies capricolum (Mcc) and M. putrefaciens. Although CA is worldwide in distribution, it has not been reported in Australia, even though studies between the 1950s and 1980s isolated each agent from sheep or goats without any clinical signs associated with it. The aim of this study was to examine sheep and goats in Victoria, Australia, for the presence of CA-associated mycoplasmas and to investigate the evolutionary relationships of these isolates by comparing their genetic differences with their counterparts from other parts of the world. METHODS: A 3-year epidemiological survey of small ruminant populations in Victoria, Australia, was conducted for the presence of CA-associated mycoplasmas and the isolates obtained were genotyped by multilocus sequence typing (MLST). RESULTS: Mmc was the only CA-associated agent isolated from the 1358 samples analysed in the study, but was not associated with CA on the property where it was found. MLST analyses of Mmc strains revealed a distinct clustering of Australian isolates into a novel clade, with the closest relatives being strains from Europe. The distinct clustering is consistent with the absence of clinical disease in Australia. CONCLUSION: The isolation of Mmc indicates that this subspecies persists in Australian small ruminant populations. However, full genome sequencing and in vitro animal experimentation are needed to unequivocally demonstrate the avirulence of Australian strains.

The role of Type 1, P and S fimbriae in binding of Escherichia coli to the canine endometrium.

Escherichia coli (E. coli) is the most commonly isolated infectious agent causing pyometra in bitches. Many E. coli strains isolated from the uteri of infected dogs carry several adhesin genes (fimH, papGIII and sfa). The objective of this study was to investigate the role of each adhesin gene product, acting alone or expressed in combination, in the bacterial binding to canine endometrium. E. coli strain P3, which was isolated from a uterus of a bitch naturally affected with pyometra, was shown by PCR to carry all three known fimbrial adhesin genes fimH, papGIII and sfa. Knockout (KO) mutants of this wildtype (P3-wt) strain were generated using insertional inactivation. Adhesion assays on anoestrous uteri of three post-pubertal bitches were undertaken. Overall, the number of bacteria adhering to canine endometrial biopsies was comparable between strains and no significant difference in the number of bound bacteria was found between the P3-wt strain and the single or double KO-strains. However, the triple knockout strain displayed less binding to the canine endometrium compared with the P3-wt strain. This study shows that a pathogenic E. coli strain (P3) isolated from the uterus of a bitch with pyometra was able to fully compensate for the loss of two of its three known adhesin genes. It was necessary to inactivate all three known adhesin genes in order to see a significant decrease in binding to canine endometrium.

Uropathogenic virulence factor FimH facilitates binding of uteropathogenic Escherichia coli to canine endometrium.

Pyometra is a potentially life-threatening condition in bitches and is often caused by Escherichia coli infection. Both pathogenic and non-pathogenic E. coli strains commonly carry the genes for type 1 fimbriae that mediate bacterial adhesion onto host epithelium. To investigate whether the type 1 fimbrial adhesin, FimH, facilitates the binding of uropathogenic E. coli to canine endometrium, the fimH gene was insertionally inactivated in a pathogenic E. coli strain. The ability of E. coli to bind to canine endometrial epithelial cells was determined in vitro using canine uterine biopsies. Binding of the fimH mutant was only 0.3% of that of the wild type. Complementation of the mutation restored the phenotype to that of the parent. This study has developed an in vitro model that allows quantitative and qualitative assessment of bacterial binding to canine endometrium and has demonstrated that the fimH gene plays a role in adherence of pathogenic E. coli to canine endometrium.

The central role of lipoproteins in the pathogenesis of mycoplasmoses.

Mycoplasmas are a diverse group of pathogens responsible for disease in a wide range of animal species. In recent years there have been considerable advances in knowledge of the proteins and structures involved in adherence in some mycoplasmas, but understanding of the biochemical functions and roles in virulence of another central feature of mycoplasmas, their lipoproteins, continues to develop. The aim of this review is to examine current knowledge of the roles of lipoproteins in the pathogenicity and the evolution of virulence in those mycoplasmas causing disease in domestic animals. Those lipoproteins that have been characterised have roles in adherence, in transport of nutrients into the mycoplasma cell, and in enzymatic interactions with the host. Furthermore they appear to play a prominent role in both inducing the host immune response to infection and in facilitating evasion of this response, particularly through the generation of dramatic levels of antigenic variation on the cell surface. Recent genomic comparisons of several pathogenic mycoplasmas have identified a further level of interaction between lipoproteins and pathogenicity. In several pathogens large scale horizontal gene transfer between distantly related mycoplasma species has resulted in the acquisition of a large number of genes, including those encoding lipoproteins thought to play a role in virulence, by one mycoplasma from another inhabiting the same host species. The interactions between these horizontally transferred genes, their new mycoplasma host and the animal that it infects may be an important contributing factor in the pathogenesis of some mycoplasmoses.

Alternative translation initiation of the Moloney murine leukemia virus mRNA controlled by internal ribosome entry involving the p57/PTB splicing factor.

Moloney murine leukemia virus (Mo-MuLV) genomic mRNA codes for two gag precursors by alternative initiations of translation. An AUG codon governs the synthesis of the retroviral capsid proteins precursor, whereas a CUG codon directs the synthesis of a glycosylated cell surface antigen, the gross cell surface antigen. Control of the relative synthesis of the two precursors is crucial for MuLV infectivity and pathology. Furthermore, the MuLV mRNA leader sequence is very long and should inhibit translation according to the classical scanning model. This suggests a different translation initiation mechanism allowing gag efficient expression. We demonstrate, by using bicistronic vectors expressed in COS-7 cells, that the Mo-MuLV mRNA leader drives translation initiation by internal ribosome entry. We have localized the internal ribosome entry site (IRES) between the two initiation codons. This 126 nucleotide long IRES implies an oligopyrimidine tract located 45 nucleotides upstream of AUG codon. UV cross-linking and affinity chromatography experiments show that the PTB/p57 splicing factor specifically interacts with this oligopyrimidine tract. The MuLV IRES controls alternative translation initiation by activating the capsid protein precursor expression. This gag translational enhancer could exist in other retroviruses.

Ralstonia solanacearum produces hrp-dependent pili that are required for PopA secretion but not for attachment of bacteria to plant cells.

As in many other Gram-negative plant pathogenic bacteria, the Ralstonia solanacearum hrp genes are involved in the production of a type III secretion apparatus that allows the translocation of PopA protein to the external medium. Here, we show that hrp genes are also involved in the biogenesis of pili that are mainly composed of the HrpY protein. These pili are produced at one pole of the bacterium and are also released into the external medium where they can form very long straight bundles. An hrpY mutant is defective in pilus production, impaired in interactions with plants and in secretion of the PopA protein but not in attachment to plant cells.

prhJ and hrpG, two new components of the plant signal-dependent regulatory cascade controlled by PrhA in Ralstonia solanacearum.

hrp gene expression in the phytopathogenic bacterium Ralstonia solanacearum GMI1000 is induced through the HrpB regulator in minimal medium and upon co-culture with plant cell suspensions. The putative outer membrane protein PrhA is specifically involved in hrp gene activation in the presence of plant cells and has been proposed to be a receptor of a plant-dependent signal transduction pathway. Here, we report on the identification of two regulatory genes, hrpG and prhJ, located at the right-hand end of the hrp gene cluster, that are required for full pathogenicity. HrpG belongs to the OmpR subclass of two-component response regulators and is homologous to HrpG, the activator of hrp genes in Xanthomonas campestris pv. vesicatoria. PrhJ is a novel hrp regulatory protein, sharing homology with the LuxR/UhpA family of transcriptional activators. As for HrpG of X. c. pv. vesicatoria, HrpG is required for hrp gene expression in minimal medium, but, in addition, we show that it also controls hrpB gene activation upon co-culture with Arabidopsis thaliana and tomato cell suspensions. In contrast, PrhJ is specifically involved in hrp gene expression in the presence of plant cells. hrpG and prhJ gene transcription is plant cell inducible through the PrhA-dependent pathway. From these results, we propose a regulatory cascade in which plant cell signal(s) sensed by PrhA are transduced to the prhJ gene, whose predicted product controls hrpG gene expression. HrpG then activates the hrpB regulatory gene, and, subsequently, the remaining hrp transcriptional units in all known inducing conditions.

PrhA controls a novel regulatory pathway required for the specific induction of Ralstonia solanacearum hrp genes in the presence of plant cells.

The Ralstonia solanacearum hrp gene cluster is organized in five transcriptional units. Expression of transcriptional units 2, 3 and 4 is induced in minimal medium and depends on the hrp regulatory gene hrpB, which belongs to unit 1. This regulatory gene also controls the expression of genes, such as popA, located to the left of the hrp cluster. Here, we show that, upon co-culture with Arabidopsis thaliana and tomato cell suspensions, the expression of the hrp transcriptional units 1, 2, 3 and 4 is induced 10- to 20-fold more than in minimal medium. This induction is not triggered by diffusible signals but requires the presence of plant cells. Moreover, we show that this specific plant cell induction of hrp genes is controlled by a gene, called prhA (plant regulator of hrp genes), located next to popA. This gene codes for a putative protein of 770 amino acids, which shows similarities with TonB-dependent outer membrane siderophore receptors. Expression of prhA and hrp genes is not regulated by iron status, and we postulate that iron is not the signal sensed by PrhA. In prhA mutants, the induction of hrpB and other hrp genes is abolished in co-culture with Arabidopsis cells, partially reduced in co-culture with tomato cells and not modified in minimal medium. prhA mutants are hypo-aggressive on Arabidopsis (accessions Col-0 and Col-5) but remain fully pathogenic on tomato plants, suggesting that the co-culture assays mimic the in planta conditions. A model suggesting that PrhA is a receptor for plant specific signals at the top of a novel hrp regulatory pathway is discussed.