Expanding the known distribution of phascolartid gammaherpesvirus 1 in koalas to populations across Queensland and New South Wales.

Koala populations across the east coast of Australia are under threat of extinction with little known about the presence or distribution of a potential pathogen, phascolartid gammaherpesvirus 1 (PhaHV-1) across these threatened populations. Co-infections with PhaHV-1 and Chlamydia pecorum may be common and there is currently a limited understanding of the impact of these co-infections on koala health. To address these knowledge gaps, archived clinical and field-collected koala samples were examined by quantitative polymerase chain reaction to determine the distribution of PhaHV-1 in previously untested populations across New South Wales and Queensland. We detected PhaHV-1 in all regions surveyed with differences in detection rate between clinical samples from rescued koalas (26%) and field-collected samples from free-living koalas (8%). This may reflect increased viral shedding in koalas that have been admitted into care. We have corroborated previous work indicating greater detection of PhaHV-1 with increasing age in koalas and an association between PhaHV-1 and C. pecorum detection. Our work highlights the need for continued surveillance of PhaHV-1 in koala populations to inform management interventions, and targeted research to understand the pathogenesis of PhaHV-1 and determine the impact of infection and co-infection with C. pecorum.

Evaluation of a biosecurity survey approach for contamination by Chlamydia pecorum in koala rehabilitation, field capture, and captive settings.

Transmission of Chlamydia pecorum between koalas is a potential risk in field capture or rehabilitation settings, where koalas are held in proximity to each other, or equipment is shared between animals. Given the impact of C. pecorum on koala welfare and population viability it is surprising that quarantine and disinfection protocols in a koala rehabilitation facility or capture settings have not previously been evaluated. This study aimed to evaluate an approach, based on the detection of chlamydial DNA and cell viability, to determine the degree of environmental contamination within a koala care facility. Various fomite sites associated with koala care at a koala rehabilitation facility in New South Wales, Australia were identified as potential sources of chlamydial contamination, following exposure to koalas known to be infected with C. pecorum. Fomite sites were swabbed following exposure, and again after decontamination procedures were carried out. Samples were tested for the presence of chlamydial DNA using qPCR and viability using both RT-qPCR and cell culture. From a total of 239 sampling events, 30 tested qPCR positive for chlamydial DNA, with 19 and 11 samples corresponding to pre-decontamination and post-decontamination events respectively. Detection of chlamydial DNA appeared to be most common in the examination room, especially on fomite sites in direct contact with koalas. Physical removal of chlamydial DNA, or its degradation by the elements, appeared to be more common on outdoor enclosures, clothing, and hands. Based on the cell culture assay, of the pre-decontamination samples with chlamydial DNA, eight had viable chlamydial cells, two of these at low levels. Of the post-decontamination samples with chlamydial DNA, one had a moderate number, and one had a very low number of viable chlamydial cells. RT-qPCR was unsuccessful in determining cell viability due to low yields of RNA and high levels of contaminants from the environmental samples. The outcomes of this study provide a knowledge base for the design of future biosecurity evaluation guidelines in captive and koala rehabilitation facilities. The higher incidence of chlamydial DNA detection by qPCR than viable organism highlights the need to use viability assays in similar studies. However, further investment is still needed to optimise these methods and improve sensitivity for complex environmental samples.

A retrospective study on antibacterial treatments for koalas infected with Chlamydia pecorum.

Chlamydiosis remains the leading infectious disease and is one of the key factors responsible for the dramatic reduction of koala populations in South-East Queensland (SEQ) and New South Wales (NSW) regions of Australia. Possible infection outcomes include blindness, infertility, painful cystitis, and death if left untreated. Studies have reported the treatment efficacy of chloramphenicol and doxycycline, which are the two most commonly administered treatments in diseased koalas, in clinical settings. However, none have directly compared the treatment efficacy of these antibacterials on koala survival. A retrospective study was essential to identify any relationships between the demographical information, and the animals' responses to the current treatment regimens. Associations were explored between six explanatory (sex; maturity; location; clinical signs, treatment; treatment duration) and two outcome variables (survival; post-treatment PCR). Results showed that female koalas had a statistical trend of lower odds of surviving when compared to males (OR = 0.36, p = 0.05). Koalas treated with chloramphenicol for ≥ 28 days had greater odds of surviving than when treated for < 28 days (OR = 8.8, p = 0.02), and those koalas administered doxycycline had greater odds of testing PCR negative when compared to chloramphenicol treatments (OR = 5.45, p = 0.008). There was no difference between the antibacterial treatments (chloramphenicol, doxycycline, and mixed/other) and the survival of koalas. Female koalas had greater odds of exhibiting UGT signs only (OR = 4.86, p < 0.001), and also greater odds of having both ocular and UGT clinical signs (OR = 5.29, p < 0.001) when compared to males. Of the koalas, 28.5% initially had no clinical signs but were PCR positive for C. pecorum. This study enables further understanding of the complex nature between chlamydial infection and response to antibacterial treatment.

Development of diagnostic and point of care assays for a gammaherpesvirus infecting koalas.

The recent listing of koala populations as endangered across much of their range has highlighted the need for better management interventions. Disease is a key threat to koala populations but currently there is no information across the threatened populations on the distribution or impact of a gammaherpesvirus, phascolarctid gammaherpesvirus 1 (PhaHV-1). PhaHV-1 is known to infect koalas in southern populations which are, at present, not threatened. Current testing for PhaHV-1 involves lengthy laboratory techniques that do not permit quantification of viral load. In order to better understand distribution, prevalence and impacts of PhaHV-1 infections across koala populations, diagnostic and rapid point of care tests are required. We have developed two novel assays, a qPCR assay and an isothermal assay, that will enable researchers, clinicians and wildlife managers to reliably and rapidly test for PhaHV-1 in koalas. The ability to rapidly diagnose and quantify viral load will aid quarantine practices, inform translocation management and guide research into the clinical significance and impacts of PhaHV-1 infection in koalas.

Evolutionarily Young African Rhinoceros Gammaretroviruses.

High-throughput sequences were generated from DNA and cDNA from four Southern white rhinoceros (Ceratotherium simum simum) located in the Taronga Western Plain Zoo in Australia. Virome analysis identified reads that were similar to Mus caroli endogenous gammaretrovirus (McERV). Previous analysis of perissodactyl genomes did not recover gammaretroviruses. Our analysis, including the screening of the updated white rhinoceros (Ceratotherium simum) and black rhinoceros (Diceros bicornis) draft genomes identified high-copy orthologous gammaretroviral ERVs. Screening of Asian rhinoceros, extinct rhinoceros, domestic horse, and tapir genomes did not identify related gammaretroviral sequences in these species. The newly identified proviral sequences were designated SimumERV and DicerosERV for the white and black rhinoceros retroviruses, respectively. Two long terminal repeat (LTR) variants (LTR-A and LTR-B) were identified in the black rhinoceros, with different copy numbers associated with each (n = 101 and 373, respectively). Only the LTR-A lineage (n = 467) was found in the white rhinoceros. The African and Asian rhinoceros lineages diverged approximately 16 million years ago. Divergence age estimation of the identified proviruses suggests that the exogenous retroviral ancestor of the African rhinoceros ERVs colonized their genomes within the last 8 million years, a result consistent with the absence of these gammaretroviruses from Asian rhinoceros and other perissodactyls. The black rhinoceros germ line was colonized by two lineages of closely related retroviruses and white rhinoceros by one. Phylogenetic analysis indicates a close evolutionary relationship with ERVs of rodents including sympatric African rats, suggesting a possible African origin of the identified rhinoceros gammaretroviruses. IMPORTANCE Rhinoceros genomes were thought to be devoid of gammaretroviruses, as has been determined for other perissodactyls (horses, tapirs, and rhinoceros). While this may be true of most rhinoceros, the African white and black rhinoceros genomes have been colonized by evolutionarily young gammaretroviruses (SimumERV and DicerosERV for the white and black rhinoceros, respectively). These high-copy endogenous retroviruses (ERVs) may have expanded in multiple waves. The closest relative of SimumERV and DicerosERV is found in rodents, including African endemic species. Restriction of the ERVs to African rhinoceros suggests an African origin for the rhinoceros gammaretroviruses.

Genetic differences in Chlamydia pecorum between neighbouring sub-populations of koalas (Phascolarctos cinereus).

Chlamydiosis, caused by Chlamydia pecorum, is regarded as an important threat to koala populations. Across the koala's geographical range, disease severity associated with C. pecorum infection varies, with pathogen diversity and strain pathogenicity being likely important factors. To examine C. pecorum diversity on a sub-population level a Multi-Locus Sequence Typing (MLST) scheme, containing the housekeeping genes; gatA, oppA_3, hflX, gidA, enoA, hemN and fumC, was used to type strains from two sub-populations of koalas from the Liverpool Plains, NSW, Australia, with different disease expressions. Typing of samples from 2015 to 2017, revealed a significant association between sequence type ST 69 and clinical disease and a significant difference in sequence type frequencies between sub-populations. Sequence type ST 69 has previously been identified in both subclinical and clinically diseased koalas indicating that these markers alone are not illustrative of pathogenicity. However, recent emergence of this sequence type in a naïve population may explain the differing disease expressions. Sequence types ST 73 and ST 69 have been described in koalas across a broad geographic range, indicating multiple introduction events and/or a limited veracity of the MLST loci to explore fine scale epidemiological investigations, particularly those examining the interface between pathogenic strain and disease outcome.

Correction to: Mosquito-borne heartworm Dirofilaria immitis in dogs from Australia.

Since publication of the original version of this article [1], it has been flagged that unfortunately there is an error in dosage units in the Discussion section, in the sentence "For example a microfilaricide, either ivermectin (50-200 mg/kg) or milbemycin oxime (500-1,000 mg/kg)".

Aqueous peat extract exposes rhizobia to sub-lethal stress which may prime cells for improved desiccation tolerance.

Inoculation of legume seed with rhizobia is an efficient and cost-effective means of distributing elite rhizobial strains to broad-acre crops and pastures. However, necessary drying steps after coating seed expose rhizobia to desiccation stress reducing survival and limiting potential nitrogen fixation by legumes. Rhizobial tolerance to desiccation varies with strain and with growth conditions prior to drying. Cells grown in peat generally survive desiccation better than cells grown in liquid broth. We aimed to identify peat-induced proteomic changes in rhizobia that may be linked to desiccation tolerance. Proteins expressed differentially after growth in peat extract when compared with a minimal defined medium were measured in four rhizobial strains. Proteins showing the greatest increase in abundance were those involved in amino acid and carbohydrate transport and metabolism. Proteins involved in posttranslational modification and cell defence mechanisms were also upregulated. Many of the proteins identified in this study have been previously linked to stress responses. In addition, analysis using nucleic acid stains SYTO9 and propidium iodide indicated that membranes had been compromised after growth in peat extract. We targeted the membrane repair protein PspA (ΔRL3579) which was upregulated in Rhizobium leguminosarum bv. viceae 3841 after growth in peat extract to validate whether the inability to repair membrane damage after growth in peat extract reduced desiccation tolerance. The ΔRL3579 mutant grown in peat extract had significantly lower survival under desiccation stress, whereas no difference in survival between wild-type and mutant strains was observed after growth in tryptone yeast (TY) or minimal medium (JMM) media. Staining mutant and wild-type strains with SYTO9 and propidium iodide indicated that membranes of the mutant were compromised after growth in peat extract and to a lesser extent in TY. This study shows that growth in peat extract causes damage to cell membranes and exposes rhizobia to sub-lethal stress resulting in differential expression of several stress-induced proteins. The induction of these proteins may prime and protect the cells when subjected to subsequent stress such as desiccation. Identifying the key proteins involved in desiccation tolerance and properties of peat that stimulate this response will be important to inform development of new inoculant technology that maximises survival of rhizobia during delivery to legume crops and pastures.

Mosquito-borne heartworm Dirofilaria immitis in dogs from Australia.

BACKGROUND: Heartworm (Dirofilaria immitis) in dogs is considered endemic in Australia, but the clinical heartworm disease caused by the heartworm is rare and prevalence is low. The mainstream prevention of the heartworm is based on macrocyclic lactone (ML) administration. The aim of this study was to confirm endemism of the heartworm under current Australian conditions using a cohort of recent microfilaria-positive dogs which were on variable heartworm prevention. METHODS: A hotspot of canine heartworm antigen-positive and microfilaria-positive dogs has been detected recently in Queensland, Australia. Blood samples from 39 dogs from Queensland and two dogs from New South Wales were investigated for canine filarioids. Rapid antigen diagnostic tests capable of detection of D. immitis and real-time PCR for quantification and differentiation between D. immitis from Acanthocheilonema reconditum with quantification of microfilariae in canine blood samples, together with D. immitis specific real-time PCR assay, were applied to microfilaria-positive dogs. The P-glycoprotein genotype was determined to test whether Australian-sourced heartworm shared the same genetic markers as those suspected of ML-resistance in North America. RESULTS: Only D. immitis was detected in the samples from Queensland and New South Wales, Australia. Using high resolution melt real-time PCR and D. immitis specific real-time PCR, the calculated microfilaria concentration ranged from 1 to 44,957 microfilariae/ml and from 7 to 60,526 microfilariae/ml, respectively. DNA sequencing of the PCR products confirmed D. immitis. Fifteen of the examined dogs were on putative, rigorous ML prevention. For the remaining dogs, compliance with heartworm prevention was unknown or reported as inconsistent. Wild-type genotype AA-GG of the P-glycoprotein locus of D. immitis sequence has been obtained for three blood samples. Due to the incomplete history, any suggestion of a loss of efficacy of MLs must be treated as 'remotely possible'. In the immediate future, records of preventative administration and annual antigen testing would be required to determine any problems with the efficacy of preventatives. CONCLUSIONS: The prevalence of canine heartworm in Australia remains poorly understood. It is generally assumed to be low by veterinary practitioners. The localised increase in the study area confirms endemism of canine heartworm and a requirement for ongoing vigilance through annual heartworm testing to better understand the changing distribution of canine heartworm, client compliance, as well as to detect any change in ML-susceptibility.

Novel genotype of Tritrichomonas foetus from cattle in Southern Africa.

Bovine trichomonosis caused by Tritrichomonas foetus is a significant reproductive disease of cattle. Preputial samples were collected using sheath washing technique in bulls in Namibia. Thirty-six trichomonad cultures were characterized using the TaqMan-probe commercial real-time polymerase chain reaction (PCR) diagnostic assay (VetMAX™-Gold Trich Detection Kit) and CYBR real-time PCR assay based on TFR3/4 primers. Diagnostic real-time PCRs and DNA sequencing of the internal transcribed region confirmed presence of T. foetus in 35 out of 36 samples. Multilocus genotyping using cysteine proteases (CP1, CP2, CP4, CP5, CP6, CP7, CP8, CP9) and malate dehydrogenase (MDH1) gene sequences demonstrate that the T. foetus in Namibia are genetically distinct from those characterized elsewhere. We report the discovery of a novel genotype of T. foetus in Namibian cattle, distinct from other T. foetus genotypes in Europe, South and North America and Australia. We suggest recognition of a 'Southern African' genotype of T. foetus. Identification of the new genotype of T. foetus demonstrates the need for wider global sampling to fully understand the diversity and origin of T. foetus causing disease in cattle or cats.

Physiological changes in rhizobia after growth in peat extract may be related to improved desiccation tolerance.

Improved survival of peat-cultured rhizobia compared to survival of liquid-cultured cells has been attributed to cellular adaptations during solid-state fermentation in moist peat. We have observed improved desiccation tolerance of Rhizobium leguminosarum bv. trifolii TA1 and Bradyrhizobium japonicum CB1809 after aerobic growth in water extracts of peat. Survival of TA1 grown in crude peat extract was 18-fold greater than that of cells grown in a defined liquid medium but was diminished when cells were grown in different-sized colloidal fractions of peat extract. Survival of CB1809 was generally better when grown in crude peat extract than in the control but was not statistically significant (P > 0.05) and was strongly dependent on peat extract concentration. Accumulation of intracellular trehalose by both TA1 and CB1809 was higher after growth in peat extract than in the defined medium control. Cells grown in water extracts of peat exhibit morphological changes similar to those observed after growth in moist peat. Electron microscopy revealed thickened plasma membranes, with an electron-dense material occupying the periplasmic space in both TA1 and CB1809. Growth in peat extract also resulted in changes to polypeptide expression in both strains, and peptide analysis by liquid chromatography-mass spectrometry indicated increased expression of stress response proteins. Our results suggest that increased capacity for desiccation tolerance in rhizobia is multifactorial, involving the accumulation of trehalose together with increased expression of proteins involved in protection of the cell envelope, repair of DNA damage, oxidative stress responses, and maintenance of stability and integrity of proteins.