Cryptosporidium-associated enteritis in captive koalas (Phascolarctos cinereus).

Cryptosporidium spp. sporadically infect a range of Australian native mammals including koalas, red kangaroos, eastern grey kangaroos, bilbies and brush tailed possums and can range from asymptomatic to fatal infections. Traditionally considered a disease of the young or immuno-compromised, and resulting in profuse diarrhoea in other species, here we report an atypical clinical syndrome associated with Cryptosporidium in a captive population of koalas. All affected animals were in-contact adults, and demonstrated anorexia, dehydration and abdominal pain in the absence of diarrhoea. Following euthanasia on welfare grounds, Cryptosporidium infection was confirmed postmortem in three of four symptomatic animals via faecal floatation and/or intestinal histopathology, with enteritis also diagnosed in the fourth koala. Further screening of the captive colony found the outbreak had been contained. Based on sequencing the cause of the infection was C. fayeri, but the source was undetermined. In conclusion, Cryptosporidium should be considered as a possible cause of generalised illness in koalas.

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.

Histological survey for oxalate nephrosis in Victorian koalas (Phascolarctos cinereus).

The Mount Lofty Ranges koala (Phascolarctos cinereus) population in South Australia has a high prevalence of the renal disease oxalate nephrosis, for which an underlying genetic cause is suspected. South Australian koalas primarily originate from those in French Island, Victoria; however, oxalate nephrosis has not previously been reported in Victorian koalas. Examination of kidney tissue sections from 63 koalas across Victoria found that nine koalas were affected by oxalate nephrosis (14.3%). These included 2/5 koalas from French Island (40%), 4/14 koalas from the western regions (29%), 2/11 Raymond Island koalas (18%), and 1/13 Cape Otway koalas (8%). There were no cases of oxalate nephrosis identified in the Strzelecki koalas (n = 12). These findings suggest that oxalate nephrosis occurs in koalas from French Island and populations that have received significant influx of koalas from French Island, but not in the Strzelecki region, which has little to no French Island input. This lends support to the theory that an inherited abnormality of oxalate metabolism could underlie the high prevalence of oxalate nephrosis in the Mount Lofty Ranges koala population, and molecular investigations are currently underway to investigate a genetic cause.

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.

Symmetric dimethylarginine values in koalas (Phascolarctos cinereus) based on oxalate nephrosis status.

Oxalate nephrosis is a prevalent renal disease in koalas (Phascolarctos cinereus) of the Mount Lofty Ranges population in South Australia. The symmetric dimethylarginine (SDMA) assay is widely used in companion animals to diagnose renal disease, particularly in the early stages. This study aimed to determine: (1) reference intervals for SDMA in koalas and (2) SDMA values of koalas with oxalate nephrosis. Blood samples were collected from 41 Mount Lofty Ranges koalas euthanased on welfare grounds. Koalas were necropsied and, based on renal histopathology, were classified as unaffected (n = 22) or affected (n = 19) by oxalate nephrosis. Serum or plasma samples were analysed for creatinine, urea and SDMA and urine samples for urine specific gravity (USG). The reference interval for SDMA in unaffected koalas was 2.4-22.9 µg/dL. In koalas with oxalate nephrosis, SDMA was elevated in 74% of cases above the upper limit of the confidence interval. SDMA was elevated in three affected koalas with normal creatinine values. A positive correlation was found between SDMA and creatinine (R = 0.775, P < 0.001) and SDMA and urea (R = 0.580, P < 0.001) and a negative correlation between SDMA and USG (R = -0.495, P = 0.027). In conclusion, SDMA correlates well with other commonly used tests of renal function in koalas and should be included as part of the standard diagnostic process to increase the accuracy of oxalate nephrosis diagnosis in koalas.

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.

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.

Induction of neutralizing antibody response against koala retrovirus (KoRV) and reduction in viral load in koalas following vaccination with recombinant KoRV envelope protein.

Koala retrovirus (KoRV) infects the majority of Australia's koalas (Phascolarctos cinereus) and has been linked to several life-threatening diseases such as lymphoma and leukemia, as well as Chlamydia and thus poses a threat to the continued survival of this species. While quarantine and antiretroviral drug treatment are possible control measures, they are impractical, leaving vaccination as the only realistic option. In this study, we examined the effect of a recombinant envelope protein-based anti-KoRV vaccine in two groups of South Australian koalas: KoRV infected or KoRV free. We report a successful vaccination response in the koalas with no vaccine-associated side effects. The vaccine induced a significant humoral immune response as well as the production of neutralizing antibodies in both groups of koalas. We also identified B-cell epitopes that were differentially recognized in KoRV-infected versus KoRV-free koalas following vaccination. Importantly, we also showed that vaccination had a therapeutic effect on koalas infected exogenously with KoRV by reducing their circulating viral load. Together, this study highlights the possibility of successfully developing a vaccine against KoRV infection in koalas.

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.

Lymphoma, Koala Retrovirus Infection and Reproductive Chlamydiosis in a Koala (Phascolarctos cinereus).

Koala retrovirus (KoRV) infection, thought to be associated with lymphoid neoplasia, and Chlamydia pecorum-related ocular and urogenital disease are both highly prevalent in eastern Australian koala (Phascolarctos cinereus) populations. However, in South Australian koalas, little is known about KoRV infection and C. pecorum-associated disease. We report the first South Australian case of lymphoma in a KoRV-A-positive female koala also affected by severe reproductive chlamydiosis. The koala was from the Mount Lofty Ranges population and was presented with hindlimb lameness. Clinical examination identified right stifle crepitus, enlarged superficial lymph nodes and paraovarian cysts. Necropsy examination revealed extensive cartilage degeneration and loss over the medial femoral condyle, solid femoral bone marrow, mesenteric and ovarian tumours, paraovarian cysts and purulent metritis. Histopathology confirmed lymphoma in the bone marrow, mesenteric lymph nodes and ovary, with infiltration and parenchymal effacement in the pancreas, adrenal glands and other tissues. Lymphoma, KoRV and chlamydiosis are being investigated further in this population.

Myalgic encephalomyelitis: International Consensus Criteria.

The label 'chronic fatigue syndrome' (CFS) has persisted for many years because of the lack of knowledge of the aetiological agents and the disease process. In view of more recent research and clinical experience that strongly point to widespread inflammation and multisystemic neuropathology, it is more appropriate and correct to use the term 'myalgic encephalomyelitis' (ME) because it indicates an underlying pathophysiology. It is also consistent with the neurological classification of ME in the World Health Organization's International Classification of Diseases (ICD G93.3). Consequently, an International Consensus Panel consisting of clinicians, researchers, teaching faculty and an independent patient advocate was formed with the purpose of developing criteria based on current knowledge. Thirteen countries and a wide range of specialties were represented. Collectively, members have approximately 400 years of both clinical and teaching experience, authored hundreds of peer-reviewed publications, diagnosed or treated approximately 50 000 patients with ME, and several members coauthored previous criteria. The expertise and experience of the panel members as well as PubMed and other medical sources were utilized in a progression of suggestions/drafts/reviews/revisions. The authors, free of any sponsoring organization, achieved 100% consensus through a Delphi-type process. The scope of this paper is limited to criteria of ME and their application. Accordingly, the criteria reflect the complex symptomatology. Operational notes enhance clarity and specificity by providing guidance in the expression and interpretation of symptoms. Clinical and research application guidelines promote optimal recognition of ME by primary physicians and other healthcare providers, improve the consistency of diagnoses in adult and paediatric patients internationally and facilitate clearer identification of patients for research studies.

Fibromyalgia syndrome improved using a mostly raw vegetarian diet: an observational study.

BACKGROUND: Fibromyalgia engulfs patients in a downward, reinforcing cycle of unrestorative sleep, chronic pain, fatigue, inactivity, and depression. In this study we tested whether a mostly raw vegetarian diet would significantly improve fibromyalgia symptoms. METHODS: Thirty people participated in a dietary intervention using a mostly raw, pure vegetarian diet. The diet consisted of raw fruits, salads, carrot juice, tubers, grain products, nuts, seeds, and a dehydrated barley grass juice product. Outcomes measured were dietary intake, the fibromyalgia impact questionnaire (FIQ), SF-36 health survey, a quality of life survey (QOLS), and physical performance measurements. RESULTS: Twenty-six subjects returned dietary surveys at 2 months; 20 subjects returned surveys at the beginning, end, and at either 2 or 4 months of intervention; 3 subjects were lost to follow-up. The mean FIQ score (n = 20) was reduced 46% from 51 to 28. Seven of the 8 SF-36 subscales, bodily pain being the exception, showed significant improvement (n = 20, all P for trend < 0.01). The QOLS, scaled from 0 to 7, rose from 3.9 initially to 4.9 at 7 months (n = 20, P for trend 0.000001). Significant improvements (n = 18, P < 0.03, paired t-test) were seen in shoulder pain at rest and after motion, abduction range of motion of shoulder, flexibility, chair test, and 6-minute walk. 19 of 30 subjects were classified as responders, with significant improvement on all measured outcomes, compared to no improvement among non-responders. At 7 months responders' SF-36 scores for all scales except bodily pain were no longer statistically different from norms for women ages 45-54. CONCLUSION: This dietary intervention shows that many fibromyalgia subjects can be helped by a mostly raw vegetarian diet.

Survey of asthma deaths in the Northern region, 1970-85.

Thirty five asthma deaths in children aged 1 to 16 years were investigated in detail. Twenty four of these children had previously been under hospital consultant care and there were seven inpatient deaths. Twenty nine cases (83%) had a history of severe asthma, 17 of whom had previously experienced a life threatening attack. The fatal outcome, however, could not have been predicted in six children (17%) with preceding mild asthma. Potentially preventable factors in management were found in 28 cases (80%). While 18 (51%) had been chronically undertreated, the major factor in 20 deaths (57%) was suboptimal management of the final attack owing to delay in seeking medical attention, inadequate medical response, or both. Only two children had received systemic corticosteroid in appropriate amounts during the final illness. If mortality is to be reduced, families of asthmatic children must be educated to recognise severe symptoms and be given an appropriate 'crisis plan'. Hospitals should permit free access and have a clear protocol for the management of children with severe asthmatic attacks.