Evaluating the genetic consequences of population subdivision as it unfolds and how to best mitigate them: A rare story about koalas.

The genetic consequences of the subdivision of populations are regarded as significant to long-term evolution, and research has shown that the scale and speed at which this is now occurring is critically reducing the adaptive potential of most species which inhabit human-impacted landscapes. Here, we provide a rare and, to our knowledge, the first analysis of this process while it is happening and demonstrate a method of evaluating the effect of mitigation measures such as fauna crossings. We did this by using an extensive genetic data set collected from a koala population which was intensely monitored during the construction of linear transport infrastructure which resulted in the subdivision of their population. First, we found that both allelic richness and effective population size decreased through the process of population subdivision. Second, we predicted the extent to which genetic drift could impact genetic diversity over time and showed that after only 10 generations the resulting two subdivided populations could experience between 12% and 69% loss in genetic diversity. Lastly, using forward simulations we estimated that a minimum of eight koalas would need to disperse from each side of the subdivision per generation to maintain genetic connectivity close to zero but that 16 koalas would ensure that both genetic connectivity and diversity remained unchanged. These results have important consequences for the genetic management of species in human-impacted landscapes by showing which genetic metrics are best to identify immediate loss in genetic diversity and how to evaluate the effectiveness of any mitigation measures.

Assessment of florfenicol as a possible treatment for chlamydiosis in koalas (Phascolarctos cinereus).

OBJECTIVE: Because of limited availability of chloramphenicol to veterinary suppliers, a preliminary study was performed to predict whether an analogue, florfenicol, is an efficacious treatment for chlamydiosis in koalas. METHODS: Florfenicol was administered to koalas with naturally occurring chlamydiosis at 20 mg/kg SC (n = 3) and at 5 mg/kg (n = 3) and 10 mg/kg (n = 3) IV. The estimated areas under the plasma concentration versus time curves (AUC) were compared with the minimum inhibitory concentration to inhibit Chlamydia pecorum. Clinical data were also examined from field trials conducted on koalas (n = 19) with naturally occurring chlamydiosis and treated with florfenicol at a range of dosages (5-20 mg/kg SC and 6-15 mg/kg IV). Florfenicol binding to proteins in plasma was also determined. RESULTS: Florfenicol was not detectable in plasma 24 h post-administration at 20 mg/kg SC. The estimated AUC0-24 h following administration at 10 mg/kg IV suggests florfenicol might be effective against Chlamydia spp. via this route. Florfenicol binding to plasma proteins was 13.0% (± 0.30 SEM). After treatment with florfenicol in field trials, 5 of 19 koalas (26%) were released without further treatment, 4 with no long-term follow-up; 6 (32%) required additional treatment with chloramphenicol to resolve chlamydiosis; 7 (36%) failed to clinically improve, of which 3 had clinical signs and/or necropsy findings suggestive of antibiotic-related gastrointestinal dysbiosis; another koala died within minutes of florfenicol administered IV at 7 mg/kg. CONCLUSION: When administered at dosages tolerable in the field, florfenicol is a problematic treatment for chlamydiosis based on equivocal outcomes and plasma concentrations below those that inhibit the pathogen.

Prevalence of koala retrovirus in geographically diverse populations in Australia.

OBJECTIVE: To determine the prevalence of koala retrovirus (KoRV) in selected koala populations and to estimate proviral copy number in a subset of koalas. METHODS: Blood or tissue samples from 708 koalas in Queensland, New South Wales, Victoria and South Australia were tested for KoRV pol provirus gene using standard polymerase chain reaction (PCR), nested PCR and real-time PCR (qPCR). RESULTS: Prevalence of KoRV provirus-positive koalas was 100% in four regions of Queensland and New South Wales, 72.2% in mainland Victoria, 26.6% on four Victorian islands and 14.8% on Kangaroo Island, South Australia. Estimated proviral copy number per cell in four groups of koalas from Queensland and Victoria showed marked variation, ranging from a mean of 165 copies per cell in the Queensland group to 1.29 × 10(-4) copies per cell in one group of Victorian koalas. CONCLUSIONS: The higher prevalence of KoRV-positive koalas in the north of Australia and high proviral loads in Queensland koalas may indicate KoRV entered and became endogenous in the north and is spreading southwards. It is also possible there are genetic differences between koalas in northern and southern Australia that affect susceptibility to KoRV infection or endogenisation, or that environmental factors affecting transmission in northern states are absent or uncommon in southern regions. Although further studies are required, the finding of proviral copy numbers orders of magnitude lower than what would be expected for the presence of a single copy in every cell for many Victorian animals suggests that KoRV is not endogenous in these animals and likely reflects ongoing exogenous infection.

Plasma concentrations of chloramphenicol after subcutaneous administration to koalas (Phascolarctos cinereus) with chlamydiosis.

Nine mature koalas with chlamydiosis, typically keratoconjunctivitis and/or urogenital tract infection, were treated with daily subcutaneous injections of chloramphenicol at 60 mg/kg for 45 days (five koalas), or for a shorter duration (four koalas). All koalas were initially positive for Chlamydia pecorum as determined by real-time polymerase chain reaction (qPCR). Plasma chloramphenicol concentrations were determined at t = 0, 1, 2, 4, 8, and 24 h after the day 1 injection (nine koalas) and after the day 15 injection (seven koalas). Chloramphenicol reached a median (and range) maximum plasma concentration of 3.03 (1.32-5.03 µg/mL) at 4 (1-8 h) after the day 1 injection and 4.82 (1.97-27.55 µg/mL) at 1 (1-2 h) after day 15. The median (and range) of AUC(0-24) on day 1 and day 15 were 48.14 (22.37-81.14 µg·h/mL) and 50.83 (28.43-123.99 µg·h/mL), respectively. The area under the moment curve (AUMC)(0-24) median (and range) for day 1 and day 15 were 530.03 (233.05-798.97 h) and 458.15 (291.72-1093.58 h), respectively. Swabs were positive for chlamydial DNA pretreatment, and all koalas except one, produced swabs negative for chlamydial DNA during treatment and which remained so, for 2-63 days after treatment, however whether chloramphenicol treatment prevented long-term recrudescence of infection was not established. At this dose and dosing frequency, chloramphenicol appeared to control mild chlamydial infection and prevent shedding, but severe urogenital disease did not appear to respond to chloramphenicol at this dosage regime. For koalas affected by severe chlamydiosis, antibiotics alone are not sufficient to effect a cure, possibly because of structural or metabolic changes associated with chronic disease and inflammation.

Using quantitative polymerase chain reaction to correlate Chlamydia pecorum infectious load with ocular, urinary and reproductive tract disease in the koala (Phascolarctos cinereus).

Complex interactions between Chlamydia pecorum infection, the immune response and disease exist in the koala. We used quantitative polymerase chain reaction to investigate the relationship between C. pecorum infectious load and ocular and urogenital tract disease. Chlamydia pecorum shedding was generally higher in animals with chronic, active disease than in animals with inactive disease. The absence of ocular disease was generally associated with low levels of shedding, but relatively high levels of shedding in the urogenital tract were detected in some koalas without clinical disease signs. These results suggest a complex disease pathogenesis and clinical course in C. pecorum-infected koalas.

The potential impact of native Australian trypanosome infections on the health of koalas (Phascolarctos cinereus).

Whole blood collected from koalas admitted to the Australian Zoo Wildlife Hospital (AZWH), Beerwah, QLd, Australia, during late 2006-2009 was tested using trypanosome species-specific 18S rDNA PCRs designed to amplify DNA from Trypanosoma irwini, T. gilletti and T. copemani. Clinical records for each koala sampled were reviewed and age, sex, blood packed cell volume (PCV), body condition, signs of illness, blood loss, trauma, chlamydiosis, bone marrow disease, koala AIDS and hospital admission outcome ('survival'/ 'non-survival') were correlated with PCR results. Overall 73.8% (439/595) of the koalas were infected with at least 1 species of trypanosome. Trypanosoma irwini was detected in 423/595 (71.1%), T. gilletti in 128/595 (21.5%) and T. copemani in 26/595 (4.4%) of koalas. Mixed infections were detected in 125/595 (21%) with co-infections of T. irwini and T. gilletti (101/595, 17%) being most common. There was a statistical association between infection with T. gilletti with lower PCV values and body condition scores in koalas with signs of chlamydiosis, bone marrow disease or koala AIDS. No association between T. gilletti infection and any indicator of health was observed in koalas without signs of concurrent disease. This raises the possibility that T. gilletti may be potentiating other disease syndromes affecting koalas.

Novel trypanosome Trypanosoma gilletti sp. (Euglenozoa: Trypanosomatidae) and the extension of the host range of Trypanosoma copemani to include the koala ( Phascolarctos cinereus).

Trypanosoma irwini was previously described from koalas and we now report the finding of a second novel species, T. gilletti, as well as the extension of the host range of Trypanosoma copemani to include koalas. Phylogenetic analysis at the 18S rDNA and gGAPDH loci demonstrated that T. gilletti was genetically distinct with a genetic distance (± s.e.) at the 18S rDNA locus of 2.7 ± 0.5% from T. copemani (wombat). At the gGAPDH locus, the genetic distance (± s.e.) of T. gilletti was 8.7 ± 1.1% from T. copemani (wombat). Trypanosoma gilletti was detected using a nested trypanosome 18S rDNA PCR in 3/139 (∼2%) blood samples and in 2/29 (∼7%) spleen tissue samples from koalas whilst T. irwini was detected in 72/139 (∼52%) blood samples and T. copemani in 4/139 (∼3%) blood samples from koalas. In addition, naturally occurring mixed infections were noted in 2/139 (∼1.5%) of the koalas tested.

Trypanosoma irwini n. sp (Sarcomastigophora: Trypanosomatidae) from the koala ( Phascolarctos cinereus).

The morphology and genetic characterization of a new species of trypanosome infecting koalas (Phascolarctos cinereus) are described. Morphological analysis of bloodstream forms and phylogenetic analysis at the 18S rDNA and gGAPDH loci demonstrated this trypanosome species to be genetically distinct and most similar to Trypanosoma bennetti, an avian trypanosome with a genetic distance of 0.9% at the 18S rDNA and 10.7% at the gGAPDH locus. The trypanosome was detected by 18S rDNA PCR in the blood samples of 26 out of 68 (38.2%) koalas studied. The aetiological role of trypanosomes in koala disease is currently poorly defined, although infection with these parasites has been associated with severe clinical signs in a number of koalas. Based on biological and genetic characterization data, this trypanosome species infecting koalas is proposed to be a new species Trypanosome irwini n. sp.

The nucleotide sequence of koala (Phascolarctos cinereus) retrovirus: a novel type C endogenous virus related to Gibbon ape leukemia virus.

A novel retrovirus, morphologically consistent with mammalian C-type retroviruses, was detected by electron microscopy in mitogen-stimulated peripheral blood mononuclear cell cultures from 163 koalas and in lymphoma tissue from 3 koalas. PCR amplified provirus from the blood and tissues of 17 wild and captive koalas, and reverse transcriptase-PCR demonstrated viral mRNA, viral genomic RNA, and reverse transcriptase activity in koala serum and cell culture supernatants. Comparison of viral sequences derived from genomic DNA and mRNA showed identity indicative of a single retroviral species-here designated koala retrovirus (KoRV). Southern blot analysis of koala tissue genomic DNA using labelled KoRV probes demonstrated banding consistent with an endogenous retrovirus. Complete and apparently truncated proviruses were detected in DNA of both clinically normal koalas and those with hematopoietic disease. KoRV-related viruses were not detected in other marsupials, and phylogenetic analysis showed that KoRV paradoxically clusters with gibbon ape leukemia virus (GALV). The strong similarity between GALV and KoRV suggests that these viruses are closely related and that recent cross-host transmission has occurred. The complete proviral DNA sequence of KoRV is reported.

The arrangement of gut-associated lymphoid tissues and lymph pathways in the koala (Phascolarctos cinereus).

Gut-associated lymphoid tissues are poorly developed in koalas. They comprise paired caecocolic lymphoid patches, and a few small mesenteric lymph nodes. The patches lie opposite one another in the lateral gut wall at the junction of the ileum, caecum and proximal colon. The lymphoid parenchyma of the patches consists of a layer of nodules and internodular parenchyma in the submucosa. Apoptosis is common in the nodules. The mucosa and lymphoid tissue of each patch is continuous over a caecocolic recess, formed by the coalescence of laminae which extend along the large intestine. Lymph sinuses between and beneath the lymphoid nodules are continuous with efferent lymph vessels in the submucosa. These then enter 2-4 small lymph nodes at the root of the mesentery. The paucity of lymphoid tissue associated with the gut may be related to germicidal activity in the Eucalypt leaves eaten by the koala.

Topography of the major superficial lymph nodes and their efferent lymph pathways in the koala (Phascolarctos cinereus).

The koala has an inguinoaxillary lymph trunk on either side of the ventral midline, and this carries efferent lymph from the superficial inguinal lymph node directly to the deep axillary lymph node. The superficial lymph nodes are large and soft compared with those of the domestic species, and each lymph centre usually contains only one or two large lymph nodes. Koalas have a rostral mandibular lymph node which has not been described in other species, but lack popliteal and subiliac lymph nodes. The superficial lymph nodes which are readily palpable in the live koala are the facial, rostral mandibular, mandibular, superficial axillary and superficial inguinal. All superficial lymph pathways terminate at the confluence of the common jugular and subclavian veins.