Damien P. Higgins

A retrospective study of admission trends of koalas to a rehabilitation facility over 30 years

To identify threats to the survival of koalas (Phascolarctos cinereus) in coastal New South Wales, Australia, we compared 3,781 admission records of koalas, admitted between 1 January 1975 and 31 December 2004 to a koala rehabilitation facility on the midnorthern coast of New South Wales, against local wild population demographics, with the use of multinomial logistic regression and chi-square analyses. Trauma, the most frequent reason for admission, affected young and male animals more frequently than other groups. Seasonal differences in the probability of males presenting as trauma cases suggest behavioral factors as an important risk factor for this group. An increasing probability of koalas presenting as a result of motor vehicle accident since 1985 strongly supports the enhanced action of local authorities to pursue traffic-calming strategies if urban koala populations are to be maintained in this area. Koalas with clinical signs of chlamydiosis made up the second most frequent admission group, and these animals were more likely to be aged. This study highlights the potential usefulness of wildlife rehabilitation centers in detailing threats to local wildlife populations, provided record keeping is efficient and focused, and the role of such studies in providing evidence for focusing threat-mitigation efforts. Continual community engagement by koala researchers is important to ensure that maximum benefit is obtained from activities of special interest groups.

Comparative Genome Analyses Reveal Distinct Structure in the Saltwater Crocodile MHC

The major histocompatibility complex (MHC) is a dynamic genome region with an essential role in the adaptive immunity of vertebrates, especially antigen presentation. The MHC is generally divided into subregions (classes I, II and III) containing genes of similar function across species, but with different gene number and organisation. Crocodylia (crocodilians) are widely distributed and represent an evolutionary distinct group among higher vertebrates, but the genomic organisation of MHC within this lineage has been largely unexplored. Here, we studied the MHC region of the saltwater crocodile (Crocodylus porosus) and compared it with that of other taxa. We characterised genomic clusters encompassing MHC class I and class II genes in the saltwater crocodile based on sequencing of bacterial artificial chromosomes. Six gene clusters spanning ∼452 kb were identified to contain nine MHC class I genes, six MHC class II genes, three TAP genes, and a TRIM gene. These MHC class I and class II genes were in separate scaffold regions and were greater in length (2–6 times longer) than their counterparts in well-studied fowl B loci, suggesting that the compaction of avian MHC occurred after the crocodilian-avian split. Comparative analyses between the saltwater crocodile MHC and that from the alligator and gharial showed large syntenic areas (>80% identity) with similar gene order. Comparisons with other vertebrates showed that the saltwater crocodile had MHC class I genes located along with TAP, consistent with birds studied. Linkage between MHC class I and TRIM39 observed in the saltwater crocodile resembled MHC in eutherians compared, but absent in avian MHC, suggesting that the saltwater crocodile MHC appears to have gene organisation intermediate between these two lineages. These observations suggest that the structure of the saltwater crocodile MHC, and other crocodilians, can help determine the MHC that was present in the ancestors of archosaurs.

Association of Uterine and Salpingeal Fibrosis with Chlamydial Hsp60 and Hsp10 Antigen-Specific Antibodies in Chlamydia-Infected Koalas

ABSTRACT Infection by Chlamydia pneumoniae or Chlamydia pecorum commonly causes chronic, fibrotic disease of the urogenital tracts of female koalas. Studies of humans have associated titers of serum immunoglobulin G (IgG) against chlamydial hsp60 and hsp10 antigens with chronic infection, salpingeal fibrosis, and tubal infertility. To determine whether a similar relationship exists in Chlamydia-infected koalas, samples were collected opportunistically from 34 wild female koalas and examined by gross pathology and histopathology, PCR, and immunohistochemistry for Chlamydia spp. and enzyme-linked immunosorbent assay for serological responses to chlamydial hsp10 and hsp60 antigens. Greater anti-hsp titers occurred in Chlamydia-infected koalas with fibrous occlusion of the uterus or uterine tube than in other Chlamydia-infected koalas (for hsp10 IgG, P = 0.005; for hsp60 IgG, P = 0.001; for hsp10 IgA, P = 0.04; for hsp60 IgA, P = 0.09). However, as in humans, some koalas with tubal occlusion had low titers. Among Chlamydia-infected koalas with tubal occlusion, those with low titers were more likely to have an active component to their ongoing uterine or salpingeal inflammation (P = 0.007), such that the assay predicted, with 79% sensitivity and 92% specificity, tubal occlusion where an active component of inflammation was absent. Findings of this study permit advancement of clinical and epidemiological studies of host-pathogen-environment interactions and pose intriguing questions regarding the significance of the Th1/Th2 paradigm and antigen-presenting and inflammation-regulating capabilities of uterine epithelial cells and the roles of latency and reactivation of chlamydial infections in pathogenesis of upper reproductive tract disease of koalas.

Absorption of enrofloxacin and marbofloxacin after oral and subcutaneous administration in diseased koalas (Phascolarctos cinereus)

Koalas (n = 43) were treated daily for up to 8 weeks with enrofloxacin: 10 mg/kg subcutaneously (s.c.), 5 mg/kg s.c., or 20 mg/kg per os (p.o.); or marbofloxacin: 1.0-3.3 mg/kg p.o., 10 mg/kg p.o. or 5 mg/kg s.c. Serial plasma drug concentrations were determined on day 1 and again at approximately 2 weeks, by liquid chromatography. The median (range) plasma maximum concentrations (C(max) ) for enrofloxacin 5 mg/kg s.c. and 10 mg/kg s.c. were 0.83 (0.68-1.52) and 2.08 (1.34-2.96) μg/mL and the median (range) T(max) were 1.5 h (1-2) and 1 h (1-2) respectively. Plasma concentrations of orally dosed marbofloxacin were too low to be quantified. Oral administration of enrofloxacin suggested absorption rate limited disposition pharmacokinetics; the median (range) C(max) for enrofloxacin 20 mg/kg p.o. was 0.94 (0.76-1.0) μg/mL and the median (range) T(max) was 4 h (2-8). Oral absorption of both drugs was poor. Plasma protein binding for enrofloxacin was 55.4 ± 1.9% and marbofloxacin 49.5 ± 5.3%. Elevations in creatinine kinase activity were associated with drug injections. Enrofloxacin and marbofloxacin administered at these dosage and routes are unlikely to inhibit the growth of chlamydial pathogens in vivo.

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.

Pharmacokinetics of meloxicam in koalas (Phascolarctos cinereus) after intravenous, subcutaneous and oral administration

The pharmacokinetic profile of meloxicam in clinically healthy koalas (n = 15) was investigated. Single doses of meloxicam were administered intravenously (i.v.) (0.4 mg/kg; n = 5), subcutaneously (s.c.) (0.2 mg/kg; n = 1) or orally (0.2 mg/kg; n = 3), and multiple doses were administered to two groups of koalas via the oral or s.c. routes (n = 3 for both routes) with a loading dose of 0.2 mg/kg for day 1 followed by 0.1 mg/kg s.i.d for a further 3 days. Plasma meloxicam concentrations were quantified by high-performance liquid chromatography. Following i.v. administration, meloxicam exhibited a rapid clearance (CL) of 0.44 ± 0.20 (SD) L/h/kg, a volume of distribution at terminal phase (Vz ) of 0.72 ± 0.22 L/kg and a volume of distribution at steady state (Vss ) of 0.22 ± 0.12 L/kg. Median plasma terminal half-life (t(1/2)) was 1.19 h (range 0.71-1.62 h). Following oral administration either from single or repeated doses, only maximum peak plasma concentration (C(max) 0.013 ± 0.001 and 0.014 ± 0.001 μg/mL, respectively) was measurable [limit of quantitation (LOQ) >0.01 μg/mL] between 4-8 h. Oral bioavailability was negligible in koalas. Plasma protein binding of meloxicam was ~98%. Three meloxicam metabolites were detected in plasma with one identified as the 5-hydroxy methyl derivative. This study demonstrated that koalas exhibited rapid CL and extremely poor oral bioavailability compared with other eutherian species. Accordingly, the currently recommended dose regimen of meloxicam for this species appears inadequate.

MHC class II diversity of koala ( Phascolarctos cinereus) populations across their range

Major histocompatibility complex class II (MHCII) genes code for proteins that bind and present antigenic peptides and trigger the adaptive immune response. We present a broad geographical study of MHCII DA β1 (DAB) and DB β1 (DBB) variants of the koala (Phascolarctos cinereus; n=191) from 12 populations across eastern Australia, with a total of 13 DAB and 7 DBB variants found. We identified greater MHCII variation and, possibly, additional gene copies in koala populations in the north (Queensland and New South Wales) relative to the south (Victoria), confirmed by STRUCTURE analyses and genetic differentiation using analysis of molecular variance. The higher MHCII diversity in the north relative to south could potentially be attributed to (i) significant founder effect in Victorian populations linked to historical translocation of bottlenecked koala populations and (ii) increased pathogen-driven balancing selection and/or local genetic drift in the north. Low MHCII genetic diversity in koalas from the south could reduce their potential response to disease, although the three DAB variants found in the south had substantial sequence divergence between variants. This study assessing MHCII diversity in the koala with historical translocations in some populations contributes to understanding the effects of population translocations on functional genetic diversity.

Characterisation of four major histocompatibility complex class II genes of the koala (Phascolarctos cinereus)

Major histocompatibility complex (MHC) class II molecules have an integral role in the adaptive immune response, as they bind and present antigenic peptides to T helper lymphocytes. In this study of koalas, species-specific primers were designed to amplify exon 2 of the MHC class II DA and DB genes, which contain much of the peptide-binding regions of the α and β chains. A total of two DA α1 domain variants and eight DA β1 (DAB), three DB α1 and five DB β1 variants were amplified from 20 koalas from two free-living populations from South East Queensland and the Port Macquarie region in northern New South Wales. We detected greater variation in the β1 than in the α1 domains as well as evidence of positive selection in DAB. The present study provides a springboard to future investigation of the role of MHC in disease susceptibility in koalas.

Genetic diversity in the plasticity zone and the presence of the chlamydial plasmid differentiates Chlamydia pecorum strains from pigs, sheep, cattle, and koalas.

BackgroundChlamydia pecorum is a globally recognised pathogen of livestock and koalas. To date, comparative genomics of C. pecorum strains from sheep, cattle and koalas has revealed that only single nucleotide polymorphisms (SNPs) and a limited number of pseudogenes appear to contribute to the genetic diversity of this pathogen. No chlamydial plasmid has been detected in these strains despite its ubiquitous presence in almost all other chlamydial species. Genomic analyses have not previously included C. pecorum from porcine hosts. We sequenced the genome of three C. pecorum isolates from pigs with differing pathologies in order to re-evaluate the genetic differences and to update the phylogenetic relationships between C. pecorum from each of the hosts.MethodsWhole genome sequences for the three porcine C. pecorum isolates (L1, L17 and L71) were acquired using C. pecorum-specific sequence capture probes with culture-independent methods, and assembled in CLC Genomics Workbench. The pairwise comparative genomic analyses of 16 pig, sheep, cattle and koala C. pecorum genomes were performed using several bioinformatics platforms, while the phylogenetic analyses of the core C. pecorum genomes were performed with predicted recombination regions removed. Following the detection of a C. pecorum plasmid, a newly developed C. pecorum-specific plasmid PCR screening assay was used to evaluate the plasmid distribution in 227 C. pecorum samples from pig, sheep, cattle and koala hosts.ResultsThree porcine C. pecorum genomes were sequenced using C. pecorum-specific sequence capture probes with culture-independent methods. Comparative genomics of the newly sequenced porcine C. pecorum genomes revealed an increased average number of SNP differences (~11 500) between porcine and sheep, cattle, and koala C. pecorum strains, compared to previous C. pecorum genome analyses. We also identified a third copy of the chlamydial cytotoxin gene, found only in porcine C. pecorum isolates. Phylogenetic analyses clustered porcine isolates into a distinct clade, highlighting the polyphyletic origin of C. pecorum in livestock.Most surprising, we also discovered a plasmid in the porcine C. pecorum genome. Using this novel C. pecorum plasmid (pCpec) sequence, a) we developed a pCpec screening assay to evaluate the plasmid distribution in C. pecorum from different hosts; and b) to characterise the pCpec sequences from available previously sequenced C. pecorum genome data. pCpec screening showed that the pCpec is common in all hosts of C. pecorum, however not all C. pecorum strains carry pCpec.ConclusionsThis study provides further insight into the complexity of C. pecorum epidemiology and novel genomic regions that may be linked to host specificity. C. pecorum plasmid characterisation may aid in improving our understanding of C. pecorum pathogenesis across the variety of host species this animal pathogen infects.

Evolution of MHC class I in the Order Crocodylia

The major histocompatibility complex (MHC) is a dynamic genomic region with an essential role in the adaptive immunity of jawed vertebrates. The evolution of the MHC has been dominated by gene duplication and gene loss, commonly known as the birth-and-death process. Evolutionary studies of the MHC have mostly focused on model species. However, the investigation of this region in non-avian reptiles is still in its infancy. To provide insights into the evolutionary mechanisms that have shaped the diversity of this region in the Order Crocodylia, we investigated MHC class I exon 3, intron 3, and exon 4 across 20 species of the families Alligatoridae and Crocodilidae. We generated 124 DNA sequences and identified 31 putative functional variants as well as 14 null variants. Phylogenetic analyses revealed three gene groups, all of which were present in Crocodilidae but only one in Alligatoridae. Within these groups, variants generally appear to cluster at the genus or family level rather than in species-specific groups. In addition, we found variation in gene copy number and some indication of interlocus recombination. These results suggest that MHC class I in Crocodylia underwent independent events of gene duplication, particularly in Crocodilidae. These findings enhance our understanding of MHC class I evolution and provide a preliminary framework for comparative studies of other non-avian reptiles as well as diversity assessment within Crocodylia.