Dataset Information

Rapid point-of-care diagnostics for the detection of Chlamydia pecorum in koalas (Phascolarctos cinereus) using loop-mediated isothermal amplification without nucleic acid purification.

ABSTRACT: Infectious disease, predominately chlamydiosis, contributes significantly to the decline in health of wild koala (Phascolarctos cinereus) populations in some regions of Australia. In this study, we describe the development and evaluation of a simple, sensitive, and specific loop-mediated isothermal amplification (LAMP) assay for the detection of Chlamydia pecorum in koalas as a point-of-care diagnostic tool that can be used in any wildlife hospital and in the field on specialized instrumentation. A set of primers targeting a 188-bp region of the C. pecorum genome was designed. 100% specificity of the LAMP assay was revealed by demonstrating no cross-reactivity with 33 nontarget pathogens, and complete correlation with qPCR results for 43 clinical swabs collected opportunistically from wildlife hospitals. In sensitivity evaluations, the technique successfully detected serial dilutions of extracted C. pecorum DNA with a detection limit of 44 IFU/ml.

SUBMITTER: Hulse LS

PROVIDER: S-EPMC6925175 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Rapid point-of-care diagnostics for the detection of Chlamydia pecorum in koalas (Phascolarctos cinereus) using loop-mediated isothermal amplification without nucleic acid purification.

Hulse Lyndal S LS McDonald Sean S Johnston Stephen D SD Beagley Kenneth W KW

MicrobiologyOpen 20190816 12

Infectious disease, predominately chlamydiosis, contributes significantly to the decline in health of wild koala (Phascolarctos cinereus) populations in some regions of Australia. In this study, we describe the development and evaluation of a simple, sensitive, and specific loop-mediated isothermal amplification (LAMP) assay for the detection of Chlamydia pecorum in koalas as a point-of-care diagnostic tool that can be used in any wildlife hospital and in the field on specialized instrumentation ...[more]

PMID: 31419041

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Project description:Koalas (Phascolarctos cinereus), an iconic Australian marsupial, are being heavily impacted by the spread of Chlamydia pecorum, an obligate intracellular bacterial pathogen. Koalas vary in their response to this pathogen, with some showing no symptoms, while others suffer severe symptoms leading to infertility, blindness or death. Little is known about the pathology of this disease and the immune response against it in this host. Studies have demonstrated that natural killer (NK) cells, key components of the innate immune system, are involved in the immune response to chlamydial infections in humans. These cells can directly lyse cells infected by intracellular pathogens and their ability to recognise these infected cells is mediated through NK receptors on their surface. These are encoded in two regions of the genome, the leukocyte receptor complex (LRC) and the natural killer complex (NKC). These two families evolve rapidly and different repertoires of genes, which have evolved by gene duplication, are seen in different species.In this study we aimed to characterise genes belonging to the NK receptor clusters in the koala by searching available koala transcriptomes using a combination of search methods. We developed a qPCR assay to quantify relative expression of four genes, two encoded within the NK receptor cluster (CLEC1B, CLEC4E) and two known to play a role in NK response to Chalmydia in humans (NCR3, PRF1).We found that the NK receptor repertoire of the koala closely resembles that of the Tasmanian devil, with minimal genes in the NKC, but with lineage specific expansions in the LRC. Additional genes important for NK cell activity, NCR3 and PRF1, were also identified and characterised. In a preliminary study to investigate whether these genes are involved in the koala immune response to infection by its chlamydial pathogen, C. pecorum, we investigated the expression of four genes in koalas with active chlamydia infection, those with past infection and those without infection using qPCR. This analysis revealed that one of these four, CLEC4E, may be upregulated in response to chlamydia infection.We have characterised genes of the NKC and LRC in koalas and have discovered evidence that one of these genes may be upregulated in koalas with chlamydia, suggesting that these receptors may play a role in the immune response of koalas to chlamydia infection.

Project description:Chlamydia pecorum is a mucosal infection, which causes debilitating disease of the urinary tract, reproductive tract and ocular sites of koalas (Phascolarctos cinereus). While antibiotics are available for treatment, they are detrimental to the koalas' gastrointestinal tract microflora leaving the implementation of a vaccine as an ideal option for the long-term management of koala populations. We have previously reported on the successes of an anti-chlamydial recombinant major outer membrane protein (rMOMP) vaccine however, recombinant protein based vaccines are not ideal candidates for scale up from the research level to small-medium production level for wider usage. Peptide based vaccines are a promising area for vaccine development, because peptides are stable, cost effective and easily produced. In this current study, we assessed, for the first time, the immune responses to a synthetic peptide based anti-chlamydial vaccine in koalas. Five healthy male koalas were vaccinated with two synthetic peptides derived from C. pecorum MOMP and another five healthy male koalas were vaccinated with full length recombinant C. pecorum MOMP (genotype G). Systemic (IgG) and mucosal (IgA) antibodies were quantified and pre-vaccination levels compared to post-vaccination levels (12 and 26 weeks). MOMP-peptide vaccinated koalas produced Chlamydia-specific IgG and IgA antibodies, which were able to recognise not only the genotype used in the vaccination, but also MOMPs from several other koala C. pecorum genotypes. In addition, IgA antibodies induced at the ocular site not only recognised recombinant MOMP protein but also, whole native chlamydial elementary bodies. Interestingly, some MOMP-peptide vaccinated koalas showed a stronger and more sustained vaccine-induced mucosal IgA antibody response than observed in MOMP-protein vaccinated koalas. These results demonstrate that a synthetic MOMP peptide based vaccine is capable of inducing a Chlamydia-specific antibody response in koalas and is a promising candidate for future vaccine development.

Project description:Chlamydia pecorum is responsible for causing ocular infection and disease which can lead to blindness in koalas (Phascolarctos cinereus). Antibiotics are the current treatment for chlamydial infection and disease in koalas, however, they can be detrimental for the koala's gastrointestinal tract microbiota and in severe cases, can lead to dysbiosis and death. In this study, we evaluated the therapeutic effects provided by a recombinant chlamydial major outer membrane protein (MOMP) vaccine on ocular disease in koalas. Koalas with ocular disease (unilateral or bilateral) were vaccinated and assessed for six weeks, evaluating any changes to the conjunctival tissue and discharge. Samples were collected pre- and post-vaccination to evaluate both humoral and cell-mediated immune responses. We further assessed the infecting C. pecorum genotype, host MHC class II alleles and presence of koala retrovirus type (KoRV-B). Our results clearly showed an improvement in the clinical ocular disease state of all seven koalas, post-vaccination. We observed increases in ocular mucosal IgA antibodies to whole C. pecorum elementary bodies, post-vaccination. We found that systemic cell-mediated immune responses to interferon-?, interleukin-6 and interleukin-17A were not significantly predictive of ocular disease in koalas. Interestingly, one koala did not have as positive a clinical response (in one eye primarily) and this koala was infected with a C. pecorum genotype (E') that was not used as part of the vaccine formula (MOMP genotypes A, F and G). The predominant MHC class II alleles identified were DAb*19, DAb*21 and DBb*05, with no two koalas identified with the same genetic sequence. Additionally, KoRV-B, which is associated with chlamydial disease outcome, was identified in two (29%) ocular diseased koalas, which still produced vaccine-induced immune responses and clinical ocular improvements post-vaccination. Our findings show promise for the use of a recombinant chlamydial MOMP vaccine for the therapeutic treatment of ocular disease in koalas.

Project description:BackgroundChlamydia pecorum is an obligate intracellular bacterium and the causative agent of reproductive and ocular disease in several animal hosts including koalas, sheep, cattle and goats. C. pecorum strains detected in koalas are genetically diverse, raising interesting questions about the origin and transmission of this species within koala hosts. While the ompA gene remains the most widely-used target in C. pecorum typing studies, it is generally recognised that surface protein encoding genes are not suited for phylogenetic analysis and it is becoming increasingly apparent that the ompA gene locus is not congruent with the phylogeny of the C. pecorum genome. Using the recently sequenced C. pecorum genome sequence (E58), we analysed 10 genes, including ompA, to evaluate the use of ompA as a molecular marker in the study of koala C. pecorum genetic diversity.ResultsThree genes (incA, ORF663, tarP) were found to contain sufficient nucleotide diversity and discriminatory power for detailed analysis and were used, with ompA, to genotype 24 C. pecorum PCR-positive koala samples from four populations. The most robust representation of the phylogeny of these samples was achieved through concatenation of all four gene sequences, enabling the recreation of a "true" phylogenetic signal. OmpA and incA were of limited value as fine-detailed genetic markers as they were unable to confer accurate phylogenetic distinctions between samples. On the other hand, the tarP and ORF663 genes were identified as useful "neutral" and "contingency" markers respectively, to represent the broad evolutionary history and intra-species genetic diversity of koala C. pecorum. Furthermore, the concatenation of ompA, incA and ORF663 sequences highlighted the monophyletic nature of koala C. pecorum infections by demonstrating a single evolutionary trajectory for koala hosts that is distinct from that seen in non-koala hosts.ConclusionsWhile the continued use of ompA as a fine-detailed molecular marker for epidemiological analysis appears justified, the tarP and ORF663 genes also appear to be valuable markers of phylogenetic or biogeographic divisions at the C. pecorum intra-species level. This research has significant implications for future typing studies to understand the phylogeny, genetic diversity, and epidemiology of C. pecorum infections in the koala and other animal species.

Dataset Information

Rapid point-of-care diagnostics for the detection of Chlamydia pecorum in koalas (Phascolarctos cinereus) using loop-mediated isothermal amplification without nucleic acid purification.

Publications

Rapid point-of-care diagnostics for the detection of Chlamydia pecorum in koalas (Phascolarctos cinereus) using loop-mediated isothermal amplification without nucleic acid purification.

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