In Chlamydia veritas.

In Chlamydia verita

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

© 2015 Jelocnik et al. Background: Chlamydia 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. Methods: Whole 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. Results: Three 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. Conclusions: This 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

Investigation of Chlamydophila spp. in dairy cows with reproductive disorders

<p>Abstract</p> <p>Background</p> <p>Reports worldwide indicate high prevalence of <it>Chlamydophila </it>spp. infection in cattle. To assess the prevalence in Sweden, 525 cows in 70 dairy herds with reproductive disorders was investigated.</p> <p>Methods</p> <p>To detect antibodies two commercially available kits were used. Moreover, 107 specimens, including vaginal swabs, organ tissues and milk were analysed by Polymerase Chain Reaction (PCR).</p> <p>Results</p> <p>Two (0.4%) cows were seropositive in the Pourquier <it>Cp. abortus </it>ELISA. The seroprevalence with the Chekit ELISA was 28% with no difference between cases and controls. Five specimens were positive in real-time PCR and further analysed by nested PCR. <it>Cp. pecorum </it>was confirmed by partial <it>omp1 </it>DNA sequencing of the nested PCR product of vaginal swabs from control cows.</p> <p>Conclusion</p> <p>The results suggest that <it>Cp. abortus </it>infection is absent or rare in Swedish cows whereas <it>Cp. pecorum </it>is probably more spread. They also suggest that <it>Chlamydophila </it>spp. are not related to reproduction disorders in Swedish cattle.</p

Chlamydia trachomatis Test-of-Cure Cannot Be Based on a Single Highly Sensitive Laboratory Test Taken at Least 3 Weeks after Treatment

Current test-of-cure practice in patients with Chlamydia trachomatis (Ct) infection is to confirm cure with a single test taken at least 3 weeks after treatment. Effectiveness of single-time-point testing however lacks a scientific evidence basis and the high sensitivity of laboratory assays nowadays in use for this purpose may compromise the clinical significance of their results. Prospectively following 59 treated Ct infections, administering care as usual, the presence of Ct plasmid DNA and rRNA was systematically assessed by multiple time-sequential measurements, i.e. on 18 samples taken per patient during 8 weeks following treatment with a single dose of 1000 mg Azythromycin. A high proportion (42%) of Ct infections tested positive on at least one of the samples taken after 3 weeks. Patients' test results showed substantial inter-individual and intra-individual variation over time and by type of NAAT used. We demonstrated frequent intermittent positive patterns in Ct test results over time, and strongly argue against current test-of-cure practice

Multicolor Combinatorial Probe Coding for Real-Time PCR

The target volume of multiplex real-time PCR assays is limited by the number of fluorescent dyes available and the number of fluorescence acquisition channels present in the PCR instrument. We hereby explored a probe labeling strategy that significantly increased the target volume of real-time PCR detection in one reaction. The labeling paradigm, termed “Multicolor Combinatorial Probe Coding” (MCPC), uses a limited number (n) of differently colored fluorophores in various combinations to label each probe, enabling one of 2n-1 genetic targets to be detected in one reaction. The proof-of-principle of MCPC was validated by identification of one of each possible 15 human papillomavirus types, which is the maximum target number theoretically detectable by MCPC with a 4-color channel instrument, in one reaction. MCPC was then improved from a one-primer-pair setting to a multiple-primer-pair format through Homo-Tag Assisted Non-Dimer (HAND) system to allow multiple primer pairs to be included in one reaction. This improvement was demonstrated via identification of one of the possible 10 foodborne pathogen candidates with 10 pairs of primers included in one reaction, which had limit of detection equivalent to the uniplex PCR. MCPC was further explored in detecting combined genotypes of five β-globin gene mutations where multiple targets were co-amplified. MCPC strategy could expand the scope of real-time PCR assays in applications which are unachievable by current labeling strategy

Evaluation of Type-Specific Real-Time PCR Assays Using the LightCycler and J.B.A.I.D.S. for Detection of Adenoviruses in Species HAdV-C

Sporadically, HAdVs from species HAdV-C are detected in acute respiratory disease outbreaks. To rapidly type these viruses, we designed real-time PCR assays that detect and discriminate between adenovirus types HAdV-C1, -C2, -C5, and -C6. Sixteen clinical isolates from the California Department of Public Health were used to validate the new assays. Type-specific TaqMan real-time PCR assays were designed and used independently to successfully identify 16 representative specimens. The lower limit of detection for our LightCycler singleplex real-time PCR assays were calculated to be 100, 100, 100, and 50 genomic copies per reaction for HAdV-C1, HAdV-C2, HAdV-C5 and HAdV-C6, respectively. The results for the singleplex J.B.A.I.D.S. assays were similar. Our assays did not cross-react with other adenoviruses outside of species HAdV-C, respiratory syncytial virus, influenza, or respiratory disease causing bacteria. These assays have the potential to be useful as diagnostic tools for species HAdV-C infection

Rapid diagnostic tests as a source of DNA for Plasmodium species-specific real-time PCR

<p>Abstract</p> <p>Background</p> <p>This study describes the use of malaria rapid diagnostic tests (RDTs) as a source of DNA for <it>Plasmodium </it>species-specific real-time PCR.</p> <p>Methods</p> <p>First, the best method to recover DNA from RDTs was investigated and then the applicability of this DNA extraction method was assessed on 12 different RDT brands. Finally, two RDT brands (OptiMAL Rapid Malaria Test and SDFK60 malaria Ag <it>Plasmodium falciparum</it>/Pan test) were comprehensively evaluated on a panel of clinical samples submitted for routine malaria diagnosis at ITM. DNA amplification was done with the 18S rRNA real-time PCR targeting the four <it>Plasmodium </it>species. Results of PCR on RDT were compared to those obtained by PCR on whole blood samples.</p> <p>Results</p> <p>Best results were obtained by isolating DNA from the proximal part of the nitrocellulose component of the RDT strip with a simple DNA elution method. The PCR on RDT showed a detection limit of 0.02 asexual parasites/μl, which was identical to the same PCR on whole blood. For all 12 RDT brands tested, DNA was detected except for one brand when a low parasite density sample was applied. In RDTs with a plastic seal covering the nitrocellulose strip, DNA extraction was hampered. PCR analysis on clinical RDT samples demonstrated correct identification for single species infections for all RDT samples with asexual parasites of <it>P. falciparum </it>(n = 60), <it>Plasmodium vivax </it>(n = 10), <it>Plasmodium ovale </it>(n = 10) and <it>Plasmodium malariae </it>(n = 10). Samples with only gametocytes were detected in all OptiMAL and in 10 of the 11 SDFK60 tests. None of the negative samples (n = 20) gave a signal by PCR on RDT. With PCR on RDT, higher Ct-values were observed than with PCR on whole blood, with a mean difference of 2.68 for OptiMAL and 3.53 for SDFK60. Mixed infections were correctly identified with PCR on RDT in 4/5 OptiMAL tests and 2/5 SDFK60 tests.</p> <p>Conclusions</p> <p>RDTs are a reliable source of DNA for <it>Plasmodium </it>real-time PCR. This study demonstrates the best method of RDT fragment sampling for a wide range of RDT brands in combination with a simple and low cost extraction method, allowing RDT quality control.</p

In Vivo Imaging of Transiently Transgenized Mice with a Bovine Interleukin 8 (CXCL8) Promoter/Luciferase Reporter Construct

One of the most remarkable properties of interleukin 8 (CXCL8/IL-8), a chemokine with known additional functions also in angiogenesis and tissue remodeling, is the variation of its expression levels. In healthy tissues, IL-8 is barely detectable, but it is rapidly induced by several folds in response to proinflammatory cytokines, bacterial or viral products, and cellular stress. Although mouse cells do not bear a clear homologous IL-8 gene, the murine transcriptional apparatus may well be capable of activating or repressing a heterologous IL-8 gene promoter driving a reporter gene. In order to induce a transient transgenic expression, mice were systemically injected with a bovine IL-8 promoter–luciferase construct. Subsequently mice were monitored for luciferase expression in the lung by in vivo bioluminescent image analysis over an extended period of time (up to 60 days). We demonstrate that the bovine IL-8 promoter–luciferase construct is transiently and robustly activated 3–5 hours after LPS and TNF-α instillation into the lung, peaking at 35 days after construct delivery. Bovine IL-8 promoter–luciferase activation correlates with white blood cell and neutrophil infiltration into the lung. This study demonstrates that a small experimental rodent model can be utilized for non-invasively monitoring, through a reporter gene system, the activation of an IL-8 promoter region derived from a larger size animal (bovine). This proof of principle study has the potential to be utilized also for studying primate IL-8 promoter regions

Genetic Resistance to Rhabdovirus Infection in Teleost Fish Is Paralleled to the Derived Cell Resistance Status

Genetic factors of resistance and predisposition to viral diseases explain a significant part of the clinical variability observed within host populations. Predisposition to viral diseases has been associated to MHC haplotypes and T cell immunity, but a growing repertoire of innate/intrinsic factors are implicated in the genetic determinism of the host susceptibility to viruses. In a long-term study of the genetics of host resistance to fish rhabdoviruses, we produced a collection of double-haploid rainbow trout clones showing a wide range of susceptibility to Viral Hemorrhagic Septicemia Virus (VHSV) waterborne infection. The susceptibility of fibroblastic cell lines derived from these clonal fish was fully consistent with the susceptibility of the parental fish clones. The mechanisms determining the host resistance therefore did not associate with specific host immunity, but rather with innate or intrinsic factors. One cell line was resistant to rhabdovirus infection due to the combination of an early interferon IFN induction - that was not observed in the susceptible cells - and of yet unknown factors that hamper the first steps of the viral cycle. The implication of IFN was well consistent with the wide range of resistance of this genetic background to VSHV and IHNV, to the birnavirus IPNV and the orthomyxovirus ISAV. Another cell line was even more refractory to the VHSV infection through different antiviral mechanisms. This collection of clonal fish and isogenic cell lines provides an interesting model to analyze the relative contribution of antiviral pathways to the resistance to different viruses