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BackgroundThe main vector and reservoir host ofRickettsia felis,an emerging human pathogen causing flea-borne spotted fever, is the cat fleaCtenocephalides felis. While cats have not been found to be infected with the organism, significant percentages of dogs from Australia and Africa are infected, indicating that they may be important mammalian reservoirs. The objective of this study was to determine the presence ofR. felisDNA in the blood of domestic dogs and cats in the USA.MethodsThree previously validated PCR assays forR. felisand DNA sequencing were performed on blood samples obtained from clinically ill domestic cats and dogs from 45 states (2008–2020) in the USA. The blood samples had been submitted for the diagnosis of various tick-borne diseases in dogs and feline infectious peritonitis virus, feline immunodeficiency virus, andBartonellaspp. in cats. Phylogenetic comparisons were performed on thegltAnucleotide sequences obtained in the study and those reported forR. felisandR. felis-like organisms.ResultsLow copy numbers ofR. felisDNA (around 100 copies/ml whole blood) were found in four cats (4/752, 0.53%) and three dogs (3/777, 0.39%). The very low levels of infection in clinically ill animals is consistent withR. felisbeing an unlikely cause of disease in naturally infected dogs and cats. The low copy numbers we found emphasize the requirement for very sensitive PCRs in prevalence studies.ConclusionsThe low prevalence of naturally infected PCR-positive cats is further evidence that cats are unlikely to be important reservoirs ofR. felis. Similarly, the low prevalence in dogs suggests they are not important reservoirs in the USA. Investigations should continue into the role other mammalian species may be playing in the epidemiology ofR. felisinfections.Graphical Abstract

Detection of anti-Chlamydia trachomatis (Ctr) antibodies is compromised by cross-reactivity and poor sensitivity of classic Ctr-antigens. We discovered 48 strongly reactive peptide antigens of Ctr-specific B-cell epitopes from 21 immunodominant proteins. In this study, we review the utility of peptide assays for diagnosis of Ctr infections. By combining many of these Ctr-specific B-cell epitopes from several proteins in separate or mixed multipeptide assays, they achieved vastly superior assay sensitivity and specificity over standard enzyme-linked immunosorbent assays. Such multipeptide assays eliminate cross-reactivities (false positives) and correct for stochastic gaps in antibody responses (false negatives). More importantly, we developed and validated a novel microarray platform in which hundreds of peptides from many proteins are spotted in a single reaction well. This offers the possibility of high-throughput screening of many candidate peptides for routine serological fingerprinting of Ctr infections. Discovery of optimal sets of antibody responses that associate with clinical pelvic inflammatory disease (PID) may identify diagnostically useful PID biomarker antigens.

Chlamydia (C.) pecorum is an obligate intracellular bacterium that infects and causes disease in a broad range of animal hosts. Molecular studies have revealed that this pathogen is genetically diverse with certain isolates linked to different disease outcomes. Limited in vitro or in vivo data exist to support these observations, further hampering efforts to improve our understanding of C. pecorum pathogenesis. In this study, we evaluated whether genetically distinct C. pecorum isolates (IPA, E58, 1710S, W73, JP-1-751) display different in vitro growth phenotypes in different mammalian epithelial and immune cells. In McCoy cells, shorter lag phases were observed for W73 and JP-1-751 isolates. Significantly smaller inclusions were observed for the naturally plasmid-free E58 isolate. C. pecorum isolates of bovine (E58) and ovine origin (IPA, W73, JP-1-751) grew faster in bovine cells compared to a porcine isolate (1710S). C. pecorum isolates could infect but appear not able to complete their developmental cycle in bovine peripheral neutrophil granulocytes. All isolates, except 1710S, could multiply in bovine monocyte-derived macrophages. These results reveal potentially important phenotypic differences that will help to understand the pathogenesis of C. pecorum in vivo and to identify C. pecorum virulence factors.

Chlamydia gallinacea is an endemic Chlamydia agent in poultry with a worldwide distribution. The aim of this study was to investigate whether C. gallinacea can be transmitted via fecal-oral, respiratory and vertical routes. After co-housing with C. gallinacea-inoculated broilers (n = 10) for 15 days, over 90.0% of SPF broilers (n = 10) became C. gallinacea-positive in their oropharyngeal and cloacal swabs. Connection of isolators with ventilation tubing resulted in transmission of infectious bronchitis virus, but not of C. gallinacea, from infected broilers in one isolator to uninfected ones in the other isolator. Chlamydia-qPCR determined that 97.6% of shells of embryonated eggs (287/294) from a breeding farm were positive for C. gallinacea. C. gallinacea positivity in egg albumen increased significantly from 7.6% (10/128) before incubating to 44.4% (8/18) of 7-day incubation, and from 5.5% (7/128) to 38.9% (7/18) in egg yolk. After incubating for 19 days, C. gallinacea DNA was detected in heart (5/55, 9.1%), liver (3/55, 5.5%), spleen (7/55, 12.7%), lung (6/55, 10.1%), kidney (8/55; 14.5%) and intestine (4/55, 7.3%) of chicken embryos. Taken together, our data indicate that C. gallinacea can be efficiently transmitted by the fecal-oral route, but not via aerosol. Additionally, vertical transmission can occur via penetration of C. gallinacea from eggshell to albumen, yolk, and the growing embryo. Our findings provide essential information for the control of C. gallinacea in poultry farms.

Chlamydia suis is an important, highly prevalent, and diverse obligate intracellular pathogen infecting pigs. In order to investigate the prevalence and diversity of C. suis in the U.S., 276 whole blood samples from feral swine were collected as well as 109 fecal swabs and 60 whole blood samples from domestic pigs. C. suis-specific peptide ELISA identified anti-C. suis antibodies in 13.0% of the blood of feral swine (26/276) and 80.0% of the domestic pigs (48/60). FRET-qPCR and DNA sequencing found C. suis DNA in 99.1% of the fecal swabs (108/109) and 21.7% of the whole blood (13/60) of the domestic pigs, but not in any of the assayed blood samples (0/267) in feral swine. Phylogenetic comparison of partial C. suis ompA gene sequences and C. suis-specific multilocus sequencing typing (MLST) revealed significant genetic diversity of the C. suis identified in this study. Highly genetically diverse C. suis strains are prevalent in domestic pigs in the USA. As crowding strongly enhances the frequency and intensity of highly prevalent Chlamydia infections in animals, less population density in feral swine than in domestic pigs may explain the significantly lower C. suis prevalence in feral swine. A future study is warranted to obtain C. suis DNA from feral swine to perform genetic diversity of C. suis between commercial and feral pigs.

Flies are well-known vectors of bacterial pathogens, but there are little data on their role in spreading microbial community and antimicrobial resistance. In this study, we compared the bacterial community, antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs) in flies with those in the feces of sympatric animals. A 16S rRNA-based microbial analysis identified 23 bacterial phyla in fecal samples and 25 phyla in flies; all the phyla identified in the fecal samples were also found in the flies. Bray–Curtis dissimilarity analysis showed that the microbiota of the flies were more similar to the microbiota of the feces of their sympatric animals than those of the feces from the three other animal species studied. The qPCR array amplified 276 ARGs/MGEs in fecal samples, and 216 ARGs/MGEs in the flies, while 198 of these genes were identified in both flies and feces. Long-term studies with larger sample numbers from more geospatially distinct populations and infection trials are indicated to further evaluate the possibility of flies as sentinels for antimicrobial resistance.

Serological analysis of Chlamydia (C.) spp. infections is still mainly based on micro-immunofluorescence and ELISA. To overcome the limitations of conventional serology, we have designed a novel microarray carrying 52 synthetic peptides representing B-cell epitopes from immunodominant proteins of all 11 chlamydial species. The new assay has been validated using monospecific mouse hyperimmune sera. Subsequently, serum samples from cattle, sheep and humans with a known history of chlamydial infection were examined. For instance, the specific humoral response of sheep to treatment with a C. abortus vaccine has been visualized against a background of C. pecorum carriership. In samples from humans, dual infection with C. trachomatis and C. pneumoniae could be demonstrated. The experiments revealed that the peptide microarray assay was capable of simultaneously identifying specific antibodies to each Chlamydia spp. The actual assay represents an open platform test that can be complemented through future advances in Chlamydia proteome research. The concept of the highly parallel multi-antigen microarray proven in this study has the potential to enhance our understanding of antibody responses by defining not only a single quantitative response, but also the pattern of this response. The added value of using peptide antigens will consist in unprecedented serodiagnostic specificity.

Objectives The objectives of this study were to determine the prevalence of feline coronavirus (FCoV) viremia, and its replication in peripheral blood using quantitative RT-PCR (qRT-PCR) methodology in a population of 205 healthy shelter cats in Southern California, as well as to assess any possible connection to longitudinal development of feline infectious peritonitis (FIP). Methods The study was performed on buffy-coat samples from EDTA-anticoagulated whole blood samples of 205 healthy shelter cats. From 50 of these cats, fecal samples were also examined. FCoV genomic and subgenomic RNA in the buffy coats was amplified by a total FCoV RNA qRT-PCR. Evidence for FCoV replication in peripheral blood and feces was obtained by M gene mRNA qRT-PCR. Results Nine of 205 cats (4.4%) were viremic by the total FCoV RNA qRT-PCR, and one of these cats had evidence of peripheral FCoV blood replication by an FCoV mRNA qRT-PCR. The single cat with peripheral blood replication had a unique partial M gene sequence distinct from positive controls and previously published FCoV sequences. Neither seven of the nine viremic cats with follow-up nor the single cat with replicating FCoV with positive qRT-PCR results developed signs compatible with FIP within 6 months of sample collection. Conclusions and relevance FCoV viremia and peripheral blood replication in healthy shelter cats have a low prevalence and do not correlate with later development of FIP in this study population, but larger case-control studies evaluating the prognostic accuracy of the qRT-PCR assays are needed.

Cross-reactivity of classical chlamydial antigens compromises Chlamydia (C.) pneumoniae serology. By testing with 185 human antisera, we expanded 18 previously discovered C. pneumoniae-specific B-cell epitopes to 48 peptide antigens from 12 C. pneumoniae immunodominant proteins. For specific detection of antibodies against C. pneumoniae, we developed novel ELISAs with strongly reactive individual peptide antigens and mixtures of these peptides. By comparison to a composite reference standard (CRS) for anti-C. pneumoniae antibody status of human sera, the top-performing CpnMixF12 peptide assay showed 91% sensitivity at 95% specificity, significantly higher than 4 commercial anti-C. pneumoniae IgG ELISAs (36-12% sensitivity at 95% specificity). Human C. pneumoniae (Cpn) and C. trachomatis (Ctr) seroreactivity was 54% biased towards co-positivity in commercial Cpn and Ctr ELISAs, but unbiased in Cpn and Ctr peptide antibody assays, suggesting severe cross-reactivity of commercial ELISAs. Using hyperimmune mouse sera against each of 11 Chlamydia spp., we confirm that commercial Cpn and Ctr ELISA antigens are cross-reactive among all Chlamydia spp., but Cpn and Ctr peptide antigens react only with antisera against the cognate chlamydial species. With simultaneously high specificity and sensitivity, and convenient use for non-specialized laboratories, these ELISAs have the potential to improve serodiagnosis of C. pneumoniae infection.

Background Chlamydia spp. serology is compromised by cross-reactivity of classical antigens. For specific detection of anti-trachomatis (Ctr) and anti-C. pneumoniae (Cpn) antibodies, we developed and validated novel peptide ELISAs. Methods Strongly reactive peptide antigens of 24 Ctr- and 48 Cpn-specific B-cell epitopes of multiple immunodominant chlamydial proteins were used in this study. For specific detection of anti-Ctr and anti-Cpn antibodies, 185 human sera were tested in colorimetric ELISAs with mixtures of 12–24 Ctr or Cpn peptide antigens using polyclonal anti-human IgG-HRP conjugates. For comparative evaluation, these sera were tested with 4 Ctr and 4 Cpn commercial IgG ELISAs. Results In commercial ELISAs, Ctr and Cpn individual serum reactivity was 54% biased towards positivity for both species (co-positivity), but unbiased in Ctr and Cpn peptide antibody assays. This finding suggested a severe specificity problem (cross-reactivity) of commercial ELISAs, but not peptide assays. Using hyperimmune mouse sera against each of 11 Chlamydia spp., we confirmed that commercial Ctr and Cpn ELISA antigens are cross-reactive among all Chlamydia spp., but Cpn and Ctr peptide antigens react specifically only with antisera against the cognate chlamydial species. By comparison at 90% specificity to a Ctr-peptide composite reference standard (CRS) for human anti-Ctr antibody status, the Ctr mixed peptide assays showed 86–83% sensitivity, significantly higher than the 59–34% sensitivity of 4 commercial anti-Ctr ELISAs. Relative to a Cpn-peptide CRS, the Cpn mixed peptide assay showed 86–80% sensitivity at 90% specificity, significantly higher than the 48–25% sensitivity of 4 commercial anti-Cpn ELISAs. Conclusion For detection of anti-Ctr and -Cpn antibodies, commercial ELISAs are not suitable due to cross-reactivity. In contrast, mixed peptide assays are accurate with simultaneous high specificity and sensitivity, and reliably determine anti-Ctr and anti-Cpn antibody prevalence. With convenient use for non-specialized laboratories, these peptide ELISAs will improve Ctr and Cpn serodiagnosis. Disclosure No significant relationships.

SUMMARYChlamydia gallinacea, a new chlamydial agent, has been reported in four European countries as well as Argentina and China. Experimentally infected chickens with C. gallinacea in previous study showed no clinical signs but had significantly reduced gains in body weight (6·5–11·4%). Slaughterhouse workers exposed to infected chickens have developed atypical pneumonia, indicating C. gallinacea is likely a zoonotic agent. In this study, FRET-PCR confirmed that C. gallinacea was present in 12·4% (66/531) of oral–pharyngeal samples from Alabama backyard poultry. Phylogenetic comparisons based on ompA variable domain showed that 16 sequenced samples represented 14 biotypes. We report for the first time the presence of C. gallinacea in North America, and this warrants further research on the organism's pathogenicity, hosts, transmission, and zoonotic potential.

Sensitive species-specific detection of anti-Chlamydia trachomatis antibodies is compromised by cross-reactivity of the C. trachomatis antigens used in standard microimmunofluorescence (MIF) testing and enzyme-linked immunosorbent assays (ELISAs). Previously, we discovered 48 strongly reactive C. trachomatis-specific B cell epitope peptides from 21 immunodominant proteins. Here we comprehensively evaluated the 11 top-ranked C. trachomatis-specific peptide antigens from 8 proteins for use in C. trachomatis serology. Sera from 125 women with nucleic acid amplification test (NAAT)-confirmed active C. trachomatis infection and from 49 healthy women with a low risk of C. trachomatis infection were used as anti-C. trachomatis antibody-positive and -negative sera. Results obtained for detection of IgG1, IgG3, and IgA1 antibodies against the 11 C. trachomatis peptide antigens were compared to results from 4 commercial anti-C. trachomatis IgG ELISAs. Using composite reference standards (CRS) of all assays for anti-C. trachomatis antibody status, commercial ELISAs detected antibodies in antibody-positive women with sensitivities of 51.5% to 64.8%. In contrast, a combination of the results of all 11 peptides detected IgG (IgG1 and IgG3) antibodies with 91.8% sensitivity, and a labor-saving combination of the 5 optimal peptides still detected antibodies in antibody-positive women with 86.5% sensitivity (all at 98% specificity). The superior performance of the combined peptide ELISAs was confirmed by area under the receiver operating characteristic curve (ROC-AUC), likelihood ratio, and predictive value analyses. The higher sensitivity of the peptide assays results from using multiple B cell epitopes of several C. trachomatis immunodominant proteins, including OmpA, compared to exclusively using the OmpA antigens used in commercial ELISAs. Thus, ELISAs with combined use of synthetic peptide antigens for C. trachomatis antibody detection have the advantage of simultaneous high sensitivity and high specificity. IMPORTANCE For detection of anti-Chlamydia trachomatis antibodies by serological assays, use of classical whole-organism chlamydial antigens results in high cross-reactivity. These antigens bind mainly antibodies against the major outer membrane protein (OmpA) and bind antibodies against other immunodominant non-OmpA proteins to a lesser extent, resulting in poor assay sensitivity. The specificity of C. trachomatis serology is also compromised by the high prevalence of cross-reactive anti-C. pneumoniae antibodies in human populations. We previously identified 48 highly specific C. trachomatis B cell epitope peptide antigens of 21 immunodominant proteins. This study validated peptide antigen-based novel ELISAs that provide highly specific and sensitive detection of anti-C. trachomatis antibodies. Compared to four commercial ELISAs that achieved only poor sensitivities (51.5% to 64.8%), the combined signals of 5 to 11 peptides provided high sensitivity (86.5% to 91.8%) at the same 98% specificity. Thus, by using multiple peptide antigens of immunodominant proteins, we created simple ELISAs with specificity and sensitivity superior to standard C. trachomatis serodiagnosis.

Background Chlamydia suis is an important, globally distributed, highly prevalent and diverse obligate intracellular pathogen infecting pigs. To investigate the prevalence and genetic diversity of C. suis in China, 2,137 nasal, conjunctival, and rectal swabs as well as whole blood and lung samples of pigs were collected in 19 regions from ten provinces of China in this study. Results We report an overall positivity of 62.4% (1,334/2,137) of C. suis following screening by Chlamydia spp. 23S rRNA-based FRET-PCR and high-resolution melting curve analysis and confirmatory sequencing. For C. suis-positive samples, 33.3 % of whole blood and 62.5% of rectal swabs were found to be positive for the C. suis tetR(C) gene, while 13.3% of whole blood and 87.0% of rectal swabs were positive for the C. suis tet(C) gene. Phylogenetic comparison of partial C. suis ompA gene sequences revealed significant genetic diversity in the C. suis strains. This genetic diversity was confirmed by C. suis-specific multilocus sequence typing (MLST), which identified 26 novel sequence types among 27 examined strains. Tanglegrams based on MLST and ompA sequences provided evidence of C. suis recombination amongst the strains analyzed. Conclusions Genetically highly diverse C. suis strains are exceedingly prevalent in pigs. As it stands, the potential pathogenic effect of C. suis on pig health and production of C. suis remains unclear and will be the subject of further investigations. Further study is also required to address the transmission of C. suis between pigs and the risk of 'spill-over' and 'spill-back' of infections to wild animals and humans. Electronic supplementary material The online version of this article (10.1186/s12917-017-1295-x) contains supplementary material, which is available to authorized users.

Ecosystem effects of polycyclic aromatic hydrocarbons (PAHs) remain under investigation following the Gulf of Mexico Deepwater Horizon oil spill. Fundulus grandis, an established indicator of aquatic ecosystem health, was investigated because this species shares genes and biochemical pathways with higher trophic–level fish and plays an important role in the gulf food chain. Oxidative stress responses including hepatic cytochrome P4501A (CYP1A) and serum antioxidant capacity were evaluated in fish exposed to PAHs. Fish were exposed to water-accommodated fractions (WAFs) of crude oil (7.0 ± 0.10 mg/L C6-C28) after which solutions were diluted below the level of detection over 8 h using 15 ppt aerated artificial seawater. Before euthanasia, fish remained in aquaria for 12 h, 24 h, or 48 h. Three replicate experiments were conducted at each time point using unexposed fish as experimental controls. Significant differences (p

Salmonella spp. are gram-negative bacteria capable of causing diseases in a wide range of aquatic and terrestrial animals, including humans. Sea and terrestrial turtles have been recognized as carriers of this zoonotic pathogen. In this project, conventional and molecular diagnostic methods were combined to investigate the prevalence of Salmonella enterica in leatherback sea turtles (Dermochelys coriacea) that used the island of St. Kitts, West Indies as a nesting ground during 2011 (n = 21). Isolates obtained from selective media were screened and colonies suspected of being Salmonella spp. were confirmed by fluorescence resonance energy transfer polymerase chain reaction. The prevalence of S. enterica within this sample population during this period was found to be 14.2%. Moreover, due to the increasing risk of antibiotic resistance in enteric bacteria, antimicrobial susceptibility was investigated in all recovered Salmonella spp. isolates utilizing the broth microdilution method. All isolates were susceptible to the lowest concentration of kanamycin, gentamicin, ciprofloxacin, enrofloxacin, nalidixic acid, and trimethoprim/sulfamethoxazole tested. Further research should be pursued to understand the interaction of this bacterial pathogen with the environment, host, and other microbial communities, and to further develop faster, more sensitive, and more specific diagnostic methods.

To investigate the prevalence and diversity of bovine Chlamydia spp. in cattle, whole blood from dairy and beef cattle in 11 provinces of China (n = 2,003) and vaginal swabs, whole blood samples, feces, milk samples from cows in a Yangzhou dairy farm (n = 108) were examined using genus- and species-specific PCRs. In cattle from 11 provinces, 2.4% (48/2,003) of whole-blood samples were positive for Chlamydia spp., and four Chlamydia species (C. pneumoniae, 41.7%, 20/48; C. psittaci, 22.9%, 11/48; C. gallinacea, 20.8%, 10/48; C. pecorum, 6.3%, 3/48) were identified. In a further study on a Yangzhou dairy farm, 64.8% (70/108) of the cows were positive for Chlamydia spp. C. pecorum was the intestinal endemic species (51/51, 100%), and C. gallinacea was the most frequent species in vaginal swabs (24/27, 88.9%), whole blood buffy coats (5/8, 62.5%) and milk (4/6, 66.7%). C. psittaci and C. pneumoniae were infrequently detected. DNA sequencing of the ompA gene demonstrated the presence of multiple in-herd C. pecorum serovars and single C. gallinacea and C. psittaci serovars which were identical with those of poultry from Yangzhou. This is the first report of C. gallinacea and C. pneumoniae in cattle. Further study is required to address the transmission of Chlamydia spp., in particular of C. gallinacea and C. pneumoniae from their natural hosts, and their potential pathogenic effect on health and production of cattle.

The persistent public health threat of animal to human transmission of influenza A virus (IAV) has stimulated interest in rapid and accurate detection of all IAV subtypes in clinical specimens of animal origin. In this study, a new set of primers and probes was designed for one-step pan-IAV reverse-transcription fluorescence resonance energy transfer (FRET)-PCR. The detection limit of one-step pan-IAV RT FRET-PCR was 10 copies of the matrix gene per reaction and proved to be equivalent or superior to virus isolation in detecting nine IAV subtypes. Application of the pan-IAV RT FRET-PCR to oral-pharyngeal and cloacal swab specimens collected from healthy poultry in 34 live bird markets in 24 provinces of China revealed that 9.2% of the animals (169/1,839) or 6.3% of their oral-pharyngeal or cloacal swabs (233/3,678) were positive for IAV and 56.8% of IAV-positive samples were of the H9N2 subtype. Paralleling detection of IAV in H9N2-infected SPF chickens and chickens from LBM showed that pan-IAV FRET-PCR had a higher detection limit than virus isolation in eggs while the results by FRET-PCR and virus isolation overall matched. It is expected that this strategy can be useful for facile surveillance for IAV in clinical samples from a variety of sources.

X-ray crystallography has shown that an antibody paratope typically binds 15–22 amino acids (aa) of an epitope, of which 2–5 randomly distributed amino acids contribute most of the binding energy. In contrast, researchers typically choose for B-cell epitope mapping short peptide antigens in antibody binding assays. Furthermore, short 6–11-aa epitopes, and in particular non-epitopes, are over-represented in published B-cell epitope datasets that are commonly used for development of B-cell epitope prediction approaches from protein antigen sequences. We hypothesized that such suboptimal length peptides result in weak antibody binding and cause false-negative results. We tested the influence of peptide antigen length on antibody binding by analyzing data on more than 900 peptides used for B-cell epitope mapping of immunodominant proteins of Chlamydia spp. We demonstrate that short 7–12-aa peptides of B-cell epitopes bind antibodies poorly; thus, epitope mapping with short peptide antigens falsely classifies many B-cell epitopes as non-epitopes. We also show in published datasets of confirmed epitopes and non-epitopes a direct correlation between length of peptide antigens and antibody binding. Elimination of short, ≤11-aa epitope/non-epitope sequences improved datasets for evaluation of in silico B-cell epitope prediction. Achieving up to 86% accuracy, protein disorder tendency is the best indicator of B-cell epitope regions for chlamydial and published datasets. For B-cell epitope prediction, the most effective approach is plotting disorder of protein sequences with the IUPred-L scale, followed by antibody reactivity testing of 16–30-aa peptides from peak regions. This strategy overcomes the well known inaccuracy of in silico B-cell epitope prediction from primary protein sequences.

To investigate the prevalence and diversity of Chlamydia spp. in domestic birds in China, oral and cloacal swabs of healthy chickens, ducks, geese and pigeons were collected nationwide from live-animal markets and examined by Chlamydia spp. 23 S rRNA gene FRET-PCR followed by high-resolution melting curve analysis and confirmatory sequencing. Overall, 26.2% of the birds (602/2,300) were positive for Chlamydia spp. and five Chlamydia spp. were identified. While occasional detection of C. suis and C. muridarum in poultry is reported here for the first time, the predominant chlamydial agent was C. gallinacea representing 63.8% of all positives (384/602) and 81.2% of positive chickens (359/442). Analysis of the C. gallinacea ompA phylogeny revealed at least 13 well segregated variants (serovars). Seven-month monitoring of C. gallinacea-infected chickens indicated that the infection was persistent. C. gallinacea-infected chickens remained without overt clinical disease, but showed body weight gains significantly reduced by 6.5–11.4% beginning in week 3 post-infection. This study indicates that C. gallinacea is the endemic chlamydial species in chickens, whereas C. psittaci dominates only in pigeons. Further studies are required to address the specific conditions under which C. gallinacea could act as an avian pathogen and possibly also a zoonotic agent.

Introduction: Although antibodies to the feline immunodeficiency virus (FIV) have been detected by SNAP assay in cats from St. Kitts, there have been no molecular studies to further confirm the infection and determine the FIV subtypes present. Methodology: Total nucleic acids were extracted from EDTA whole blood specimens from 35 cats, followed by quantitative fluorescence resonance energy transfer (FRET) PCR under a six-channel LightCycler 2.0 Instrument with Software version 4.1. Results: Four of 11 stray cats (36 %) but none of 24 owned cats were FIV positive by real-time PCR. High-resolution melting curve analysis indicated that all four positive cats were infected with FIV subtype-B. Conclusions: This is the first molecular characterization of FIV subtypes on St. Kitts and the results confirm the high prevalence of FIV infection in stray cats on the island.

Feline immunodeficiency virus (FIV) is among the most common infectious agents of cats. Five well-characterized FIV subtypes, A, B, C, D, and E, are recognized worldwide. As in HIV diagnosis, serum antibodies against FIV classically serve as an indicator of infection status. After the introduction of an inactivated FIV vaccine, this approach has become problematic, since antibodies generated by vaccination are indistinguishable from antibodies in response to infection. However, PCR detection of host-cell-integrated FIV DNA will differentiate infection-derived antibody from vaccination-derived positivity because presumably the RNA of inactivated vaccine virus will not integrate into the host genome. In this study, we established a gag gene-based dual-emission fluorescence resonance energy transfer (FRET) real-time PCR that amplifies single-target copies of all known FIV strains and differentiates five FIV subtypes. All blood samples from experimentally FIV-infected cats ( n = 5) were antibody positive and highly positive in the FIV PCR. In contrast, nine cats became antibody positive after FIV vaccination but remained negative in the FIV PCR. Of 101 FIV antibody-positive feline blood specimens submitted for FIV PCR diagnosis, 61 were positive (60%). A total of 23 of the positive PCRs identified subtype A, 11 identified subtype B1, 11 identified subtype B2/E, and 16 identified subtype C. FIV subtype D was not detected in any submitted specimens even though 13 blood specimens were from cats known to have received the FIV vaccine, which contains FIV subtype A and D inactivated virions. Therefore, this PCR quantitatively identifies FIV subtypes and unambiguously discriminates between FIV-vaccinated and FIV-infected cats.

Babesia gibsoni and Babesia canis are the etiological agents of canine babesiosis, a protozoal hemolytic disease with global significance. Canine babesiosis has been diagnosed by microscopic identification of intra-erythrocytic trophozoites in blood smear, and by serological testing. Here we developed a quantitative fluorescence resonance energy transfer (FRET)-PCR that amplifies a fragment of the Babesia spp. 18S rRNA gene with high sensitivity and specificity. Melting curve analysis differentiates B. gibsoni, B. canis canis/B. canis vogeli, and B. canis rossi by the disassociation temperature of the fluorescent probes. Babesia gibsoni infection was detected in 8 of 48 canine breeds (17%) and 24 of a total of 235 specimens (10.2%) submitted from 22 states of the continental United States of America. A potential blood donor was positive for B. canis vogeli infection. In Hong Kong (China), B. gibsoni infection was detected in 30 of 64 specimens (46.9%) from 15 of the 24 breeds (63%). While the frequency of canine babesiosis did not associate with seasonal change in Hong Kong, positivity in the USA for Babesia spp. infection was higher in Spring and Summer than in Autumn and Winter. The data suggest that environmental factors associated with tick vector exposure rather than genetic susceptibility determine the incidence of canine babesiosis. Babesia spp. burdens in blood declined significantly with increasing age of the infected dogs, and therapy with atovaquone and tilmicosin eliminated B. gibsoni while doxcycline and berenil did not. This demonstrates that high-resolution real-time PCR analysis may advance diagnosis and therapy monitoring of canine babesiosis.

Hepatozoon (H.) americanum and H. canis are the etiological agents of canine hepatozoonosis, a disease that is found worldwide and is also prevalent in the southeastern United States. Current laboratory diagnosis of canine hepatozoonosis caused by H. americanum is usually dependent on visual identification of Hepatozoon "onion skin cysts" in muscle biopsies, an approach that requires invasive sampling and can result in false negatives. We have developed a diagnostic method for detection of Hepatozoon spp. DNA that integrates nucleic acid extraction with extensive agitation to maximize DNA extraction efficiency. The DNA extracted from canine EDTA-whole blood is subjected to real-time PCR, and fluorescence resonance energy transfer (FRET) probes detect a signature polymorphism in the amplified DNA. This PCR method amplifies a fragment of the Hepatozoon 18S rDNA gene, detects as few as 7 genomic copies of Hepatozoon spp. per ml of blood with high specificity, and differentiates between H. americanum and H. canis amplicons. A surprising 300-fold increase of H. americanum 18S rDNA targets occurred during 3-0 days of storage of positive blood specimens. Examination of 614 EDTA-blood samples submitted mostly from the southeastern Unites States from dogs with suspected hepatozoonosis identified H. americanum in 167 samples (27.2%). An additional 14 samples (2.3%) were positive for H. canis, and 14 samples (2.3%) were positive for both H. americanum and H. canis. These results suggest that the Hepatozoon spp. 18S rDNA quantitative PCR may be a valuable tool that can improve diagnosis and therapy of canine hepatozoonosis.

A mouse model was used to study the genetic control of differential host response to pulmonary infection with Chlamydia pneumoniae. The A/J and C57BL/6 strains show differential response to intranasal infection with respect to their ability to clear pulmonary bacterial load and the extent of lung pathology developed by 2 weeks post infection. The genetic basis of this interstrain difference was studied by whole-genome scan in an informative [A/J x C57BL/6J] F2 cross using the pulmonary microbial load as a phenotypic readout of host response. We detected a highly significant linkage (LOD score=11.5) on chromosome 17 that overlaps with the major histocompatibility (MHC) locus. This quantitative trait locus (QTL) accounts for approximately 30% of the phenotypic variance with B6 alleles conferring susceptibility and inherited in a recessive fashion. Significant linkage was also detected to chromosome 5 in female mice, while chromosome 6 showed suggestive linkage in male mice, pointing to additional complexity in the genetic control of the difference in susceptibility observed in A/J and C57BL/6J.

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. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-2053-8) contains supplementary material, which is available to authorized users.

Urgently needed species-specific enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies againstChlamydiaspp. have been elusive due to high cross-reactivity of chlamydial antigens. To identifyChlamydiaspecies-specific B cell epitopes for such assays, we ranked the potential epitopes of immunodominant chlamydial proteins that are polymorphic among allChlamydiaspecies. High-scoring peptides were synthesized with N-terminal biotin, followed by a serine-glycine-serine-glycine spacer, immobilized onto streptavidin-coated microtiter plates, and tested with mono-specific mouse hyperimmune sera against eachChlamydiaspecies in chemiluminescent ELISAs. For each of nineChlamydiaspecies, three to nine dominant polymorphic B cell epitope regions were identified on OmpA, CT618, PmpD, IncA, CT529, CT442, IncG, Omp2, TarP, and IncE proteins. Peptides corresponding to 16- to 40-amino-acid species-specific sequences of these epitopes reacted highly and with absolute specificity with homologous, but not heterologous,Chlamydiamonospecies-specific sera. Host-independent reactivity of such epitopes was confirmed by testing of sixC. pecorum-specific peptides from five proteins withC. pecorum-reactive sera from cattle, the natural host ofC. pecorum. The probability of cross-reactivity of peptide antigens from closely related chlamydial species or strains correlated with percent sequence identity and declined to zero at Chlamydiaspp. We anticipate that these peptide antigens will improve chlamydial serology by providing easily accessible assays to nonspecialist laboratories. Our approach also lends itself to the identification of relevant epitopes of other microbial pathogens.

© 2015 Elsevier GmbH. The family Chlamydiaceae (order Chlamydiales, phylum Chlamydiae) comprises important, obligate intracellular bacterial pathogens of humans and animals. Subdivision of the family into the two genera Chlamydia and Chlamydophila has been discussed controversially during the past decade. Here, we have revisited the current classification in the light of recent genomic data and in the context of the unique biological properties of these microorganisms. We conclude that neither generally used 16S rRNA sequence identity cut-off values nor parameters based on genomic similarity consistently separate the two genera. Notably, no easily recognizable phenotype such as host preference or tissue tropism is available that would support a subdivision. In addition, the genus Chlamydophila is currently not well accepted and not used by a majority of research groups in the field. Therefore, we propose the classification of all 11 currently recognized Chlamydiaceae species in a single genus, the genus Chlamydia. Finally, we provide emended descriptions of the family Chlamydiaceae, the genus Chlamydia, as well as the species Chlamydia abortus, Chlamydia caviae and Chlamydia felis., PMB was supported by Sexually Transmitted Infections Cooperative Research Center grant U19 AI084044 from the National Institute of Health, National Institute of Allergy and Infectious Diseases. MH acknowledges support from the European Research Council (ERC StG EVOCHLAMY, no. 281633)

The inactive X chromosome of female mammals displays several properties of heterochromatin including late replication, histone H4 hypoacetylation, histone H3 hypomethylation at lysine-4, and methylated CpG islands. We show that cre-Lox-mediated excision of 21 kb from both Xist alleles in female mouse fibroblasts led to the appearance of two histone modifications throughout the inactive X chromosome usually associated with euchromatin: histone H4 acetylation and histone H3 lysine-4 methylation. Despite these euchromatic properties, the inactive X chromosome was replicated even later in S phase than in wild-type female cells. Homozygosity for the deletion also caused regions of the active X chromosome that are associated with very high concentrations of LINE-1 elements to be replicated very late in S phase. Extreme late replication is a property of fragile sites and the 21-kb deletions destabilized the DNA of both X chromosomes, leading to deletions and translocations. This was accompanied by the phosphorylation of p53 at serine-15, an event that occurs in response to DNA damage, and the accumulation of γ-H2AX, a histone involved in DNA repair, on the X chromosome. The Xist locus therefore maintains the DNA stability of both X chromosomes.

Research on intracellular bacteria of the family Chlamydiaceae, and the diseases they cause, requires large amounts of infectious elementary bodies (EB). We describe an approach that maximizes the generation of Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia abortus, or Chlamydia pecorum EBs in several replication cycles over approximately 10 days or more in a saturated equilibrium monolayer cell culture system. Buffalo Green Monkey Kidney (BGMK) cells, Human Epidermoid Carcinoma-2 (HEp-2) cells, or mouse McCoy cells were tested. BGMK cells best supported C. pneumoniae replication when cultivated in Iscove's Modified Dulbecco's Medium. From day 1 to day 9 after inoculation, C. pneumoniae genomes per ml culture medium increased from 10(5.1) to 10(8.6) in BGMK, from 10(5.6) to 10(8.1) in HEp-2, and remained at 10(5.2) in McCoy cell cultures. Three-month pre-inoculation maintenance of BGMK cells in different culture media did not influence C. pneumoniae yields. Inoculation at multiplicities of infection (MOI) of 10 or higher and supplementation of the cell culture medium on day 7 after inoculation with 0.1% glucose enhanced C. pneumoniae EB yields in harvested cell culture medium. For purification, EBs in medium were concentrated by sedimentation, followed by low-speed centrifugation for removal of host cell nuclei, and by step-gradient centrifugation of the supernatant in a 30% RenoCal-76-50% sucrose step-gradient. Extensive sonication increased yield and infectivity of chlamydial EB. The combined method typically produced from 1000 ml infected BGMK culture medium 10 ml homogeneous, single-cell, highly infectious EB stock containing approximately 5x10(11) C. pneumoniae genomes equivalent to 4-5x10(11) inclusion forming units.

Classical methods for detection of Chlamydophila species, and of antibodies against these agents, have indicated that these bacteria are highly prevalent in cattle and associated with numerous disease conditions. These methods demonstrated acute Chlamydophila-induced diseases such as epizootic bovine abortion, as well as worldwide variable, but generally high, Chlamydophila seroprevalence. However, it was impossible to consistently detect the low levels of these organisms which were suspected to be present in endemic infections. Application of highly sensitive real-time PCR and ELISA methods for detection of Chlamydophila spp. DNA and of antibodies against Chlamydophila spp., respectively, in a series of prospective cohort studies revealed a high prevalence of Chlamydophila spp. genital infections in female calves (61%) and adult heifers (53%). These infections were acquired by extragenital transmission in the first weeks of life, and infection frequency was increased by crowding of the animals. A challenge study demonstrated that infection with C. abortus resulted in decreased fertility of heifers. The experimental use of a C. abortus vaccine provided evidence for immunoprotection against C. abortus-induced suppression of bovine fertility. The results of these investigations suggest that bovine Chlamydophila infection should be viewed more as pervasive, low-level infection of cattle than as rare, severe disease. Such infections proceed without apparent disease or with only subtle expressions of disease, but potentially have a large impact on bovine herd health and fertility.

Detection and titration of chicken anemia virus (CAV)-neutralizing antibodies has relied on tedious, time-consuming passaging of infected cells, or subjective recognition of cytopathic effect in individual cells, because CAV replicates in culture only in lymphoblastoid cell lines, and thus generates no plaques. This paper describes a rapid method, in which CAV genomes in infected cells are quantitated by qPCR 3-4 days postinfection (p.i.), without passaging cells. Three sera, weakly positive with a commercial CAV ELISA kit, from broiler chickens immunized with a commercial CAV vaccine, were used to develop the assay. Virus neutralization titers of these sera were determined using two different CAV-susceptible cell lines (MDCC-MSB1 and MDCC-CU147) by the conventional method of passaging cells infected with 10,000 TCID(50) CAV per well, and by qPCR-based methods using cells infected with 100 or 10,000 TCID(50) per well in 24-well or 96-well plates. The method was also adapted to conventional PCR. The positive sera exhibited virus neutralization activity at dilutions ranging from 1:10 to 1:320 by the various assays. Although virus neutralization titers differed somewhat depending on the assay conditions used, the relative order of the titers of the three positive sera was the same for all assays. The qPCR-based assays are as sensitive and more rapid for detection of neutralizing antibody than the conventional assay based on passaging infected cells, and more sensitive for detection of low-level CAV antibodies than a commercial blocking ELISA.

Real-time PCR methods have become widely used within the past few years. However, real-time PCR is rarely used to study chronic diseases with low pathogen loads, presumably because of insufficient sensitivity. In this report, we developed an integrated nucleic acid isolation and real-time PCR platform that vastly improved the sensitivity of the quantitative detection of the intracellular bacterium, Chlamydia spp., by fluorescence resonance energy transfer realtime PCR. Determinants of the overall detection sensitivity were analyzed by extracting nucleic acids from bovine milk specimens spiked with low amounts of chlamydial organisms. Nucleic acids were optimally preserved and recovered by collection in guanidinium stabilization buffer, binding to a matrix of glass fiber fleece, and elution in low volume. Step-down thermal cycling and an excess of hot-start Taq polymerase vastly improved the robustness and sensitivity of the real-time PCR while essentially maintaining 100% specificity. The amplification of Chlamydia 23S rRNA allowed for the differentiation of chlamydial species and was more robust at low target numbers than amplification of the omp1 gene. The best combined method detected single targets per a 100-μL specimen equivalent in a 5-μL real-time PCR input. In an initial application, this high-sensitivity realtime PCR platform demonstrated a high prevalence of chlamydial infection in cattle.