Chlamydia trachomatis Seroassays Used in Epidemiologic Research: A Narrative Review and Practical Considerations.

Chlamydia trachomatis (CT) is a sexually transmitted infection that can lead to adverse reproductive health outcomes. CT prevalence estimates are primarily derived from screening using nucleic acid amplification tests (NAATs). However, screening guidelines in the United States only include particular subpopulations, and NAATs only detect current infections. In contrast, seroassays identify past CT infections, which is important for understanding the public health impacts of CT, including pelvic inflammatory disease and tubal factor infertility. Older seroassays have been plagued by low sensitivity and specificity and have not been validated using a consistent reference measure, making it challenging to compare studies, define the epidemiology of CT, and determine the effectiveness of control programs. Newer seroassays have better performance characteristics. This narrative review summarizes the "state of the science" for CT seroassays that have been applied in epidemiologic studies and provides practical considerations for interpreting the literature and employing seroassays in future research.

Multipeptide Assays for Sensitive and Differential Detection of Anti-Chlamydia Trachomatis Antibodies.

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.

Inadequate Reference Datasets Biased toward Short Non-epitopes Confound B-cell Epitope Prediction.

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.

From genomes to genotypes: molecular epidemiological analysis of Chlamydia gallinacea reveals a high level of genetic diversity for this newly emerging chlamydial pathogen.

BACKGROUND: Chlamydia (C.) gallinacea is a recently identified bacterium that mainly infects domestic chickens. Demonstration of C. gallinacea in human atypical pneumonia suggests its zoonotic potential. Its prevalence in chickens exceeds that of C. psittaci, but genetic and genomic research on C. gallinacea is still at the beginning. In this study, we conducted whole-genome sequencing of C. gallinacea strain JX-1 isolated from an asymptomatic chicken, and comparative genomic analysis between C. gallinacea strains and related chlamydial species. RESULTS: The genome of C. gallinacea JX-1 was sequenced by single-molecule, real-time technology and is comprised of a 1,059,522-bp circular chromosome with an overall G + C content of 37.93% and sequence similarity of 99.4% to type strain 08-1274/3. In addition, a plasmid designated pJX-1, almost identical to p1274 of the type strain, except for two point mutations, was only found in field strains from chicken, but not in other hosts. In contrast to chlamydial species with notably variable polymorphic membrane protein (pmp) genes and plasticity zone (PZ), these regions were conserved in both C. gallinacea strains. There were 15 predicted pmp genes, but only B, A, E1, H, G1 and G2 were apparently intact in both strains. In comparison to chlamydial species where the PZ may be up to 50 kbp, C. gallinacea strains displayed gene content reduction in the PZ (14 kbp), with strain JX-1 having a premature STOP codon in the cytotoxin (tox) gene, while tox gene is intact in the type strain. In multilocus sequence typing (MLST), 15 C. gallinacea STs were identified among 25 strains based on cognate MLST allelic profiles of the concatenated sequences. The type strain and all Chinese strains belong to two distinct phylogenetic clades. Clade of the Chinese strains separated into 14 genetically distinct lineages, thus revealing considerable genetic diversity of C. gallinacea strains in China. CONCLUSIONS: In this first detailed comparative genomic analysis of C. gallinacea, we have provided evidence for substantial genetic diversity among C. gallinacea strains. How these genetic polymorphisms affect C. gallinacea biology and pathogenicity should be addressed in future studies that focus on phylogenetics and host adaption of this enigmatic bacterial agent.

Asymptomatic infections with highly polymorphic Chlamydia suis are ubiquitous in pigs.

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.

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

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.

Salmonella enterica prevalence in leatherback sea turtles (Dermochelys coriacea) in St. Kitts, West Indies.

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.

Comparative Evaluation of GS-441524, Teriflunomide, Ruxolitinib, Molnupiravir, Ritonavir, and Nirmatrelvir for In Vitro Antiviral Activity against Feline Infectious Peritonitis Virus.

Feline infectious peritonitis (FIP), caused by feline coronavirus (FcoV), is considered one of the most enigmatic diseases in cats. Developing effective drugs for FIP is crucial due to its global prevalence and severity. In this study, six antiviral drugs were tested for their cytotoxicity, cell viability, and antiviral efficacies in Crandell-Reese feline kidney cells. A cytotoxicity assay demonstrated that these drugs were safe to be used with essentially no cytotoxicity with concentrations as high as 250 µM for ruxolitinib; 125 µM for GS441524; 63 µM for teriflunomide, molnupiravir, and nirmatrelvir; and 16 µM for ritonavir. GS441524 and nirmatrelvir exhibited the least detrimental effects on the CRFK cells, with 50% cytotoxic concentration (CC50) values of 260.0 µM and 279.1 µM, respectively, while ritonavir showed high toxicity (CC50 = 39.9 µM). In the dose-response analysis, GS441524, nirmatrelvir, and molnupiravir demonstrated promising results with selectivity index values of 165.54, 113.67, and 29.27, respectively, against FIPV. Our study suggests that nirmatrelvir and molnupiravir hold potential for FIPV treatment and could serve as alternatives to GS441524. Continued research and development of antiviral drugs are essential to ensure the well-being of companion animals and improve our preparedness for future outbreaks of coronaviruses affecting animals and humans alike.

Genome sequence of the obligate intracellular animal pathogen Chlamydia pecorum E58.

Chlamydia pecorum is an obligate intracellular bacterial pathogen that causes diverse disease in a wide variety of economically important mammals. We report the finished complete genome sequence of C. pecorum E58, the type strain for the species.

Dual-emission fluorescence resonance energy transfer (FRET) real-time PCR differentiates feline immunodeficiency virus subtypes and discriminates infected from vaccinated cats.

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.

Identification of Rickettsia felis DNA in the blood of domestic cats and dogs in the USA.

BACKGROUND: The main vector and reservoir host of Rickettsia felis, an emerging human pathogen causing flea-borne spotted fever, is the cat flea Ctenocephalides 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 of R. felis DNA in the blood of domestic dogs and cats in the USA. METHODS: Three previously validated PCR assays for R. felis and 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, and Bartonella spp. in cats. Phylogenetic comparisons were performed on the gltA nucleotide sequences obtained in the study and those reported for R. felis and R. felis-like organisms. RESULTS: Low copy numbers of R. felis DNA (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 with R. felis being 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. CONCLUSIONS: The low prevalence of naturally infected PCR-positive cats is further evidence that cats are unlikely to be important reservoirs of R. 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 of R. felis infections.

Comparison of microbiota, antimicrobial resistance genes and mobile genetic elements in flies and the feces of sympatric animals.

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.

Mucosal Vaccination with UV-Inactivated Chlamydia suis in Pre-Exposed Outbred Pigs Decreases Pathogen Load and Induces CD4 T-Cell Maturation into IFN-γ+ Effector Memory Cells.

Chlamydia trachomatis (Ct) infections are the most frequent bacterial sexually transmitted disease, and they can lead to ectopic pregnancy and infertility. Despite these detrimental long-term sequelae, a vaccine is not available. Success in preclinical animal studies is essential for vaccines to move to human clinical trials. Pigs are the natural host to Chlamydia suis (Cs)-a chlamydia species closely related to Ct, and are susceptible to Ct, making them a valuable animal model for Ct vaccine development. Before making it onto market, Ct vaccine candidates must show efficacy in a high-risk human population. The high prevalence of human Ct infection combined with the fact that natural infection does not result in sterilizing immunity, results in people at risk likely having been pre-exposed, and thus having some level of underlying non-protective immunity. Like human Ct, Cs is highly prevalent in outbred pigs. Therefore, the goal of this study was to model a trial in pre-exposed humans, and to determine the immunogenicity and efficacy of intranasal Cs vaccination in pre-exposed outbred pigs. The vaccine candidates consisted of UV-inactivated Cs particles in the presence or absence of an adjuvant (TriAdj). In this study, both groups of vaccinated pigs had a lower Cs burden compared to the non-vaccinated group; especially the TriAdj group induced the differentiation of CD4+ cells into tissue-trafficking CCR7- IFN-γ-producing effector memory T cells. These results indicate that Cs vaccination of pre-exposed pigs effectively boosts a non-protective immune response induced by natural infection; moreover, they suggest that a similar approach could be applied to human vaccine trials.

Peptide ELISA and FRET-qPCR Identified a Significantly Higher Prevalence of Chlamydia suis in Domestic Pigs Than in Feral Swine from the State of Alabama, USA.

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.

Multi-peptide ELISAs overcome cross-reactivity and inadequate sensitivity of conventional Chlamydia pneumoniae serology.

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.

In vitro analysis of genetically distinct Chlamydia pecorum isolates reveals key growth differences in mammalian epithelial and immune cells.

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.

Efficient fecal-oral and possible vertical, but not respiratory, transmission of emerging Chlamydia gallinacea in broilers.

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.

Temporal delay of peak T-cell immunity determines Chlamydia pneumoniae pulmonary disease in mice.

Severe chlamydial disease typically occurs after previous infections and results from a hypersensitivity response that is also required for chlamydial elimination. Here, we quantitatively dissected the immune and disease responses to repeated Chlamydia pneumoniae lung infection by multivariate modeling with four dichotomous effects: mouse strain (A/J or C57BL/6), dietary protein content (14% protein and 0.3% L-cysteine-0.9% L-arginine, or 24% protein and 0.5% L-cysteine-2.0% L-arginine), dietary antioxidant content (90 IU alpha-tocopherol/kg body weight versus 450 IU alpha-tocopherol/kg and 0.1% g L-ascorbate), and time course (3 or 10 days postinfection). Following intranasal C. pneumoniae challenge, C57BL/6 mice on a low-protein/low-antioxidant diet, but not C57BL/6 mice on other diets or A/J mice, exhibited profoundly suppressed early lung inflammatory and pan-T-cell (CD3delta(+)) and helper T-cell (CD45) responses on day 3 but later strongly exacerbated disease on day 10. Contrast analyses characterized severe C. pneumoniae disease as being a delayed-type hypersensitivity (DTH) response with increased lung macrophage and Th1 cell marker transcripts, increased Th1:Th2 ratios, and Th1 cytokine-driven inflammation. Results from functional analyses by DTH, enzyme-linked immunospot, and immunohistofluorescence assays were consistent with the results obtained by transcript analysis. Thus, chlamydial disease after secondary infection is a temporal dysregulation of the T-cell response characterized by a profoundly delayed T-helper cell response that results in a failure to eliminate the pathogen and provokes later pathological Th1 inflammation. This delayed T-cell response is under host genetic control and nutritional influence. The mechanism that temporally and quantitatively regulates the host T-cell population is the critical determinant in chlamydial pathogenesis.

Diversity of Hepatozoon species in naturally infected dogs in the southern United States.

Hepatozoon americanum is a protozoan that causes American canine hepatozoonosis (ACH) in the southern United States; Hepatozoon canis, the causative agent of canine hepatozoonosis in Africa, Asia, Europe, and South America, has not previously been definitively identified in dogs in the United States. To characterize the diversity of Hepatozoon spp. in domestic dogs from Oklahoma, blood samples collected from dogs residing in an endemic area of the state, clinical cases presented to veterinarians with symptoms of ACH, and dogs housed at a local shelter were evaluated by a nested PCR designed to amplify a variable region of the 18S rRNA gene of blood ampicomplexa, including Hepatozoon spp. Hepatozoon sequences recovered from a dog from an area where ACH is endemic, from clinically ill dogs, and from one shelter dog most closely resembled H. americanum. However, two other shelter dogs had evidence of infection with H. canis or a closely related organism. A subsequent review of real-time PCR results from the Molecular Diagnostics Laboratory at Auburn University revealed that the majority of samples submitted from dogs from across the United States which tested positive for Hepatozoon spp. had H. americanum. However, some submissions were also found which contained DNA sequence of H. canis. Mixed H. americanum and H. canis-like infections also were detected. Our data suggest that H. americanum, H. canis, as well as H. canis-like organisms are present and may cause disease in dogs in the southern U.S.

Diagnosis of canine Hepatozoon spp. infection by quantitative PCR.

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.