Dongya Gao

Quantitative Detection of Chlamydia psittaci and C. pecorum by High-Sensitivity Real-Time PCR Reveals High Prevalence of Vaginal Infection in Cattle

ABSTRACT Bovine vaginal cytobrush specimens were analyzed for the presence of Chlamydia spp. by a high-sensitivity, high-specificity quantitative PCR. The 53% prevalence of low-level Chlamydia psittaci and C. pecorum genital infection detected in virgin heifers suggests predominantely extragenital transmission of Chlamydia in cattle and conforms to the high seroprevalence of anti-Chlamydia antibodies.

The quantity of nitric oxide released by macrophages regulates Chlamydia-induced disease

Intracellular bacteria of the genus Chlamydia cause numerous typically chronic diseases, frequently with debilitating sequelae. Genetic determinants of disease susceptibility after infection with Chlamydia bacteria are unknown. C57BL/6 mice develop severe pneumonia and poor immunity against Chlamydia after moderate respiratory infection whereas BALB/c mice are protected from disease and develop vigorous Th1 immunity. Here we show that infected C57BL/6 macrophages release more NO synthesized by NO synthase 2 (NOS2) than BALB/c macrophages and have lower mRNA concentrations of arginase II, a competitor of NOS2 for the common substrate, l-arginine. Reduction, but not elimination, of NO production by incomplete inhibition of NOS2 abolishes susceptibility of C57BL/6 mice to Chlamydia-induced disease. Thus, the quantity of NO released by infected macrophages is the effector mechanism that regulates between pathogenic and protective responses to chlamydial infection, and genes controlling NO production determine susceptibility to chlamydial disease.

One-step real-time duplex reverse transcription PCRs simultaneously quantify analyte and housekeeping gene mRNAs

We developed a one-step real-time duplex reverse transcription PCR (RT-PCR) method using the LightCycler platform. This method allows simultaneous reverse transcription and real-time PCR amplification of two mRNAs of specific genes of interest (analyte genes) and mRNA of constantly transcribed genes (housekeeping genes) in a single-tube reaction. Specimen total nucleic acids were used because eukaryotic cDNA is discriminated from genomic DNA using exon-spanning primers and/or fluorescence resonance energy transfer (FRET) probes. Transcripts of murine arginase I and hypoxanthine-phosphoribosyl transferase (HPRT; housekeeping gene) or murine arginase II analyte and porphobilinogen deaminase (PBGD; housekeeping gene) were quantified in such duplex RT-PCRs. Twenty-minute reverse transcription reactions at 55 degrees C followed by 18 high-stringency step-down thermal cycles and 25 relaxed-stringency fluorescence acquisition cycles produced sensitive and accurate RT-PCR results. Fluorescent signal spillover between channels was fully compensated. A matrix of duplex PCRs at variable ratios of target standards yielded equations for factors that correct PCR-specific target ratio-dependent deviations in quantification. The one-step real-time duplex RT-PCRs reliably and accurately determined 10-10,000 copies of each target over a 100,000-fold range of target copy ratios (analyte to housekeeping mRNA = 10(-2.5)-10(2.5)) in a single assay.

Frequency and therapy monitoring of canine Babesia spp. infection by high-resolution melting curve quantitative FRET-PCR.

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.

Acute Chlamydia pneumoniae Reinfection Accelerates the Development of Insulin Resistance and Diabetes in Obese C57BL/6 Mice

BACKGROUND Epidemiological and pathological evidence links highly prevalent pathogens to chronic inflammatory diseases, such as type 2 diabetes. Animal models contribute critically to the mechanistic understanding of infectious enhancement of inflammatory diseases, which share insulin resistance as the central pathophysiological defect. METHODS With use of a mouse model, we examined insulin resistance progression and the influence of infection (Chlamydia pneumoniae-infected vs. uninfected control mice), genetic background (C57BL/6 vs. A/J mice), dietary fat concentration (27% vs. 5%), and time (2, 5, 9, or 15 weeks after inoculation). RESULTS In obese C57BL/6 mice, C. pneumoniae infection induced significantly increased insulin resistance that persisted long after bacterial clearance. Circulating tumor necrosis factor (TNF)-alpha produced in response to acute C. pneumoniae lung colonization exacerbated insulin resistance but not TNF-alpha released in situ during secondary chlamydial infection. Azithromycin or anti-TNF-alpha antibody prevented infection-exacerbated insulin resistance but significantly enhanced chlamydial dissemination to the heart. Azithromycin-treated mice did not eliminate C. pneumoniae from lungs by 3 weeks after inoculation but had significantly lower loads (42 genomes per 100 mg) than did control mice (219 genomes per 100 mg) or anti-TNF-alpha antibody-treated mice (3090 genomes per 100 mg). CONCLUSIONS Murine C. pneumoniae infection enhanced insulin resistance development in a genetically and nutritionally restricted manner via circulating mediators. The relevance for the current human diabetes epidemic remains to be determined, but this finding is potentially important because of the high prevalence of human C. pneumoniae infection worldwide.

Real-time PCR of the mammalian hydroxymethylbilane synthase (HMBS) gene for analysis of flea (Ctenocephalides felis) feeding patterns on dogs

BackgroundPrecise data on quantitative kinetics of blood feeding of fleas, particularly immediately after contact with the host, are essential for understanding dynamics of flea-borne disease transmission and for evaluating flea control strategies. Standard methods used are inadequate for studies that simulate early events after real-life flea access to the host.MethodsHere, we developed a novel quantitative polymerase chain reaction targeting mammalian DNA within fleas to quantify blood consumption with high sensitivity and specificity. We used primers and fluorescent probes that amplify the hydroxymethylbilane synthase (HMBS) gene, an evolutionary divergent gene that is unlikely to be detected in insects by mammalian-specific primers and probes. To validate this assay, fleas were placed on dogs, allowed to distribute in the hair, and removed at specific time points with single-use combs. Fleas were then immediately homogenized by vigorous shaking with ceramic beads in guanidinium-based DNA preservation buffer for DNA extraction.ResultsThe specificity of this assay was ascertained by amplification of canine, feline and equine blood with differential product melting temperatures (Tm), and lack of amplification of bovine and porcine blood and of adult fleas reared from larvae fed with bovine blood. Sensitivity of the assay was established by limiting dilution and detection of single copies of HMBS DNA equivalent to 0.043 nL blood. Application of the assay indicated that after 15 minutes on a dog, male and female fleas had ingested low, but similar amounts of approximately 1.1. nL blood. Saturation uptake of 118 and 100 nL blood per flea was found at 30 and 60 min on the dog, respectively.ConclusionsThe HMBS PCR method developed here offers the advantages of both exquisite sensitivity and specificity that make it superior to other approaches for quantification of blood ingested by fleas. The capability to detect minute quantities of blood in single fleas, particularly immediately after colonization of the host, will provide a superior tool for studying flea-host interactions, flea-borne disease transmission, and flea control strategies.

Diagnosis of Canine Leptospirosis by a Highly Sensitive FRET-PCR Targeting the lig Genes

Canine leptospirosis is underdiagnosed due to its wide spectrum of clinical presentations and the lack of a rapid and sensitive test for the accurate diagnosis of acute and chronic infections. In this study, we developed a highly sensitive and specific fluorescence resonance energy transfer (FRET)-PCR to detect common pathogenic leptospires in dogs, including Leptospira interrogans serovars Autumnalis, Canicola, Copenhageni (Icterohaemorrhagiae serogroup) and Pomona, and Leptospira kirschneri serovar Grippotyphosa. This PCR targets the lig genes, exclusively found in the pathogenic Leptospira species but not in saprophytic species (L. biflexa). A robust, high-stringency step-down real-time platform was coupled to the highly specific detection of leptospiral DNA by fluorescently labeled FRET probes. This enabled the detection of a single copy of the lig gene in a PCR containing DNA from up to 50 µL canine blood or 400 µL urine. Sensitivity determination by use of limiting serial dilutions of extracted leptospiral DNA indicated that the lig FRET-PCR we established was almost 100-fold more sensitive than the widely accepted lipL32 SYBR assay and 10-fold more sensitive than a 16S rRNA TaqMan assay. Application of this method to 207 dogs with potential leptospiral infection enabled us to diagnose three cases of canine leptospirosis characterized by low amounts of leptospiral DNA in body fluids. Detection of canine leptospirosis with the lig FRET-PCR was more sensitive with the lig FRET-PCR than with the 16S rRNA TaqMan PCR, which detected only 2 of the 3 cases, and the lipL32 SYBR PCR, which detected none of the 3 dogs with leptospirosis.

Dual-Emission Fluorescence Resonance Energy Transfer (FRET) Real-Time PCR Differentiates Feline Immunodeficiency Virus Subtypes and Discriminates Infected from Vaccinated Cats

ABSTRACT 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.

Novel Chlamydia pneumoniae vaccine candidates confirmed by Th1-enhanced genetic immunization

Identification of highly immunogenic antigens is critical for the construction of an efficacious subunit vaccine against Chlamydia pneumoniae infections. A previous project used a genome-wide screen to identify 12 protective C. pneumoniae candidate genes in an A/J mouse lung disease model (Li et al. [14]). Due to insufficient induction of Th1 immunity, these genes elicited only modest protection. Here, we used the Escherichia coli heat-labile enterotoxin as a Th1-enhancing genetic adjuvant, and re-tested these 12 genes, in parallel with six genes identified by other investigators. Vaccine candidate genes cutE and Cpn0420 conferred significant protection by all criteria evaluated (prevention of C. pneumoniae-induced death, reduction of lung disease, elimination of C. pneumoniae). Gene oppA_2 was protective by disease reduction and C. pneumoniae elimination. Four other genes were protective by a single criterion. None of the six genes reported elsewhere protected by reduction of lung disease or elimination of C. pneumoniae, but three protected by increasing survival.

Genome-wide association analysis of intra-specific QTL associated with the resistance for enteric septicemia of catfish

Disease resistance is one of the most important traits for aquaculture industry. For catfish industry, enteric septicemia of catfish (ESC), caused by the bacterial pathogen Edwardsiella ictaluri, is the most severe disease, causing enormous economic losses every year. In this study, we used three channel catfish families with 900 individuals (300 fish per family) and the 690K catfish SNP array, and conducted a genome-wide association study to detect the quantitative trait loci (QTL) associated with ESC resistance. Three significant QTL, with two of located on LG1 and one on LG26, and three suggestive QTL located on LG1, LG3, and LG21, respectively, were identified to be associated with ESC resistance. With a well-assembled- and -annotated reference genome sequence, genes around the involved QTL regions were identified. Among these genes, 37 genes had known functions in immunity, which may be involved in ESC resistance. Notably, nlrc3 and nlrp12 identified here were also found in QTL regions of ESC resistance in the channel catfish × blue catfish interspecific hybrid system, suggesting this QTL was operating within both intra-specific channel catfish populations and interspecific hybrid backcross populations. Many of the genes of the Class I MHC pathway, for mediated antigen processing and presentation, were found in the QTL regions. The positional correlation found in this study and the expressional correlation found in previous studies indicated that Class I MHC pathway was significantly associated with ESC resistance. This study validated one QTL previously identified using the second and fourth generation of the interspecific hybrid backcross progenies, and identified five additional QTL among channel catfish families. Taken together, it appears that there are only a few major QTL for ESC disease resistance, making marker-assisted selection an effective approach for genetic improvements of ESC resistance.