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Intracellular bacteria of the genusChlamydiacause numerous typically chronic diseases, frequently with debilitating sequelae. Genetic determinants of disease susceptibility after infection withChlamydiabacteria are unknown. C57BL/6 mice develop severe pneumonia and poor immunity againstChlamydiaafter 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 toChlamydia-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.

International audience; Chlamydia (C.) pecorum, an obligate intracellular bacterium, may cause severe diseases in ruminants, swine and koalas, although asymptomatic infections are the norm. Recently, we identified genetic polymorphisms in the ompA, incA and ORF663 genes that potentially differentiate between high-virulence C. pecorum isolates from diseased animals and low-virulence isolates from asymptomatic animals. Here, we expand these findings by including additional ruminant, swine, and koala strains. Coding tandem repeats (CTRs) at the incA locus encoded a variable number of repeats of APA or AGA amino acid motifs. Addition of any non-APA/AGA repeat motif, such as APEVPA, APAVPA, APE, or APAPE, associated with low virulence (P

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.

Reverse-transcription PCR (RT-PCR) is the gold standard for mRNA quantification. Efficient, rapid, and high-throughput mRNA extraction is a prerequisite to ensure PCR sensitivity and precision, particularly for quantification of low-abundance mRNAs, and for large numbers of samples. Many mRNA extraction methods entail meticulous handling of individual samples, and are not well suited for large sample numbers. To achieve simple separation of mRNA binding matrix and the medium from which mRNA is to be isolated, oligo (dT)(20)-coated silica beads were used. Simple centrifugation and decanting steps can be used throughout the extraction procedure to separate supernatant fluids from the silica beads. DNase treatment reduced clumping of sedimented beads, thus facilitating bead resuspension and avoiding repeated agitation. DNase treatment also significantly reduced contaminating DNA, increased mRNA purity, and enhanced mRNA PCR readout by approximately 5-fold. The number of target transcripts per sample aliquot was higher in DNase-treated mRNA than in non-treated mRNA or in total nucleic acids. Thus, use of DNase-treated mRNA increased sensitivity of detection and quantification of low-copy transcripts. In conclusion, we describe here a simple, rapid, and cost-effective method that facilitates convenient extraction of high-quality mRNA by minimizing cumbersome mechanical disruption and pipetting steps.

In a previous immunohistological study, chlamydiae were detected in 5 out of 139 cases of swine abortion, and a possible implication of C. psittaci serovar 1 was suggested. The present study sought to classify the chlamydiae found in the fetal organs of these abortions. DNA extracted from 15 paraffin-embedded tissue speciments (10 livers and 5 lungs, obtained from 10 fetuses from 9 cases of abortion) was amplified in a nested PCR with Chlamydia ompl genus-specific primers. Chlamydia DNA was amplified in 9 liver and 2 lung specimens. Eight of the amplification products were cloned, and 5 clones of each amplification were sequenced. Sequence analysis demonstrated in 7 specimens the simultaneous presence of porcine C. trachomatis S45 and C. pecorum 1710S ompl genotypes. All DNA fragments of 1 amplification were identical to the ruminant C. psittaci B577 ompl genotype (serovar 1). The results suggest that mixed infections with porcine C. trachomatis and C. pecorum dominate chlamydial infections associated with abortion in swine, but ruminant abortigenic C. psittaci are also found.

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 0.05) in CYP1A induction were observed in exposed versus control fish at 24 h. Expression of CYP1A increased by 25%, 66%, and 23% in exposed fish at 12 h, 24 h, and 48 h, respectively. Significant increases were observed in antioxidant capacity of nonenzymatic antioxidants in exposed versus control fish at each time point. Given the activity of CYP1A, radicals formed during PAH detoxification likely resulted in increased oxidant load requiring elevated antioxidant defenses. Research is needed to determine the duration of oxidative stress responses considering the potential for lipid oxidation in exposed fish or species feeding on exposed fish.

Background Precise 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. Methods Here, 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. Results The specificity of this assay was ascertained by amplification of canine, feline and equine blood with differential product melting temperatures (T m), 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. Conclusions The 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.

Intracellular Chlamydia (C.) bacteria cause in cattle some acute but rare diseases such as abortion, sporadic bovine encephalomyelitis, kerato-conjunctivitis, pneumonia, enteritis and polyarthritis. More frequent, essentially ubiquitous worldwide, are low-level, asymptomatic chlamydial infections in cattle. We investigated the impact of these naturally acquired infections in a cohort of 51 female Holstein and Jersey calves from birth to 15 weeks of age. In biweekly sampling, we measured blood/plasma markers of health and infection and analyzed their association with clinical appearance and growth in dependence of chlamydial infection intensity as determined by mucosal chlamydial burden or contemporaneous anti-chlamydial plasma IgM. Chlamydia 23S rRNA gene PCR and ompA genotyping identified only C. pecorum (strains 1710S, Maeda, and novel strain Smith3v8) in conjunctival and vaginal swabs. All calves acquired the infection but remained clinically asymptomatic. High chlamydial infection associated with reduction of body weight gains by up to 48% and increased conjunctival reddening (P

Epidemiological data indicate that infection of cattle with chlamydiae such as Chlamydophila (C.) pecorum, C. abortus, C. psittaci and Chlamydia suis, is ubiquitous with mixed infections occurring frequently. The apparent lack of association between infection and clinical disease has resulted in debate as to the pathogenic significance of these organisms, and their tendency to sub-clinical and/or persistent infection presents a challenge to the study of their potential effects. However, recent evidence indicates that chlamydial infections have a substantial and quantifiable impact on livestock productivity with chronic, recurrent infections associated with pulmonary disease in calves and with infertility and sub-clinical mastitis in dairy cows. Data also suggest these infections manifest clinically when they coincide with a number of epidemiological risk factors. Future research should: (1) use relevant animal models to clarify the pathogenesis of bovine chlamydioses; (2) quantify the impact of chlamydial infection at a herd level and identify strategies for its control, including sub-unit vaccine development; and (3) evaluate the zoonotic risk of bovine chlamydial infections which will require the development of species-specific serodiagnostics.

Identification of highly immunogenic antigens is critical for construction of an efficacious subunit vaccine against C. pneumoniae infections. A previous project used a genome-wide screen to identify twelve protective C. pneumoniae candidate genes in an A/J mouse lung disease model (Li et al., Vaccine 24:2917, 2006). Due to insufficient induction of Th1 immunity, these genes elicited only modest protection. Here, we used the E. coli heat-labile enterotoxin as a Th1-enhancing genetic adjuvant, and re-tested these twelve 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.

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.

Fluoroquinolones are used to treat infections caused by Escherichia coli in canine and feline veterinary patients, particularly those infecting the urinary tract. The gyrA gene is a primary target causing fluoroquinolone resistance in gram negative coliforms, with mutations in codons 83 and 87 generally associated with high-level of resistance E. coli clinical isolates. We have developed a fluorescence resonance energy transfer (FRET) quantitative PCR to identify enrofloxacin-resistance in clinical E. coli isolates that carry mutations in codons 83 and 87 of gyrA. This real-time quantitative PCR assay is rapid, economical, and sensitive compared with cultured antimicrobial susceptibility testing. The assay identified as few as four genome copies per reaction from culture and 19 genome copies in urine. For the 70 isolates tested, the sensitivity was 87.5% (95% CI=75-95.3%) (n=42/48), specificity was 100% (95% CI=87.3-100%) (n=22/22), whereas accuracy was 91.4% (95% CI=82.3-97%) (n=64/70). Furthermore, we were able to accurately differentiate between the wild type and mutants E. coli directly from infected canine urine samples (n=5) within 2 h. These results were confirmed by sequence alignments of the PCR products and comparison with the susceptibility testing. The FRET-PCR assay appears to have promising clinical application as an early diagnostic tool for rapid and sensitive detection and differentiation of the level of fluoroquinolone resistance among clinical E. coli isolates that may facilitate design of the dosing regimen.

Infections with the intracellular bacterium Chlamydophila (C.) pecorum are highly prevalent worldwide in cattle. These infections cause significant diseases such as polyarthritis, pneumonia, enteritis, genital infections and fertility disorders, and occasionally sporadic bovine encephalomyelitis. Subclinical respiratory infections of calves with C. pecorum have been associated with airway obstruction, pulmonary inflammation, and reduced weight gains. This investigation examined four chlamydial strains with biological properties of C. pecorum isolated from feces of clinically normal cattle, from calves with pneumonia, and from bulls with posthitis. The objective was to characterize the evolutionary relationships of these bovine chlamydial isolates to other chlamydiae by genetic analysis of the ompA gene, and by the immunological cross-reactivities in Western immunoblot analysis. PCR typing of the ompA gene identified these isolates as C. pecorum. The OmpA-deduced amino acid dissimilarities between these four strains spanned 10-20%. In phylogenetic analysis, the four isolates clustered with C. pecorum ruminant, porcine, and koala strains of different geographic origins rather than with each other. All four isolates showed different patterns of Western immunoblot reactivity with antiserum against bovine C. pecorum strain LW63, and, interestingly, no cross-reactivity of the OmpA proteins with the anti-LW613 OmpA antibodies. These data underscore the polyphyletic population structure of C. pecorum and suggest that the spectrum of C. pecorum OmpA proteins in a host species can occupy the entire evolutionary bandwidth within C. pecorum. The variant immunoblot reactivities support the notion of considerable genomic plasticity of C. pecorum.

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 repeatedChlamydia pneumoniaelung 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 α-tocopherol/kg body weight versus 450 IU α-tocopherol/kg and 0.1% gl-ascorbate), and time course (3 or 10 days postinfection). Following intranasalC. pneumoniaechallenge, 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 (CD3δ+) and helper T-cell (CD45) responses on day 3 but later strongly exacerbated disease on day 10. Contrast analyses characterized severeC. pneumoniaedisease 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.

In Chlamydiaceae , the nucleotide sequence between the 5S rRNA gene and the gene for subunit F of the Na + -translocating NADH-quinone reductase ( nqrF or dmpP ) has varied lengths and gene contents. We analyzed this site in 45 Chlamydiaceae strains having diverse geographical and pathological origins and including members of all nine species.

Infections with Chlamydophila abortus and C. pecorum are highly prevalent in cattle and have been associated with bovine mastitis. A prospective cohort study was conducted with a herd of 140 Holstein dairy cows to investigate the influence of Chlamydophila infection on subclinical inflammation of the bovine mammary gland as characterized by somatic cell numbers in milk. PCR detection of C. abortus and low serum antibody levels against Chlamydophila spp. were significantly associated with subclinical mastitis. To examine the effect of the infection by response modification, immune perturbation was done by two subcutaneous administrations of an experimental vaccine preparation of inactivated C. abortus and C. pecorum elementary bodies. Vaccination against Chlamydophila highly significantly decreased milk somatic cell numbers, thus reducing bovine mastitis, and increased antibody levels against Chlamydophila but did not eliminate shedding of C. abortus in milk as detected by PCR. The protective effect peaked at 11 weeks after vaccination and lasted for a total of 14 weeks. Vaccination with the Chlamydophila vaccine, a mock vaccine, or a combination vaccine against bovine viral diseases highly significantly increased C. abortus shedding in milk for 1 week, presumably mediated by the vaccine adjuvant. In summary, this study shows an etiological involvement of the widespread Chlamydophila infections in bovine mastitis, a herd disease of critical importance for the dairy industry. Furthermore, this investigation shows the potential for temporary improvement of chlamydial disease by therapeutic vaccination. Chlamydophila vaccination of cattle might serve as a testing ground for vaccines against human chlamydial infections.

The polymerase chain reaction (PCR) has become one of the most important tools in molecular diagnostics, providing exquisite sensitivity and specificity for detection of nucleic acid targets. Real-time monitoring of PCR has simplified and accelerated PCR laboratory procedures and has increased information obtained from specimens including routine quantification and differentiation of amplification products. Clinical diagnostic applications and uses of real-time PCR are growing exponentially, real-time PCR is rapidly replacing traditional PCR, and new diagnostic uses likely will emerge. This review analyzes the scope of present and potential future clinical diagnostic applications of this powerful technique. Critical discussions focus on basic concepts, variations, data analysis, instrument platforms, signal detection formats, sample collection, assay design, and execution of real-time PCR.

An inbred A/J mouse respiratory challenge model was validated for vaccine testing against Chlamydia (C.) pneumoniae and used to screen the C. pneumoniae genome for vaccine candidates by expression library immunization (ELI). Biolistic delivery of genetic vaccine constructs elicited Th2-like immunity that was associated with inefficient elimination of C. pneumoniae. Delivery by injection elicited protective Th1-like responses. Since biolistic delivery of pools of ORFs was used in first round screening, the screen presumably selected against potent immunogens. Nevertheless, it was sufficiently accurate to identify three weakly protective antigens among all putative C. pneumoniae ORFs. The results suggest ELI discovery of highly protective C. pneumoniae vaccine candidates requires tight control of the Th1 immunity elicited by the genetically delivered library of test antigens.

The polymerase chain reaction (PCR) has become one of the most important tools in molecular diagnostics, providing exquisite sensitivity and specificity for detection of nucleic acid targets. Real-time monitoring of PCR has simplified and accelerated PCR laboratory procedures and has increased information obtained from specimens including routine quantification and differentiation of amplification products. Clinical diagnostic applications and uses of real-time PCR are growing exponentially, real-time PCR is rapidly replacing traditional PCR, and new diagnostic uses likely will emerge. This review analyzes the scope of present and potential future clinical diagnostic applications of this powerful technique. Critical discussions focus on basic concepts, variations, data analysis, instrument platforms, signal detection formats, sample collection, assay design, and execution of real-time PCR.

We investigated the acquisition and prevalence of Chlamydophila sp. infection in calves. Specimens were collected at weekly intervals from birth to week 12 postpartum from 40 female Holstein calf-dam pairs in a dairy herd. Real-time PCR detected, quantified, and differentiated Chlamydophila 23S rRNA gene DNA from vaginal cytobrush swabs and milk samples. Chemiluminescence enzyme-linked immunosorbent assay with lysed Chlamydophila abortus or Chlamydophila pecorum elementary body antigens quantified antibodies against Chlamydophila spp. in sera. Chlamydophila sp. DNA was found in 61% of calves and 20% of dams in at least one positive quantitative PCR. In calves, clinically inapparent C. pecorum infection with low organism loads was fivefold more prevalent than C. abortus infection and was most frequently detected by vaginal swabs compared to rectal or nasal swabs. In dams, C. abortus dominated in milk and C. pecorum dominated in the vagina. The group size of calves correlated positively ( P < 0.01) with Chlamydophila infection in quadratic, but not linear, regression. Thus, a doubling of the group size was associated with a fourfold increase in frequency and intensity of Chlamydophila infection. For groups of 14 or 28 calves, respectively, logistic regression predicted a 9 or 52% probability of infection of an individual calf and a 52 or 99.99% probability of infection of the group. Anti- Chlamydophila immunoglobulin M antibodies in Chlamydophila PCR-positive calves and dams and in dams that gave birth to calves that later became positive were significantly higher than in PCR-negative animals ( P ≤ 0.02). Collectively, crowding strongly enhances the frequency and intensity of highly prevalent Chlamydophila infections in cattle.

This study investigated the effects of controlled reinfection on fertility of cattle naturally preexposed toChlamydophila abortus. All animals had high prechallenge levels of immunoglobulin M (IgM), IgG, IgG1, and IgG2 serum antibodies against ruminantC. abortusin a chemiluminescent enzyme-linked immunosorbent assay. Twenty virgin heifers were estrus synchronized with prostaglandin F2, artificially inseminated 2 to 3 days later, and challenged immediately by intrauterine administration of 0, 104, 105, 106, or 108inclusion-forming units (IFU) ofC. abortus. Ten heifers were estrus synchronized, inseminated, and uterine challenged 2 weeks later. These animals were also indirectly exposed toC. abortusinfection (cohort challenged) by contact with their previously challenged cohorts. Pregnancy was determined by rectal palpation 42 days after insemination. All anti-C. abortusantibody isotypes increased in heifers following uterine challenge with 108IFU. A total of 11, 83, 50, 66, and 0% of heifers were pregnant after uterine challenge with 0, 104, 105, 106, and 108IFU ofC. abortus, respectively. A total of 50 and 65% of heifers were pregnant with and without cohort challenge, respectively. Uterine inoculum dose and cohort challenge (or, alternatively, a negative pregnancy outcome [infertility]) correlated highly significantly with a rise in postchallenge anti-C. abortusIgM levels over prechallenge levels. Logistic regression modeled fertility, with uterine challenge dose and cohort challenge or prechallenge IgM as predictors (P< 0.05). The models predict that the uterineC. abortusinoculum causing infertility is 8.5-fold higher for heifers without cohort exposure and 17-fold higher for heifers with high IgM levels than for heifers with cohort exposure or with low IgM levels.

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°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 = 10−2.5–102.5) in a single assay.

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.

Quantitative detection of intracellular bacteria of the genus Chlamydia by the standard cell culture method is cumbersome and operator dependent. As an alternative, we adapted hot-start PCR to the glass capillary quantitative PCR format of the LightCycler®. The optimized PCR was consistently more efficient than commercially available pre-assembled PCRs. Detection by quantitative PCR of as few as single copies of DNA of Chlamydia spp. was accomplished by SYBR® Green fluorescence of the dsDNA product and by fluorescence resonance energy transfer (FRET) hybridization probes. The PCRs were 15-fold more sensitive than the cell culture quantitative assay of C. psittaci B577 infectious stock. The number of chlamydial genomes detected by C. psittaci B577 FRET PCR correlated well with cell culture determination of inclusion forming units (IFUs) (r = 0.96, P < 0.0008). When infected tissue samples were analyzed by cell culture and PCR, the correlation coefficient between IFUs and chlamydial genomes was higher with C. psittaci B577 FRET PCR (r = 0.90, P < 0.0004) than with Chlamydia omp1 SYBR Green PCR (r = 0.85, P < 0.002).

Protection against infections with the intracellular bacterium Chlamydia spp. requires Th1-polarized CD4+ T cell immunity. In BALB/c mouse lung infections, immediate innate and nascent Chlamydia-specific immune responses following intranasal inoculation of Chlamydia psittaci strain B577 were modulated by 7-day i.p. administration of murine rIL-12, the initiation cytokine for Th1 immunity. Treatment with IL-12 reduced the severity of chlamydial pneumonia, abolished mortality (37.5% in untreated mice), and significantly reduced numbers of chlamydial organisms in lungs. On day 4 after inoculation, the neutrophil:macrophage ratio in bronchointerstitial pneumonias was 1.96 in untreated mice and 0.51 in IL-12-treated mice. This immediate, IL-12-mediated shift in innate inflammatory phenotype was correlated with a significant reduction of lung concentrations of the neutrophil chemoattractant macrophage inflammatory protein (MIP)-2 (putative murine homologue of human IL-8), monocyte chemotactic protein-1, and TNF-α; and a reduction in MIP-1α and IFN-γ, at high-dose infection only, and IL-12-independent IL-10 levels. Chlamydia-specific Ab titers and Ig isotype ratios indicated an IL-12-dependent Th1 shift. Recall responses of IL-12-primed mice to secondary chlamydial lung infection eliminated chlamydiae more effectively and generated a lung cytokine profile conducive to perpetuation of the Th1 memory population. These data support the hypothesis that genetic differences in endogenous IL-12 production and response pathways could determine disease outcomes characterized by poor chlamydial clearance and a purulent inflammatory infiltrate vs effective elimination of chlamydiae in a macrophage-dominated response.

Background Although tick-borne diseases are important causes of morbidity and mortality in dogs in tropical areas, there is little information on the agents causing these infections in the Caribbean. Methodology We used PCRs to test blood from a cross-section of dogs on St Kitts for Ehrlichia (E.) canis, Babesia (B.) spp., Anaplasma (A.) spp. and Hepatozoon (H.) spp. Antibodies against E. canis and A. phagocytophilum/platys were detected using commercial immunochromatography tests. Records of the dogs were examined retrospectively to obtain clinical and laboratory data. Principal findings There was serological and/or PCR evidence of infections of dogs with E. canis (27%; 46/170), Babesia spp. (24%; 90/372) including B. canis vogeli (12%; 43/372) and B. gibsoni (10%; 36/372), A. platys (11%; 17/157) and H. canis (6%; 15/266). We could not identify the Babesia sp. detected in nine dogs. There was evidence of multiple infections with dual infections with E. canis and B. canis vogeli (8%; 14/179) or B. gibsoni (7%; 11/170) being the most common. There was agreement between immunochromatography and PCR test results for E. canis for 87% of dogs. Only 13% of exposed dogs had signs of a tick-borne disease and 38% had laboratory abnormalities. All 10 dogs presenting for a recheck after treatment of E. canis with doxycycline were apparently healthy although all remained seropositive and six still had laboratory abnormalities despite an average of two treatments with the most recent being around 12 months previously. Infections with Babesia spp. were also mainly subclinical with only 6% (4/67) showing clinical signs and 13% (9/67) having laboratory abnormalities. Similarly, animals with evidence of infections with A. platys and H. canis were largely apparently healthy with only occasional laboratory abnormalities. Conclusions Dogs are commonly infected with tick-borne pathogens in the Caribbean with most having no clinical signs or laboratory abnormalities.

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. © 2011, American Society for Microbiology.

Chronic inflammatory diseases caused by obesity represent critical public health concerns worldwide. In these diseases such as metabolic syndrome, diabetes and atherosclerosis, adipose tissue acts as an endocrine organ that releases large quantities of inflammatory mediators into circulation. Besides classically recognized effectors on the development of obesity and resultant conditions, infection has attracted attention as an enhancer of chronic inflammatory diseases. Infectious diseases have long been associated with obesity, metabolic syndrome, diabetes and atherosclerosis. However, the infectious hypothesis for chronic inflammatory diseases has been challenged by inconclusive clinical trials. Nevertheless, the large body of evidence accumulated over decades on the association of infectious diseases with obesity, diabetes and cardiovascular disease should not be disregarded. Instead, re-formulation of hypotheses of the mechanisms by which microbes affect obesity-associated diseases may be required with an emphasis on the early events in the progression of such diseases and the multifactorial nature of pathogen-host interactions. This review focuses on pathogens that directly promote obesity and on pathogens that cause chronic infections and thereby enhance metabolic diseases in obese patients. A new perspective on the interaction between infections and obesity-related diseases may improve management of chronic inflammatory diseases that rank high among global threats to human health.

Recent advances in molecular biology are providing new opportunities for the diagnosis, prognosis and treatment of cancer. At two decades from its discovery, PCR with its hundreds of variants and improvements is still the keystone of molecular techniques which gave rise to such a revolution. Novel methods and techniques have been introduced in recent years which promise further breakthroughs. Unfortunately, in veterinary medicine, the unaffordable cost of new instrumentations has delayed their application and practical use in clinical settings. Nevertheless, thanks to PCR, the exciting era of molecular medicine has also begun in veterinary medicine. Due to the limited space available, this chapter cannot deal with all the potential applications of PCR in the cancer battlefield. Thus, the authors have focused their attention on those concerned with PCR applications for the diagnosis and prognosis of cancer in pets which are already currently available, albeit not diffusely, at both academic and private laboratories around the world. In some cases, such as in c-KIT somatic mutations, for the first time in veterinary medicine, a consensus panel of specialists has recommended the inclusion of a molecular assay in the staging work-up of a neoplastic disease (canine mast cell tumors). In addition to the role of the molecular biologist in developing, implementing and refining the molecular classification for routine clinical practice, it is necessary to discover and validate new targets able to provide accurate information regarding diagnosis, prognosis, treatment resistance, susceptibility or predisposition to toxicity, or the prediction of a therapeutic response.