Development of a Chlamydia suis-specific antibody enzyme-linked immunosorbent assay based on the use of a B-cell epitope of the polymorphic membrane protein C.

Chlamydia suis infections lead to economic loss in the pork industry. Chlamydia suis infections could be successfully treated with tetracyclines until the appearance of a tetracycline resistant phenotype, which was acquired via horizontal gene transfer of the tet(C) gene. Given the importance of C. suis as a swine pathogen and as a recently emerged tetracycline resistant pathogen with zoonotic potential, our aim was to develop a sensitive C. suis-specific antibody ELISA based on the polymorphic membrane proteins (Pmps). Chlamydia Pmps are important virulence factors and candidate antigens for serodiagnosis. We identified nine Pmps (PmpA to I) in C. suis strain MD56 using a recently developed Hidden-Markov model. PmpC was the most promising candidate for the development of a C. suis-specific antibody ELISA as the protein was absent in C. abortus, C. pecorum and C. psittaci which also infect pigs and as the protein contained C. suis-specific amino acid regions, absent in C. trachomatis PmpC. We identified an immunodominant B-cell epitope in C. suis PmpC using experimental porcine sera. The sensitivity and specificity of the PmpC ELISA was compared to the complement fixation test (CFT) and to a recombinant MOMP ELISA using experimental sera. The PmpC ELISA detected all positive control sera and was in contrast to CFT and the rMOMP ELISA 100% C. suis specific as positive control sera against other Chlamydia species did not react in the PmpC ELISA. The test was successfully validated using slaughterhouse sera and sera from clinically affected pigs. The PmpC ELISA could assist in diminishing the spread of C. suis infections in the pork industry.

Evidence that human Chlamydia pneumoniae was zoonotically acquired.

Zoonotic infections are a growing threat to global health. Chlamydia pneumoniae is a major human pathogen that is widespread in human populations, causing acute respiratory disease, and has been associated with chronic disease. C. pneumoniae was first identified solely in human populations; however, its host range now includes other mammals, marsupials, amphibians, and reptiles. Australian koalas (Phascolarctos cinereus) are widely infected with two species of Chlamydia, C. pecorum and C. pneumoniae. Transmission of C. pneumoniae between animals and humans has not been reported; however, two other chlamydial species, C. psittaci and C. abortus, are known zoonotic pathogens. We have sequenced the 1,241,024-bp chromosome and a 7.5-kb cryptic chlamydial plasmid of the koala strain of C. pneumoniae (LPCoLN) using the whole-genome shotgun method. Comparative genomic analysis, including pseudogene and single-nucleotide polymorphism (SNP) distribution, and phylogenetic analysis of conserved genes and SNPs against the human isolates of C. pneumoniae show that the LPCoLN isolate is basal to human isolates. Thus, we propose based on compelling genomic and phylogenetic evidence that humans were originally infected zoonotically by an animal isolate(s) of C. pneumoniae which adapted to humans primarily through the processes of gene decay and plasmid loss, to the point where the animal reservoir is no longer required for transmission.

Genomic analysis of an attenuated Chlamydia abortus live vaccine strain reveals defects in central metabolism and surface proteins.

Comparative genomic analysis of a wild-type strain of the ovine pathogen Chlamydia abortus and its nitrosoguanidine-induced, temperature-sensitive, virulence-attenuated live vaccine derivative identified 22 single nucleotide polymorphisms unique to the mutant, including nine nonsynonymous mutations, one leading to a truncation of pmpG, which encodes a polymorphic membrane protein, and two intergenic mutations potentially affecting promoter sequences. Other nonsynonymous mutations mapped to a pmpG pseudogene and to predicted coding sequences encoding a putative lipoprotein, a sigma-54-dependent response regulator, a PhoH-like protein, a putative export protein, two tRNA synthetases, and a putative serine hydroxymethyltransferase. One of the intergenic mutations putatively affects transcription of two divergent genes encoding pyruvate kinase and a putative SOS response nuclease, respectively. These observations suggest that the temperature-sensitive phenotype and associated virulence attenuation of the vaccine strain result from disrupted metabolic activity due to altered pyruvate kinase expression and/or alteration in the function of one or more membrane proteins, most notably PmpG and a putative lipoprotein.

Genome sequence of Chlamydophila caviae (Chlamydia psittaci GPIC): examining the role of niche-specific genes in the evolution of the Chlamydiaceae.

The genome of Chlamydophila caviae (formerly Chlamydia psittaci, GPIC isolate) (1 173 390 nt with a plasmid of 7966 nt) was determined, representing the fourth species with a complete genome sequence from the Chlamydiaceae family of obligate intracellular bacterial pathogens. Of 1009 annotated genes, 798 were conserved in all three other completed Chlamydiaceae genomes. The C.caviae genome contains 68 genes that lack orthologs in any other completed chlamydial genomes, including tryptophan and thiamine biosynthesis determinants and a ribose-phosphate pyrophosphokinase, the product of the prsA gene. Notable amongst these was a novel member of the virulence-associated invasin/intimin family (IIF) of Gram-negative bacteria. Intriguingly, two authentic frameshift mutations in the ORF indicate that this gene is not functional. Many of the unique genes are found in the replication termination region (RTR or plasticity zone), an area of frequent symmetrical inversion events around the replication terminus shown to be a hotspot for genome variation in previous genome sequencing studies. In C.caviae, the RTR includes several loci of particular interest including a large toxin gene and evidence of ancestral insertion(s) of a bacteriophage. This toxin gene, not present in Chlamydia pneumoniae, is a member of the YopT effector family of type III-secreted cysteine proteases. One gene cluster (guaBA-add) in the RTR is much more similar to orthologs in Chlamydia muridarum than those in the phylogenetically closest species C.pneumoniae, suggesting the possibility of horizontal transfer of genes between the rodent-associated Chlamydiae. With most genes observed in the other chlamydial genomes represented, C.caviae provides a good model for the Chlamydiaceae and a point of comparison against the human atherosclerosis-associated C.pneumoniae. This crucial addition to the set of completed Chlamydiaceae genome sequences is enabling dissection of the roles played by niche-specific genes in these important bacterial pathogens.

Role of mobile DNA in the evolution of vancomycin-resistant Enterococcus faecalis.

The complete genome sequence of Enterococcus faecalis V583, a vancomycin-resistant clinical isolate, revealed that more than a quarter of the genome consists of probable mobile or foreign DNA. One of the predicted mobile elements is a previously unknown vanB vancomycin-resistance conjugative transposon. Three plasmids were identified, including two pheromone-sensing conjugative plasmids, one encoding a previously undescribed pheromone inhibitor. The apparent propensity for the incorporation of mobile elements probably contributed to the rapid acquisition and dissemination of drug resistance in the enterococci.

Expression of two novel proteins in Chlamydia trachomatis during natural infection.

Genes for a putative membrane associated protein (mvi -homologue) and a 48 kDa protein (ctr48) in Chlamydia trachomatis were characterized. The mvi -homologue has 12 transmembrane domains and shows considerable homology to the members of this gene family in various organisms. The ctr48 has a leader sequence and the C-proximal half is tryptophan-rich. The latter region shares 65% identity with the N-proxima third of C. pneumoniae 76 kDa protein over an overlap of 231 amino acid residues. The genes for the mvi -homologue and the ctr48 are present in the B, Ba, D, E, J and L2 serotypes of C. trachomatis. Immediately downstream from the ctr48 gene are multiple stop codons which are followed by a functional rho-independent terminator. The mvi -homologue and ctr48 genes are independently transcribed, albeit poorly in serotype B. However, protein products corresponding to these genes could not be detected by western blotting in HEp2 cells infected with C. trachomatis. Nevertheless, antibodies to peptides corresponding to these proteins were detected in sera with high micro-immunofluorescence titre against C. trachoImatic, collected from a Chlamydia -endemic population. These results suggest that the mvi -homologue and ctr48 are expressed by C. trachomatis during natural infection.

Ion chromatographic analysis of glucose, fructose, and sucrose concentrations in raw and processed vegetables.

To meet the demands for improved carbohydrate analysis techniques, a method of sugar extraction and analysis has been developed using ion chromatography. The sample preparation method, which uses filtration and extraction steps, has been shown to adequately isolate glucose, fructose, and sucrose from other food components in raw and processed vegetables. Resulting sample preparations were successfully analyzed by ion chromatography using pulsed amperometric detection, which is a sensitive, rapid, and accurate technique. Sugar concentrations in a number of different vegetable products were successfully determined in this way.

Evaluation of aortic stenosis by spectral analysis of the murmur.

A relation between the peak transaortic pressure gradient and the frequency content of the murmur (r = 0.79) was demonstrated in a prospective "test" set of 50 patients with the clinical diagnosis of aortic stenosis. After heart sounds were recorded and digitized, three segments of the systolic murmur were isolated and analyzed by fast Fourier transform technique. An average frequency spectrum was quantitated by a previously described empiric spectral estimator. Clinical data and spectral ratio were correlated with the transaortic pressure gradient and aortic valve area was calculated from cardiac catheterization data. The best prediction of the transaortic pressure gradient was obtained when a 170 ms murmur segment was analyzed and when the predictive algorithm also included the aortic dimension (r = 0.87). The aortic valve area was poorly predicted (r = -0.48) unless estimates of blood flow and valvular calcification were included in the algorithm (r = 0.84). Further refinement of this technique may provide a non-invasive and clinically useful method for the estimation of aortic valve stenosis.

Detection of aortic porcine valve dysfunction by maximum entropy spectral analysis.

A high-resolution method of spectral analysis, of the class generally called "maximum entropy method," was used in a study of aortic porcine valve closing sounds in 37 patients (ages 19 to 76). Spectra from 27 normal xenografts, implanted from 2 weeks to 61 months previously, were characterized by a dominant frequency peak, F1, at 89 +/- 15 Hz (mean +/- SD), with a lower amplitude peak, F2, at 154 +/- 25 Hz. Eight of nine patients with aortic porcine valve dysfunction were proved surgically to have leaflet degeneration or infection and had either F1 (139 +/- 54 Hz) and/or F2 (195 +/- 74 Hz) significantly higher than normal (p less than .001). In two patients with paravalvar leak but no leaflet abnormality, F1 and F2 were in the normal range. Estimation of F1 and F2 was highly reproducible and was unaffected by duration of implant up to 5 years. Spectral analysis of aortic porcine valve closing sounds by the maximum entropy method may be useful for detection of intrinsic xenograft dysfunction.

The effect of protein-energy malnutrition on appositional bone growth in the rat.

During protein-energy malnutrition appositional bone growth in the seventh caudal vertebra of the rat slows and finally ceases. During rehabilitation appositional growth begins agains and attains a rate in excess of that of the controls. This may account for alterations in skeletal proportions resulting from malnutrition.

Myocardial function in hypothyroidism. Relation to disease severity and response to treatment.

Systolic time intervals (ST) were used to evaluate myocardial function prospectively in 29 hypothyroid patients. The patients were divided into three categories of disease severity: (1) severe hypothyroidism, (2) mild hypothyroidism, and (3) decreased thyroid reserve or "prehypothyroidism." Groups 1 and 2 showed decreased myocardial contractility with a prolonged preejection period (PEP), shortened left ventricular ejection time (LVET), and increase PEP/LVET, compared with normal controls. The STI were more abnormal (P less than .05) in group 1 than in group 2, suggesting that the severity of myocardial dysfunction correlates with the severity of the hypothyroidism. Group 3 had normal STI. Ten patients were restudied when euthyroid and showed complete normalization of their STI, supporting the thesis that hypothyroidism was the sole cause of the initial myocardial dysfunction.

Characterization of normal and abnormal pulmonary surface by reflected ultrasound.

The use of reflected ultrasound in the diagnosis of pulmonary disease has been limited due to the strong reflection and high absorption of acoustic energy by alveoli containing air. Our study reports the ultrasonic characteristics of normal and diseased pulmonary surface. Two measurements, the coefficient of pulmonary reflection and the tissue frequency signature, were studied with specially calibrated instuments. The coefficient of pulmonary reflection in 23 normal subjects was - 19.6 +/- 0.1 dB at 5.5 MHz. In ten patients with documented pulmonary emboli, the coefficient of pulmonary reflection was -47.4 +/- 0.1 dB, significantly less than normal (P less than 0.1). The tissue frequency signature in 18 normal subjects was specular at higher and nonspecular at lower frequencies, with a typical dip at 5.2 MHz. In patients with chronic obstructive pulmonary disease, the tissue frequency signature was flat, and the typical dip was absent. In patients with pulmonary emboli, the characteristic shape of the tissue frequency signature was preserved, but there was a generalized loss of magnitude of the reflection. Thus, our preliminary data indicate that reflected ultrasound provides a noninvasive method for differentiating normal from diseased pulmonary surfaces.

Interobserver variability in echocardiography.

High-quality echocardiograms were performed on 146 normal individuals whose ages ranged from 3 to 73 years (mean 27 years). Normal values for mitral diastolic excursion and E-F slope, the chamber dimensions of the right ventricle, left atrium, and left ventricle, the aortic root dimension, and thickness of the interventricular septum and left ventricular posterior wall were determined. Each tracing was then read independently by two experienced echocardiographers. The extent of interobserver variability was calculated and expressed as a percent of the mean. The 95 per cent confidence limits for these estimates were calculated. Small but significant interobserver variability was found for all nine of these commonly measured echocardiographic parameters. Observer variability is a small but potentially important consideration in investigative echocardiography.

Echocardiographic spectrum of posterior systolic motion of the mitral valve in the general population.

The prevalence of mitral valve prolapse has been established in selected groups of patients but not in the general population. The present study was designed to define the echocardiographic spectrum of mitral valve motion in a population of young individuals without clinical evidence of significant cardiac disease or hypertension. Echocardiograms were performed on 136 normal volunteers. Six subjects (4.4 per cent) had mitral valve prolapse. Eighteen subjects (13.2 per cent) had a lesser degree of posterior systolic motion of the mitral valve leaflets which was suggestive but not diagnostic of prolapse. Minor degrees of posterior systolic mitral valve motion may represent a variant of normal. Caution should be exercised in making the echocardiographic diagnosis of mitral prolapse until this question is settled.

The value and limitations of echocardiography in recording mitral valve vegetations.

The echocardiographic findings and case reports of three patients with active Streptococcal endocarditis and severe congestive heart failure are presented. All three had pathologically proven vegetations on the mitral valve; however, only the two with calcification of the vegetations were successfully demonstrated on echocardiography. Clinical and pathological differences are highlighted and prior case reports in the literature are reviewed. The nonspecific nature of echoes recorded from valvular vegetation is stressed and factors in their echocardiographic detection are discussed.

Detection of left atrial myxoma by gated radionuclide cardiac imaging.

Gated radionuclide cardiac blood pool scans (GCS) of end-systole and end-diastole or eight images subtending the entire cardiac cycle were performed on seven patients with left atrial myxomas documented by pulmonary cineangiography with left atrial follow-through. The ethocardiogram was either suggestive or diagnostic in all patients. In addition to demonstration of the tumor (6 patients), the GCS detected three patterns of tumor motion: 1) a defect which moved from the left atrium in end systole to the left ventricle in end diastole (2 patients); 2) a defect which remained within the region of the left atrium but decreased in size between end diastole and end systole (3); and 3) a defect which was observed within the region of the left ventricle in end diastole but disappeared in end systole (1). Thus, the GCS is a noninvasive method for detection and evaluation of motion of left atrial myxomas.

Noninvasive evaluation of cardiac function in hypothyroidism. Response to gradual thyroxine replacement.

Left ventricular performance was studied in 15 patients with severe, primary hypothyroidism (mean serum total thyroxine of 0.8 mug per 100 ml and serum thyrotropin of 160 muU per milliliter). Pretreatment systolic-time intervals were characterized by prolongation of the pre-ejection period (delta PEP = +30) and reduction of the left ventricular ejection period (delta LVET = -23) with a resultant increase in the PEP/LVET ratio (0.47). Nine of 14 patients demonstrated pericardial effusions. These abnormalities were reversed with physiologic thyroxine replacement. Further reductions of the delta PEP and PEP/LVET ratio occurred with supraphysiologic doses (200 to 300 mug per day). During therapy, delta PEP was inversely correlated with serum thyroxine (P less than 0.001) and directly correlated with serum thyrotropin (P less than 0.001). Thus physiologic thyroid hormone replacement, appropriately adjusted to need, appears necessary in hypothyroidism for optimal left ventricular function.

Hypertrophic cardiomyopathy. Evaluation by gated cardiac blood pool scanning.

The gated radionuclide cardiac blood pool scan (GCS) can be used to visualize the entire profile of the interventricular septum and left ventricular contraction. Twenty-two patients with hypertrophic cardiomyopathy, nine with valvular aortic stenosis and six normals, underwent echocardiography and GCS. All patients with hypertrophic cardiomyopathy had asymmetric septal hypertrophy and 14 of 22 had resting systolic anterior motion of the anterior leaflet of the mitral valve on echocardiogram. In eight patients with aortic stenosis with adequate echocardiograms, two had asymmetric septal hypertrophy and none had systolic anterior motion. The GCS demonstrated disproportionate upper septal thickening in 11; septal flattening in 16; cavity obliteration in 17; and a filling defect in the region of the left ventricular outflow tract in 16 of the 22 patients with hypertrophic cardiomyopathy. In the nine patients with valvular aortic stenosis, two demonstrated septal flattening, two cavity obliteration, two an outflow tract defect, and none disproportionate upper septal thickening. Both patients with cavity obliteration demonstrated asymmetric septal hypertrophy on echocardiogram. One normal control patient had septal flattening. Thus the gated cardiac blood pool scan provides an atraumatic technique for the evaluation of patients with hypertrophic cardiomyopathy which complements the echocardiogram.