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The Chlamydiaceae are Gram-negative bacteria causing diseases in humans and in both, endothermic (mammals and birds) and poikilothermic (e.g. reptiles, amphibians) animals. As most chlamydial species described today were isolated from humans and endothermic animals, the commonly used culturing temperature in vitro is 37 °C, although the centrifugation temperature during experimental infection, a technique necessary to improve the infection rate, may vary from 25 to 37 °C. The aim of this study was to investigate the influence of different centrifugation (28° or 33 °C) and incubation temperatures (28 °C or 37 °C) on the average inclusion size, infectivity and ultrastructural morphology of human and animal chlamydial strains, as well as two recently described species originating from snakes, C. poikilothermis and C. serpentis, in LLC-MK2 cells at 48 h post infection. Infectivity and average inclusion size was reduced at an incubation temperature of 28 °C compared to 37 °C for all strains including C. poikilothermis, although the latter formed larger, fully matured inclusions at 28 °C in comparison to the other investigated Chlamydia species. C.psittaci displayed a shorter developmental cycle than the other species confirming previous studies. Higher centrifugation temperature increased the subsequent inclusion size of C. trachomatis, C. abortus and C. suis but not their infectivity, while the incubation temperature had no discernable effect on the morphology, inclusion size and infectivity of the other chlamydial strains. In conclusion, we found that all Chlamydia species are viable and can grow at low incubation temperatures, although all strains grew better and more rapidly at 37 °C compared to 28 °C.

Until recently, Chlamydia psittaci was considered to be the only etiological agent of avian chlamydiosis, but two new avian species, Chlamydia gallinacea and Chlamydia avium, have recently been described in poultry and pigeons or psittacine birds, respectively. The aim of this study was to explore the occurrence of C. psittaci and C. gallinacea in backyard chickens in Italy. Cloacal swabs were taken from 160 asymptomatic chickens reared in 16 backyard farms. Samples were tested for C. psittaci and C. gallinacea by specific real-time polymerase chain reaction assays, with 24 (15%) of the 160 chickens resulting positive for C. gallinacea. To attempt chlamydial isolation, new samples were obtained from two farms harboring a high prevalence (60% and 70%, respectively) of C. gallinacea-positive chickens. In total, eight C. gallinacea and one C. psittaci isolates were successfully recovered from 13 chickens. C. gallinacea was confirmed to be the endemic chlamydial species in chickens, with a high ompA intraspecies diversity. The presence of viable C. psittaci and C. gallinacea demonstrated by isolation from chickens in backyard farms poses a potential public health problem.

Background Simkania negevensis is an obligate intracellular bacterium belonging to the family Simkaniaceae in the Chlamydiales order. It is considered an ubiquitous microorganism and aquatic environments may be involved as a source of infection for humans. It was just isolated in samples from domestic water supplies and from mains water supplies, like spa water or swimming pool water, confirming its ability to resist to the common chlorination treatments. Evidence indicates a possible role of the microorganism in respiratory tract infections, in gastroenteric disorders and in the pathogenesis of cardiovascular disease, furthermore it has hypothesized that it could play a role in lung transplant rejection. Prevalence and possible effects in nephrology are unknown. Methods We examined the occurrence of Simkania negevensis in two differents populations, both characterized by a high susceptibility to infectious complications: 105 hemodialysis patients, 105 renal transplant recipients and 105 healthy subjects through the IgG and IgA response to Simkania negevensis in their sera. Serum antibodies to Simkania negevensis were detected by a homemade ELISA performed according to the Kahane’s protocol. Furthermore water samples from hemodialytic circuit were collected, to evaluate Simkania negevensis resistance to usual treatment of disinfection. Results Our results were unexpected, showing a higher seroprevalence of antibodies against Simkania negevensis in the hemodialysis patients, compared to renal transplant patients (IgG 22% vs 9% - IgA 9% vs 3%). S. negevensis was isolated in all water samples analyzed. Conclusions Our study detected for the first time the occurrence of S. negevensis in hemodialysis and in renal transplant patients. Our findings suggest that water used in hemodialysis could be one of the possible sources of S. negevensis infection, without clinical involvement risk for patients.

Background: Simkania negevensis is an obligate intracellular Gram-negative bacterium (family Simkaniaceae, order Chlamydiales) that has been isolated from domestic and mains water supplies, is able to infect human macrophages, and can induce an inflammatory response in the host. Methods: From June to December 2016, in a single-center observational study, colonic Crohn’s disease patients and controls (subjects undergoing screening for colorectal cancer) underwent blood tests to identify serum-specific immunoglobulinG (IgG) and immunoglobulinA (IgA) to S.negevensis and a colonoscopy with biopsies for detection of S.negevensis DNA by polymerase chain reaction (PCR). Results: Forty-three Crohn’s disease patients and 18 controls were enrolled. Crohn’s disease patients had higher prevalence of IgA antibodies to S.negevensis compared with controls (20.9% versus 0%, p = 0.04). Simkaniaceae negevensis DNA was detected in 34.9% and 5.6% of intestinal biopsies in Crohn’s disease patients and controls, respectively (p = 0.02). All Crohn’s disease patients with PCR-positive biopsies for S.negevensis were IgG seropositive, with specific IgA in 60% of them (p < 0.001). Immunosuppressive therapies, extraintestinal manifestations, or disease activity did not influence the presence of S.negevensis in the Crohn’s disease population. Conclusions: We identified S.negevensis in Crohn’s disease patients by demonstrating the presence of S.negevensis mucosal DNA and seropositivity to the bacterium. These results could support the presence of an acute or persistent S.negevensis infection and suggest a possible role in the pathogenesis of Crohn’s disease.

Chlamydia species have recently been recognized as emerging pathogens in snakes. However, isolation of novel snake chlamydiae is critical and their growth characteristics are largely unknown. In this study, two novel chlamydial species are described: Chlamydia serpentis and Chlamydia poikilothermis, isolated after attempts on 23 cloacal and choanal swabs from 18 PCR-positive captive snakes originating from different Swiss snake collections. Isolation success, growth curve and infectivity rates over a 48-hour time period were dependent on temperature (37 °C for C. serpentis, 28 °C for C. poikilothermis). C. serpentis and C. poikilothermis were sensitive to tetracycline and moxifloxacin during evaluation by in vitro antibiotic susceptibility assay but intermediate to resistant (2–4 μg/ml) to azithromycin. Whole genome sequencing of the isolates provided proof of the novel species status, and gives insights into the evolution of these branches of genus Chlamydia.

Castanea sativa Mill (ENC®), containing tannins against 33 Chlamydia strains, was compared to SMAP-29 with inhibitory effect against C. trachomatis and C. pneumoniae. The ENC® activity against Chlamydia spp. was evaluated determining the lowest concentration to achieve more than half reduction of intact chlamydial inclusions versus controls. ENC® reduced all Chlamydia strains tested at 1 µg/mL, while SMAP-29 induced reductions of C. trachomatis and C. pneumoniae infectivity at 10 µg/mL. A great reduction of C. trachomatis, C. pneumoniae, and C. abortus infectivity was achieved with a 10 µg/mL ENC® concentration, whereas their infectivity was almost inhibited at 100 µg/mL ENC® concentration.

OBJECTIVE: Chlamydia pneumoniae has been linked to atherosclerosis, strictly associated with hyperlipidemia. The liver plays a central role in the regulation of lipid metabolism. Since in animal models C. pneumoniae can be found at hepatic level, this study aims to elucidate whether C. pneumoniae infection accelerates atherosclerosis by affecting lipid metabolism. METHODS: Thirty Balb/c mice were challenged intra-peritoneally with C. pneumoniae elementary bodies and thirty with Chlamydia trachomatis, serovar D. Thirty mice were injected with sucrose-phosphate-glutamate buffer, as negative controls. Seven days after infection, liver samples were examined both for presence of chlamydia and expression of genes involved in inflammation and lipid metabolism. RESULTS: C. pneumoniae was isolated from 26 liver homogenates, whereas C. trachomatis was never re-cultivated (P < 0.001). C. pneumoniae infected mice showed significantly increased serum cholesterol and triglycerides levels compared both with negative controls (P < 0.001 and P = 0.0197, respectively) and C. trachomatis infected mice (P < 0.001). Liver bile acids were significantly reduced in C. pneumoniae compared to controls and C. trachomatis infected mice. In C. pneumoniae infected livers, cholesterol 7α-hydroxylase (Cyp7a1) and low-density lipoprotein receptor (Ldlr) mRNA levels were reduced, while inducible degrader of the low-density lipoprotein receptor (Idol) expression was increased. Hypertriglyceridemia was associated to reduced expression of hepatic carnitine palmitoyltransferase-1a (Cpt1a) and medium chain acyl-Coenzyme A dehydrogenase (Acadm). Pro-inflammatory cytokines gene expression was increased compared to negative controls. Conversely, in C. trachomatis infected animals, normal serum lipid levels were associated with elevated pro-inflammatory cytokines gene expression, linked to only a mild disturbance of lipid regulatory genes. CONCLUSION: Our results indicate that C. pneumoniae mouse liver infection induces dyslipidemic effects with significant modifications of genes involved in lipid metabolism.

A mouse model for Chlamydia suis genital infection was developed. Ninety-nine mice were randomly divided into three groups and intravaginally inoculated with chlamydia: 45 mice (group 1) received C. suis purified elementary bodies (EBs), 27 (group 2) were inoculated with C. trachomatis genotype E EBs and 27 mice (group 3) with C. trachomatis genotype F EBs. Additionally, 10 mice were used as a negative control. At seven days post-infection (dpi) secretory anti-C. suis IgA were recovered from vaginal swabs of all C. suis inoculated mice. Chlamydia suis was isolated from 93, 84, 71 and 33% vaginal swabs at 3, 5, 7 and 12 dpi. Chlamydia trachomatis genotype E and F were isolated from 100% vaginal swabs up to 7 dpi and from 61 and 72%, respectively, at 12 dpi. Viable C. suis and C. trachomatis organisms were isolated from uterus and tubes up to 16 and 28 dpi, respectively. The results of the present study show the susceptibility of mice to intravaginal inoculation with C. suis. A more rapid course and resolution of C. suis infection, in comparison to C. trachomatis, was highlighted. The mouse model could be useful for comparative investigations involving C. suis and C. trachomatis species.

Chlamydia pneumoniae and human cytomegalovirus (HCMV) are intracellular pathogens able to infect hepatocytes, causing an increase in serum triglycerides and cholesterol levels due to the production of inflammatory cytokines. We investigated whether these pathogens could interfere with cholesterol metabolism by affecting activity of hepatic cholesterol 7α-hydroxylase ( CYP7A1 ) promoter. CYP7A1 is the rate-limiting enzyme responsible for conversion of cholesterol to bile acids, which represents the main route of cholesterol catabolism. A straightforward dual-reporter bioluminescent assay was developed to simultaneously monitor CYP7A1 transcriptional regulation and cell viability in infected human hepatoblastoma HepG2 cells. C. pneumoniae and HCMV infection significantly decreased CYP7A1 promoter activity in a dose-dependent manner, with maximal inhibitions of 33 ± 10% and 32 ± 4%, respectively, at a multiplicity of infection of 1. To support in vitro experiments, serum cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides and glucose levels were also measured in Balb/c mice infected with C. pneumoniae. Serum cholesterol and triglycerides also increased in infected mice compared with controls. Although further investigation is required, this work presents the first experimental evidence that C. pneumoniae and HCMV inhibit CYP7A1 gene transcription in the cultured human hepatoblastoma cell line.

The aims of the present study were to assess the prevalence of Chlamydia suis in an Italian pig herd, determine the tetracycline susceptibility of C. suis isolates, and evaluate tet(C) and tetR(C) gene expression. Conjunctival swabs from 20 pigs were tested for C. suis by real-time polymerase chain reaction, and 55% (11) were positive. C. suis was then isolated from 11 conjunctival swabs resampled from the same herd. All positive samples and isolates were positive for the tet(C) resistance gene. The in vitro susceptibility to tetracycline of the C. suis isolates showed MIC values ranging from 0.5 to 4 μg/mL. Tet(C) and tetR(C) transcripts were found in all the isolates, cultured both in the absence and presence of tetracycline. This contrasts with other Gram-negative bacteria in which both genes are repressed in the absence of the drug. Further investigation into tet gene regulation in C. suis is needed.

It is known that neutralizing species-specific or serovar-specific antibodies are produced in response to chlamydial infection in humans and in some animal species. In a previous study, a strong in vitro neutralizing activity to Chlamydia suis in 80% of sera from C. suis-infected pigs had been observed. In view of the close relationship between C. suis and Chlamydia trachomatis, in the present study, the neutralizing activity against D-K C. trachomatis and C. suis purified elementary bodies (EBs) in sera collected from C. trachomatis-infected patients and C. suis-infected pigs was evaluated. A neutralizing activity of 50-70% was observed in the human sera against the homologous serovar and one to five heterologous C. trachomatis serovars. These sera were also able to neutralize C. suis EBs. The pig sera showed a strong neutralizing activity (70-100%) against C. suis EBs and all eight urogenital C. trachomatis serovars. These results suggested the presence of common immunogenic antigens in C. trachomatis and C. suis. Immunoblot analysis, performed to elucidate the target of this neutralizing activity, showed a clear reactivity in human and pig sera against two proteins of 150 and 40 kDa MW, when tested either with C. trachomatis or with C. suis EBs.

Despite several decades of intensive studies, no vaccines againstChlamydia trachomatis, an intracellular pathogen causing serious ocular and urogenital diseases, are available yet. Infection-induced immunity in both animal models and humans strongly supports the notion that for a vaccine to be effective a strong CD4+Th1 immune response should be induced. In the course of our vaccine screening program based on the selection of chlamydial proteins eliciting cell-mediated immunity, we have found that CT043, a protein annotated as hypothetical, induces CD4+Th1 cells both in chlamydia-infected mice and in human patients with diagnosedC. trachomatisgenital infection. DNA priming/protein boost immunization with CT043 results in a 2.6-log inclusion-forming unit reduction in the murine lung infection model. Sequence analysis of CT043 fromC. trachomatishuman isolates belonging to the most representative genital serovars revealed a high degree of conservation, suggesting that this antigen could provide cross-serotype protection. Therefore, CT043 is a promising vaccine candidate againstC. trachomatisinfection.

BACKGROUND: An increasing number of studies suggest that chlamydiae can infect immune cells. The altered immune cell function could contribute to the progression of several chronic inflammatory diseases.The aim of this study was to comparatively evaluate Chlamydia pneumoniae (CP) and Chlamydia trachomatis (CT) interactions with in vitro infected human blood monocytes. RESULTS: Fresh isolated monocytes were infected with viable CP and CT elementary bodies and infectivity was evaluated by recultivating disrupted monocytes in permissive epithelial cells.The production of reactive oxygen and nitrogen species was studied in the presence of specific fluorescent probes. Moreover, TNF-α, INF-α, INF-β and INF-γ gene expression was determined. CT clearance from monocytes was complete at any time points after infection, while CP was able to survive up to 48 hours after infection. When NADPH oxydase or nitric oxide synthase inhibitors were used, CT infectivity in monocytes was restored, even if at low level, and CT recovery's rate was comparable to CP one.CT-infected monocytes produced significantly higher levels of reactive species compared with CP-infected monocytes, at very early time points after infection. In the same meanwhile, TNF-α and INF-γ gene expression was significantly increased in CT-infected monocytes. CONCLUSIONS: Our data confirm that CP, but not CT, is able to survive in infected monocytes up to 48 hours post-infection. The delay in reactive species and cytokines production by CP-infected monocytes seems to be crucial for CP survival.

AVIAN chlamydiosis is primarily caused by the intracellular bacterium Chlamydia psittaci , belonging to the Chlamydiaceae family. Depending on the species and age of the bird and the virulence of the infectious bacterial strain, avian chlamydiosis can be subclinical or characterised by respiratory, digestive, or systemic disorders (Knittler and others 2014). Seven C. psittaci outer-membrane protein A ( omp A) genotypes (A-F and E/B), have been initially detected in birds. All these genotypes can be transmitted to humans by contact with contaminated faeces or feathers or by inhalation of an infectious aerosol, causing a mild flu-like illness or severe atypical pneumonia. Recently, six additional C. psittaci omp A genotypes, all occurring in wildlife birds, have been proposed (Sachse and others 2008). Recent studies suggested that more chlamydial agents, beyond C. psittaci, can be involved in avian chlamydiosis. In this respect, Chlamydia abortus , Chlamydia pecorum, Chlamydia trachomatis and Chlamydia pneumoniae have been detected in birds (Pantchev and others 2009, Sachse and others 2012, Frutos and others 2015). Recently, two new bacterial species belonging to the Chlamydiaceae family have been described: Chlamydia avium from pigeons and psittacine birds and Chlamydia gallinacea from poultry (Sachse and others 2014). In addition, a novel candidate species, named Chlamydia ibidis, …

We investigated the Chlamydia spp. occurrence in Eurasian Collared Doves (Streptopelia decaocto) from urban and suburban areas in northern Italy. Among 76 doves screened, prevalence of Chlamydia spp. was 61%. Chlamydia psittaci genotype E was identified in 33 of the 46 positive samples. The multilocus sequence typing pattern of one highly positive sample showed a new allelic combination. The same molecular features were observed in a C. psittaci strain subsequently isolated from a live dove. Our results reveal a high C. psittaci prevalence in S. decaocto. The spread of this zoonotic pathogen from collared doves to other birds or humans seems to be a potential risk.

Chlamydia are intracellular bacteria associated to serious human disease. A vaccine has proved difficult to obtain so far, and current opinions agree that multi-antigen combinations may be required to induce optimal protective responses. In order to identify new potential vaccine candidates, we recently screened the Chlamydia pneumoniae (Cpn) genome and described 53 recombinant proteins which elicited antibodies binding to purified Cpn cells. We now report that six proteins in this group can also induce in vitro neutralizing antibodies. Antibody specificity for the corresponding antigens was assessed by immunoblot analysis of 2DE Cpn protein maps. Furthermore, four of the six in vitro neutralizing antigens (Pmp2, Pmp10, OmpH-like and enolase) could inhibit Cpn dissemination in a hamster model. The results show that these Cpn proteins are immunoaccessible in infectious EBs, and recommend further investigation on their value as vaccine components.

Chlamydia trachomatis pgp3 DNA immunized (no. 300) and non-immunized (no. 300) C3H/HeN mice were infected by vaginal inoculation with infectious C. trachomatis serotype D elementary bodies (EBs) and the spread of infection to the salpinges was assessed by cell culture isolation from tissue homogenates 7, 14, 21, 28, 35 and 42 days post-infection (p.i.). Overall, the pgp3-DNA immunization prevented salpinx infection in 94 (56%) mice, if compared with the 168 positive animals found among the non-immunized animals (P < 0.001). A group of negative control animals (i.e. mice immunized with plasmid DNA containing an irrelevant insert) was not protected, whereas all the mice of a positive immune control group (mice that had resolved a primary genital C. trachomatis infection) were resistant to re-infection. Pgp3 DNA immunization induced both humoral and mucosal anti-pgp3 antibodies.

Chlamydia suis is a natural pathogen of pigs ( Sus scrofa ) and causes conjunctivitis, pneumonia, enteritis, and various reproductive disorders that adversely impact this economically important animal. Here, we report the first C. suis genome, that of C. suis MD56, isolated from a conjunctival swab of a weaned piglet.

In the present study we evaluated the relation among histology, H. pylori, IgG to H. pylori, gastric emptying, and acid secretion in 43 patients with fundic atrophic gastritis. On the basis of gastric acid secretion, patients were divided into three subgroups: patients with preserved acid secretion (Group 1), patients with hypochlorhydria (Group 2), and patients with achlorhydria (Group 3). Fundic glandular atrophy was more severe in hypoachlorhydric patients than in those with preserved acid secretion (P < 0.05 vs Group 2, P < 0.005 vs Group 3). H. pylori colonization was found in 94% of patients in Group 1, in 61% of patients in Group 2, and in only 8% of patients in Group 3 (P < 0.001 vs Group 1, P < 0.05 vs Group 2). Conversely, serological positivity to H. pylori was high in all three subgroups of patients (100% in Group 1, 77% in Group 2, 92% in Group 3). Gastric emptying was delayed in atrophic patients, particularly in those with hypoachlorhydria. Our data suggest that fundic atrophic gastritis represents a possible end stage of H. pylori infection, characterized by a progressive disappearance of the bacterium and a progressive deterioration of gastric functions.

Thirty patients with dyspepsia, with histological diagnosis of gastritis, and with endoscopic diagnosis of peptic ulcer disease (PUD) (n 5 13) or nonulcer dyspepsia (NUD) (n 5 17) were admitted to the study. Helicobacter pylori vacuolating cytotoxin-producing strains (Tox 1 ) were isolated from 14 (46.7%) patients, whereas non-cytotoxin-producing (Tox 2 ) H. pylori strains were isolated from the remaining patients. Of 30 patients studied, 20 (66.7%) had serum cytotoxin neutralizing activity in vitro. Fourteen patients with Tox 1 H. pylori strains showed serum cytotoxin neutralizing activity and serum immunoglobulin G (IgG) and IgA antibodies reactive with both 87-kDa H. pylori vacuolating cytotoxin (VacA) and 128-kDa cytotoxin-associated gene product (CagA) by immunoblotting using native enriched preparations of VacA and CagA proteins from H. pylori culture supernatants as the antigens. A 94-kDa antigen cross-reacting with the 87-kDa VacA protein could be demonstrated in culture supernatant with immune sera from humans and animals. All patients (n 5 10) lacking serum neutralizing activity were also negative for IgG or IgA against VacA antigen, whereas 6 of the 10 patients showed IgG serum antibody responses against CagA antigen. The prevalence of antibodies to VacA and CagA antigens was significantly (P < 0.001) higher in patients with gastritis (20 and 26 patients for VacA and CagA, respectively, of 30 patients) than in H. pylori culture-negative controls (0 of 27 for both VacA and CagA) and in randomly selected blood donors (17 and 21 for VacA and CagA, respectively, of 120 subjects). All patients with PUD had antibodies to CagA, whereas 13 of 17 (76.5%) patients with NUD had anti-CagA antibodies. Serum IgG antibodies to VacA were present in 9 (69.2%) patients with PUD of 13 patients and in 11 (64.7%) patients with NUD of 17 patients. Anti-CagA antibodies seemed to correlate better with PUD than anti-VacA antibodies. Helicobacter pylori has been established as a major cause of chronic active gastritis and peptic ulcer disease (PUD), also associated with gastric cancer (2, 26). However, the clinical diagnosis of Helicobacter infection may be difficult due to the absence of either specific symptoms or specific endoscopic findings. In addition, the results of cultures, histological tests, and urease test on biopsies may be highly variable depending on the size and number of the specimens obtained. Therefore, immunological tests may be critical for the reliable diagnosis of H. pylori infections. Several serological studies using enzyme-linked immunosorbent assay (ELISA) or Western immunoblotting (WB) have been performed to detect antibodies against H. pylori. Most of the available data have been obtained by ELISA for serum immunoglobulins against H. pylori (17, 22, 24), where whole bacterial cells have been used as the antigen. However, the presence of serological cross-reactions between H. pylori and other gram-negative bacteria such as Pseudomonas aeruginosa, Campylobacter jejuni, and Haemophilus influenzae (3, 19) has called into question the specificity of the results obtained by ELISAs using the whole bacterial cell as the antigen. More recently, recombinant CagA has been used as an antigen by ELISA for specific serological diagnosis of H. pylori infection

Chlamydiae are obligate intracellular bacteria that cause serious diseases in a wide range of hosts. Chlamydia trachomatis is one of the leading sexually transmitted pathogens in the world. Because vaccines are not currently available, effective drugs are essential. In both animals and humans, chlamydial infections are often treated with tetracycline or its derivatives. A stable tetracycline-resistant phenotype was described in Chlamydia suis strains from pigs in the USA and in Europe. In humans, there are reports of tetracycline treatment failure and the in vitro adaptability of C. trachomatis to evolve to antibiotic resistance has been described, suggesting the pressing need to search for alternative and effective classes of antimicrobial drugs. Host defense peptides (HDPs) are known as direct antimicrobial agents as well as innate immune modulators. Being active against multidrug-resistant bacteria, HDPs are attractive candidates as templates for new drugs. A number of studies evaluated the activity of natural and synthetic HDPs against Chlamydia spp., showing C. trachomatis to be the most sensitive among chlamydia species tested. Protegrins and α-helical peptides were the most active among the HDPs assessed.

Analysis by two-dimensional gel electrophoresis of the N-laurylsarkosinate(Sarkosyl)-insoluble envelope complexes of L-[35]S-cysteine-labeled elementary bodies of Chlamydia pneumoniae strain IOL-207, Chlamydia trachomatis serovar LGV2, D, and F, and Chlamydia psittaci strain 6BC showed differences in the molecular charges of chlamydial outer membrane proteins. The apparent isoelectric point (pI) of the major outer membrane protein of C. pneumoniae strain IOL-207 was 6.4, whereas the pI of the major outer membrane protein of the C. trachomatis and C. psittaci strains differed little from one another, ranging from 5.3 to 5.5. The 60-kDa cysteine-rich protein of C. pneumoniae was the only 60-kDa chlamydial protein with a pI value (5.9) more acidic than that of the corresponding major outer membrane protein. As a general rule, the charges of both the 60-kDa and the low-molecular-mass (12-15 kDa) cysteine-rich proteins were widely variable, depending on the strain. However, in each individual strain, the variation of the charge of the 60-kDa protein had a compensatory change in the low-molecular-mass cysteine-rich protein.

In pigs, Chlamydia suis has been associated with respiratory disease, diarrhea and conjunctivitis, but there is a high rate of inapparent C. suis infection found in the gastrointestinal tract of pigs. Tetracycline resistance in C. suis has been described in the USA, Italy, Switzerland, Belgium, Cyprus and Israel. Tetracyclines are commonly used in pig production due to their broad-spectrum activity and relatively low cost. The aim of this study was to isolate clinical C. suis samples in cell culture and to evaluate their antibiotic susceptibility in vitro under consideration of antibiotic treatment on herd level. Swab samples (n = 158) identified as C. suis originating from 24 farms were further processed for isolation, which was successful in 71% of attempts with a significantly higher success rate from fecal swabs compared to conjunctival swabs. The farms were divided into three treatment groups: A) farms without antibiotic treatment, B) farms with prophylactic oral antibiotic treatment of the whole herd consisting of trimethoprime, sulfadimidin and sulfathiazole (TSS), or C) farms giving herd treatment with chlortetracycline with or without tylosin and sulfadimidin (CTS). 59 isolates and their corresponding clinical samples were selected and tested for the presence or absence of the tetracycline resistance class C gene [tet(C)] by conventional PCR and isolates were further investigated for their antibiotic susceptibility in vitro. The phenotype of the investigated isolates was either classified as tetracycline sensitive (Minimum inhibitory concentration [MIC] < 2 μg/ml), intermediate (2 μg/ml ≤ MIC < 4 μg/ml) or resistant (MIC ≥ 4 μg/ml). Results of groups and individual pigs were correlated with antibiotic treatment and time of sampling (beginning/end of the fattening period). We found clear evidence for selective pressure as absence of antibiotics led to isolation of only tetracycline sensitive or intermediate strains whereas tetracycline treatment resulted in a greater number of tetracycline resistant isolates.

The aim of this study was to investigate Chlamydia suis in a pig farm with an outbreak of conjunctivitis and diarrhea. Eye swabs and pooled fecal samples were investigated for the presence of C. suis by real-time PCR and ArrayTube microarray. Samples positive for C. suis by ArrayTube microarray assay were further tested for the presence of the tet(C) resistance gene by PCR. In the first examination, C. suis was identified in 12 six-week-old pigs showing conjunctivitis. Of these, the tet(C) gene-coding region was amplified in one pooled fecal sample and one eye swab, respectively. After oral treatment with tetracycline, clinical symptoms disappeared. Subsequently, all eye swabs investigated from 10 healthy pigs were positive for C. suis and the tet(C) gene-coding region. The present study reports rapid selection for tetracycline-resistant C. suis after antibiotic treatment.

A molecular biological approach, based on preproregion homology in the precursors of several diverse antibacterial peptides, was used to clone a pig bone marrow cDNA encoding a novel 167-residue polypeptide. The preproregion of this polypeptide is highly similar to corresponding regions in congeners from pig, cattle and rabbit. It is followed by a unique, cationic, 37-residue sequence, which was predicted to have a high propensity for an α-helical conformation. A peptide, termed PMAP-37, corresponding to this sequence, was chemically synthesized and shown to undergo a transition from a random coil to an ordered, mainly helical, conformation on addition of trifluoroethanol. This behaviour is typical of an amphipathic α helix, a structure common to several membrane-active, antimicrobial peptides. In vitro experiments showed that PMAP-37 strongly inhibits the growth of several strains of Gram-negative and Gram-positive bacteria, with minimal inhibitory concentrations ranging over 1–4 μM, and permeabilizes the inner membrane of Escherichia coll. Interestingly, the 15–32 stretch of PMAP-37 show a remarkable similarity to N-terminal stretches in cecropins B and A from Drosophila melanogaster and Cecropia hyalophora, respectively. This affords an uncommon example of sequence convergence.

A systematic evaluation of the susceptibility of all Chlamydia trachomatis urogenital serovars (D through K) to levofloxacin, erythromycin, doxycycline, clarithromycin, and azithromycin was performed. All C. trachomatis serovars had comparable susceptibilities with respect to the various antimicrobials tested, thus confirming the homogeneous data so far obtained regarding the susceptibility of C. trachomatis to antimicrobial agents.

Introduction. Chlamydophila pneumoniae (C. pneumoniae) is one of the most common respiratory pathogen, with an incidence of infection varying from 6% to 20%. The present study aimed to assess the incidence of C. pneumoniae infections in patients with acute respiratory diseases using a RealTime PCR (RT-PCR) method. Methods. In the period January 2007-December 2008 279 biological samples coming from patients (190 males and 89 females) with acute respiratory infections was collected and tested. Samples have been extracted using NucliSens easyMag Biomerieu according to manufacturer’s instructions and amplified by LightCycler Real-Time PCR Roche for the detection of C. pneumoniae DNA. Results. Data analysis revealed a higher prevalence of C. pneumoniae infections in male patients (7.9%) than in females (5.6%). In addition, it is interesting to note that the incidence of C. pneumoniae infection was higher 28.6% in the period February - April. Conclusions. The results obtained in this study confirm the utility of molecular techniques in laboratory diagnosis and epidemiological investigations of respiratory infection caused by C. pneumoniae. RT-PCR have proved to be a rapid and a reliable technique to monitor and treat opportunely C. pneumoniae infections to avoid short and medium/long term complications.

Cathelicidins are antimicrobial peptides, stored by mammalian leukocytes, showing an antimicrobial activity against bacteria, fungi, protozoa and enveloped viruses. In accordance with other authors, we reported in a previous study that the protegrin-1 (PG-1), at 80 microg mL(-1), inhibited the in vitro growth of Chlamydia trachomatis serovars D, H and L2; however, we observed an increased infectivity of some animal chlamydial species after their treatment with the same PG-1 concentration. In this study, the treatment of LLC-MK2 cells with PG-1 before chlamydial infection resulted in an increased infectivity of Chlamydophila abortus probably due to their easier entry into the host cells, whereas no increase in S26/3 infectivity was detected in LLC-MK2 cells treated with PG-1 postchlamydial infection.

In 2008, 50 samples (BAL), coming from hospital patients, with acute respiratory symptoms have been investigated using two real‐time PCR methods: one assay for the single detection of Chlamydophila pneumoniae and Mycoplasma pneumoniae DNA and one commercially available real‐time duplex PCR assay for the detection of C. pneumoniae and M. pneumoniae DNA. Both techniques used here showed compliant results, with 100% concordance for detection of C. pneumoniae and 98% for detection of M. pneumoniae. The positive results obtained agreed with the clinical suspicion of such infections in some cases and with the presence of IgM specific for C. pneumoniae and M. pneumoniae in all cases of acute infection. J. Clin. Lab. Anal. 23:206–209, 2009. © 2009 Wiley‐Liss, Inc.

Specific antibodies to plasmid-encoded protein pgp3 are known to be encountered in human Chlamydia (C.) trachomatis infections. In order to verify whether antibodies to this protein could be developed in animals infected with plasmid-carrying chlamydial strains, 454 animal sera were examined using a home-made pgp3 protein ELISA and Western blots (WB) of recombinant pgp3 protein from Chlamydophila (Cp.) psittaci. Likewise, 50 human sera were tested by ELISA and WB of recombinant pgp3 from C. trachomatis. The reactivity against pgp3 protein was compared to the reactivity against chlamydial elementary bodies (EBs) detected by microimmunofluorescence (MIF) test. The presence of pgp3-specific antibodies was demonstrated in most ducks and pigeons with Cp. psittaci infection detected by MIF, as well as in the majority of symptomatic cats and pigs infected with Cp. felis and C. suis, respectively, which reacted at high titres to Cp. felis and C. suis EBs by MIF. Moreover, most of the sera collected from patients with C. trachomatis culture-confirmed infection and seropositive to C. trachomatis by MIF, presented antibodies specific to C. trachomatis pgp3 recombinant protein. Therefore, pgp3 protein could be a useful marker of chlamydial infections in animals, as well as in humans.

The distribution of Chlamydia trachomatis serovars among 157 heterosexual male patients with urethritis and the presence of coinfections with other sexually transmitted infections were studied. One hundred seventeen (74.5%) patients, with a mean age of 33.7 years, were Italians, whereas 40 (25.5%) were immigrants coming from eastern European countries, Africa, and South America. All the immigrants and 82 (70.0%) Italian patients reported sex with prostitutes. Out of 157 patients, 73 (46.5%) were found positive for C. trachomatis in urethral secretions and eight different C. trachomatis serovars were identified. The most common serovars were E (n = 18; 24.7%), D (n = 15; 20.5%), G (n = 14;19.2%), and F (n = 12; 16.4%). The sequencing data showed a high degree of conservation of the omp1 gene. Thirty-six (46.7%) out of the 73 C. trachomatis-positive patients were coinfected with another sexually transmitted infection. The most common coinfection was gonorrhoea detected in 22 (30.1%) patients, followed by condyloma in eight (8.2%) patients, syphilis in five (6.8%), and HIV in three (4.1%).

Summary Background: [18F]-FDG is a widely used tracer for the non-invasive evaluation of hypermetabolic processes like cancer and inflammation. However, [18F]-FDG is considered inaccurate for the diagnosis of urinary tract and genital infections because of its urinary excretion. Since the 1970s, Gallium scintigraphy is a well established test that has been used for the evaluation of inflammation and infection in human patients. Aim: The aim of this study was to assess the feasibility of 68Ga-Chloride small animal PET for the analysis of an animal model of genital infection, induced after the vaginal inoculum of Chlamydia muridarum. Material and methods: Thirty mice were infected by placing 15 μl of sucrose phosphate glutamic acid (SPG) 107 inclusion forming units of C. muridarum into the vaginal vault. As controls of inflammation, three animals were challenged with 15 μl of SPG and one healthy animal was used to assess the tracer biodistribution. Four animals died during the experiment. Eleven animals were evaluated with 68Ga-Chloride small animal PET (GE, eXplore Vista) 3–5, 10–12, 17–19 days after infection, as well as three controls of inflammation and one healthy animal. Infection was monitored by obtaining cervical-vaginal swabs from all the animals on the day of each PET procedure. Moreover, five groups of three animals each were killed at 6, 13, 20, 27 and 34 days after infection were studied. Results: 68Ga-PET turned out positive in all the infected animals, concordantly to data obtained by the cervical swabs and by the ex vivo analysis. The tumour-to-background ratio (TBR) decreased over time as the inflammation tended to naturally extinguish. The controls showed a slightly increased uptake of tracer due to the aseptic inflammation caused by SPG and frequent cervical swabs. The healthy control did not show any pelvic uptake. Conclusion: 68Ga-Chloride is a promising tracer for the assessment of genital infection in a mouse animal model.

Feral pigeons (Columba livia domestica) which thrive in most European towns and cities are commonly infected with the zoonotic bacterium Chlamydophila psittaci, the agent of psittacosis (also known as ornithosis) in humans. A number of surveys carried out in the last thirty years across Europe allowed to detect high seropositivity values and high percentages of infection in feral pigeon populations. Overall, considering data from 11 European countries, seropositivity values to C. psittaci in the sampled populations ranged from 19.4 to 95.6 %. In most surveys, antibodies were detected with the complement fixation test in a percentage of samples from 19.4 to 66.3 %, with a median of 46.1 %. An indirect immunofluorescence test and an ELISA were employed less frequently, and allowed to detect higher percentages of seropositivity (23.7 – 67.7 % and 56 – 95.6 %, respectively). Attempts to grow C. psittaci in cell cultures or in embryonated chicken eggs were successful in 2 – 42.3 % and 0 – 57.1 % of samples respectively, antigen detection methods were positive in 2.3 – 40% of samples, while conventional PCR and real-time PCR with different genomic targets detected the organism in 3.4 – 50 % of samples. Twenty-five C. psittaci strains were typed as genotype B (n=14), E (n=10) and E/B (n=1). The huge increase of feral pigeon populations in Europe is a major cause of concern for the detrimental effect of pigeon droppings to environmental hygiene, in addition to the extensive damage due to the fouling of buildings and monuments. The most important pathogenic organism transmissible from feral pigeons to humans is C. psittaci, with 101 cases of disease reported in the literature. Exposure to C. psittaci-contaminated dust, direct contact with pigeons through handling and, to a lesser extent, through pigeon feeding have been identified as hazardous exposures in more than half cases of human disease, while loose or transient contacts with feral pigeons have been referred in about 40 % cases. Education initiatives as to the communication of a health risk deriving from contact with pigeons and pigeon excreta should primarily be targeted to workers that may be exposed to C. psittaci-contaminated dust, such as demolition/construction labourers. Recommendations to this category of workers include wearing protective clothes with hood, boots, gloves and air face filter masks when removing pigeon faeces from roofs, garrets and buildings, especially if working indoors. Monitoring for C. psittaci infections over time should also be considered in these workers. Children should be warned not to handle sick or dead pigeons and immunocompromised individuals should be educated to carefully limit their contact with feral pigeons. The culling of pigeons by shooting or poisoning is both unethical and useless as the place of the killed birds in the population is quickly filled by new juveniles or by birds immigrating from neighbouring areas. Pigeon-deterring systems such as nets and plastic or metal spikes applied to buildings and monuments will prevent their fouling, and the administration of contraceptive drugs may allow to resize the pigeon populations, but the measure that will ultimately lead to their permanent reduction and to establish healthy sustainable populations is the restriction of their indiscriminate feeding by pigeon lovers. The building of dovecotes and artificial breeding facilities should be considered for providing shelter and a balanced diet to the birds, as well as a chance of interaction for pigeon lovers in a hygienically-controlled environment.

A prospective surveillance study of legionnaires disease and an environmental survey of Legionella species were performed simultaneously in a general hospital. During a period of 3 years, 705 patients with pneumonia were screened with a Legionella urinary antigen test, and pneumonia was confirmed by culture and serological tests. Twelve cases of legionnaires disease were identified, none of which were hospital acquired, despite the fact that 60% of hospital water samples were contaminated with Legionella pneumophila at a concentration of more than 103 colony-forming units/L. The probable source of infection was identified for only 2 community-acquired cases. The results show that environmental contamination alone is not able to predict the risk of legionnaires disease. If no cases are present, monitoring of hospital water systems is of little significance; clinical surveillance is much more important.

The BfaI digestion of PCR-based restriction fragment length polymorphism analysis of the plasmid orf3 of Chlamydia trachomatis and Chlamydia psittaci provided evidence for two distinct restriction patterns, respectively. The nucleotide sequences of orf3 genes confirmed these differences. Serum antibodies against recombinant C. psittaci protein (pgp3) encoded by orf3 were detected both in pigeons with C. psittaci infection and in a human patient with psittacosis.

AIM: To develop an animal model of liver infection with Chlamydia pneumoniae (C. pneumoniae) in intraperitoneally infected mice for studying the presence of chlamydiae in Kupffer cells and hepatocytes. METHODS: A total of 80 BALB/c mice were inoculated intraperitoneally with C. pneumoniae and sacrificed at various time points after infection. Chlamydiae were looked for in liver homogenates as well as in Kupffer cells and hepatocytes separated by liver perfusion with collagenase. C. pneumoniae was detected by both isolation in LLC-MK2 cells and fluorescence in situ hybridization (FISH). The releasing of TNFA-alpha by C. pneumoniae in vitro stimulated Kupffer cells was studied by enzyme-linked immunosorbent assay. RESULTS: C. pneumoniae isolation from liver homogenates reached a plateau on d 7 after infection when 6 of 10 animals were positive, then decreased, and became negative by d 20. C. pneumoniae isolation from separated Kupffer cells reached a plateau on d 7 when 5 of 10 animals were positive, and became negative by d 20. The detection of C. pneumoniae in separated Kupffer cells by FISH, confirmed the results obtained by culture. Isolated hepatocytes were always negative. Stimulation of Kupffer cells by alive C. pneumoniae elicited high TNF-alpha levels. CONCLUSION: A productive infection by C. pneumoniae may take place in Kupffer cells and C. pneumoniae induces a local pro-inflammatory activity. C. pneumoniae is therefore, able to act as antigenic stimulus when localized in the liver. One could speculate that C. pneumoniae infection, involving cells of the innate immunity such as Kupffer cells, could also trigger pathological immune reactions involving the liver, as observed in human patients with primary biliary cirrhosis.

Nine Chlamydia suis isolates, obtained from pigs with conjunctivitis, were molecularly characterized by ompA sequencing and their in vitro susceptibility to six cathelicidin peptides (SMAP-29, BAC-7, BMAP-27, BMAP-27, BMAP-28, PG-1, LL-37) determined in cell culture. SMAP-29 was the most active peptide, reducing the intracellular inclusion number by ≥50% at a concentration of 10 μg/ml (3 μM) in six of the nine isolates tested. Three molecularly identical isolates were insensitive at a concentration as high as 80 μg/ml (25 μM). Of the remaining cathelicidin peptides tested, BAC-7 and BMAP-27 were active against six C. suis isolates at a concentration of 80 μg/ml (25 and 26 μM, respectively). Cathelicidins LL-37 and PG-1 did not show any anti-chlamydial activity at 80 μg/ml.

The aim of this study was to develop a new experimental model of Chlamydophila pneumoniae infection in the hamster. Intraperitoneal injection of C. pneumoniae purified elementary bodies (EBs) in the hamsters caused a systemic infection, since it was possible to isolate viable chlamydiae from several organs up to 14 days after infection. In particular, spleen infection was detectable up to 7 days post infection in 100% of animals. In contrast, cultures of the organs obtained from intranasally infected animals were far less frequently positive. Systemic infection probably occurred via macrophages, as demonstrated by the presence of intracellular chlamydial inclusions in peritoneal macrophages of peritoneally inoculated animals four days after infection. Furthermore, by infecting LLC-MK2 cells with supernatant preparations obtained from these macrophages, it was possible to observe the development of chlamydial intra-cytoplasmic inclusions after 96 h. Immunization of 18 hamsters with heat-inactivated purified EBs completely protected 16 animals and substantially reduced infection levels in the remaining two. Sera obtained from immunized hamsters prior to challenge reacted mainly against two C. pneumoniae proteins of about 60 kDa, when tested by immunoblot.

The TprI antigen of Treponema pallidum subsp. pallidum is a putative virulence factor predicted to be located in the outer membrane of the syphilis spirochete. In this study, we analyzed the immune response against TprI and its subunits in sera collected both from rabbits experimentally infected with the Nichols strain and from patients with syphilis, showing a different pattern of reactivity toward the antigen in these two groups of samples. The protective ability of recombinant TprI and its hypothetical outer membrane location were also investigated. Although no rabbit was protected after challenge, immunoelectron microscopy results, to be further investigated, were compatible with the outer membrane location of the antigen.

The in vitro activity of six cathelicidin peptides against 25 strains of Chlamydia was investigated. SMAP-29 proved to be the most active peptide, reducing the inclusion numbers of all 10 strains of Chlamydia trachomatis tested by ≥50% at 10 μg/ml. This peptide was also active against C. pneumoniae and C. felis.

Helicobacter pylori causes gastritis and peptic ulcer disease and has been associated with gastric malignancies. During recent years, non invasive diagnostic tests have gained in significance.Well-established non invasive tests are serology and the [13C] urea breath test. Serology, however, is not suitable for an early follow-up examination due to the slow reduction of the anti-H. pylori antibody titer after successful treatment. The [13C] urea breath test-delivering accurate results both in the pretreatment examination of infected individuals and in the early post treatment control-fulfills the demands for such a test. However, expensive instrumentation and a specialized technician are required. Since infected individuals excrete H. pylori in stool specimens, a sufficiently accurate test using feces would be an important alternative to [13C] urea breath test. In this study we compared the diagnostic performances of the immunocromatographic test “Helicobacter pylori Antigen Onestep Test” with those of two enzyme linked immunoassay tecniques “Fecal-clean H. pylori Ag” and “Amplified IDEIATM Hp StARTM” for detection of H. pylori antigens in stool specimens.The values of agreement ranged from 89.6% to 93.6%.

We report that intraperitoneal injection of Chlamydia pneumoniae purified elementary bodies (EBs) in the hamster causes a systemic infection allowing the isolation of viable chlamydiae from several organs for several days post-infection (p.i.). In particular, spleen infection occurred up to Day 7 p.i. in 100% of animals. Systemic infection probably occurs via macrophages as intraperitoneally injected chlamydiae which are taken up by the hamster macrophages remain viable and can infect in vitro cell cultures. Immunization of 18 hamsters with heat-inactivated purified EBs, completely protected the spleens of 16 animals and substantially reduced infection levels in the remaining two. This model, therefore, provides a robust screening tool for the assessment of the protective activity of potential vaccine candidates. In a pilot study on five recombinant antigens recently described as EB surface proteins, three gave results undistinguishable from non-immunized, or mock-immunized controls; however two antigens, derived, respectively, from the product of the lcrE gene (a component of the putative TTSS of C. pneumoniae) and the product of Cpn0498 open reading frame, proved to be capable of inducing protective immune responses.

Chlamydiae are obligate intracellular bacteria, parasitizing eukaryotic cells. Chlamydia trachomatis, C. psittaci and C. pneumoniae are the three species of chlamydiae pathogenic to humans. C. trachomatis shows a tropism for the genital and conjunctival epithelia and consists of 19 different serovars which are pathogenic predominantly for the urogenital tract.A distinguishing feature of chlamydiae is their transition between the infectious elementary body that enters the host cell and the non-infectious reticulate body that replicates intracellularly within an inclusion that does not fuse with lysosomes. Chlamydiae depend for some functions upon the host cell; in particular, chlamydiae have little capacity for generating energy. The complete sequence of the 1000-kb chromosome of C. trachomatis is known, as are most of the genes located on the 7.5-kb cryptic plasmid. Recently, several concepts about the biology and the metabolic pathways of C. trachomatis have been revised in relation to the genome sequence, and different novel proteins have been described.