Kristel Verminnen

Chlamydophila psittaci Transmission from Pet Birds to Humans

We studied zoonotic transmission of Chlamydophila psittaci in 39 breeding facilities for Psittaciformes (cockatoos, parrots, parakeets, lories) that frequently used antimicrobial drugs. Genotypes A or E/B were detected in 14.9% of humans at these facilities. Information on antimicrobial drug use in Psittaciformes and a C. psittaci vaccine are urgently required.

Chlamydial infections in duck farms associated with human cases of psittacosis in France.

Five severe cases of psittacosis in individuals associated with duck farms were notified in France between January and March 2006. Diagnostic examination included serology and/or molecular detection by PCR from respiratory samples. As a consequence, we investigated all duck flocks (n=11) that were housed in the three farms where human infections occurred. While serology by complement fixation test was negative for all samples, cloacal and/or tracheal chlamydial excretion was detected by PCR in all three units. Notably, one duck flock was tested strongly positive in 2 of the 3 affected farms, and Chlamydophila (C.) psittaci strains were isolated from cloacal and/or tracheal swab samples from both farms. Human samples and duck isolates exhibited the same PCR-RFLP restriction pattern, which appeared to be an intermediate between genotypes A and B. Analysis of ompA gene sequences and comparison to those of the type strains showed that the isolates could not be strictly assigned to any of the generally accepted genotypes of C. psittaci. Further analysis by MLVA of the PCR-positive human samples revealed two distinct patterns, which were related to previously isolated C. psittaci duck strains.

Evaluation of a Chlamydophila psittaci Infection Diagnostic Platform for Zoonotic Risk Assessment

ABSTRACT Reports on zoonotic transmission of Chlamydophila psittaci originating from poultry are incidentally published. During recent studies in European turkeys we isolated C. psittaci genotypes A, B, D, E, F, and E/B, all considered potentially dangerous for humans. This encouraged us to analyze the zoonotic risk on a Belgian turkey farm, from production onset until slaughter, using a Chlamydophila psittaci diagnostic platform. Twenty individually marked hens, as well as the farmer and two scientists, were monitored medically. Bioaerosol monitoring, serology, isolation, and nested PCR demonstrated chlamydiosis on the farm leading to symptomatic psittacosis in all 3 persons involved. ompA sequencing confirmed the zoonotic transmission of C. psittaci genotype A. Strangely, two different antibody microimmunofluorescence (MIF) tests remained negative in all infected persons. The results demonstrate the value of the currently used diagnostic platform in demonstrating C. psittaci infections in both birds and humans but raise questions regarding use of the MIF test for diagnosing human psittacosis. In addition, our results suggest the underestimation of psittacosis in the poultry industry, stressing the need for a veterinary vaccine and recommendations for zoonotic risk reduction strategies.

Prevalence of Chlamydophila psittaci infections in a human population in contact with domestic and companion birds

Chlamydophila psittaci infections in humans are underestimated. We investigated the occurrence of C. psittaci in a Belgian population of 540 individuals. Data were from a population survey (n=2524) of apparently healthy community-dwelling subjects aged 35-55 years. Pharyngeal swabs and blood were taken. Individuals completed a questionnaire on professional and nonprofessional activities, smoking habits, medical history and contact frequency with different bird species. Swabs were analysed by a C. psittaci-specific and a Chlamydophila pneumoniae-specific PCR. Sera were tested by a recombinant C. psittaci major outer-membrane protein-based ELISA, a C. psittaci whole organism-based ELISA (Serion) and a micro-immunofluorescence test (Focus Diagnostics). Results confirmed our suspicion about the underestimation of psittacosis in Belgium. Psittaciformes and racing pigeons were the main infection source. Women with excessive alcohol intake defined as a mean intake of >2 units daily were more frequently infected than men. We analysed the effect of seropositivity and/or PCR positivity on inflammation (white blood cell count, high-sensitivity C-reactive protein, fibrinogen). In general, seropositivity showed a trend to slightly higher levels of inflammatory variables (all non-significant), whilst PCR positivity showed a trend to no effect or even lower inflammatory levels.

Use of a nested PCR-enzyme immunoassay with an internal control to detect Chlamydophila psittaci in turkeys

BackgroundLaboratory diagnosis of Chlamydophila psittaci, an important turkey respiratory pathogen, is difficult. To facilitate the diagnosis, a nested PCR-enzyme immunoassay (PCR-EIA) was developed to detect the Cp. psittaci outer membrane protein A (ompA) gene in pharyngeal swabs.MethodsThe fluorescein-biotin labelled PCR products were immobilized on streptavidin-coated microtiter plates and detected with anti-fluorescein peroxidase conjugate and a colorimetric substrate. An internal inhibition control was included to rule out the presence of inhibitors of DNA amplification. The diagnostic value of the ompA nested PCR-EIA in comparison to cell culture and a 16S-rRNA based nested PCR was assessed in pharyngeal turkey swabs from 10 different farms experiencing respiratory disease.ResultsThe sensitivity of the nested PCR-EIA was established at 0.1 infection forming units (IFU). Specificity was 100%. The ompA nested PCR-EIA was more sensitive than the 16S-rRNA based nested PCR and isolation, revealing 105 out of 200 (52.5%) positives against 13 and 74 for the latter two tests, respectively. Twenty-nine (23.8%) out of 122 ompA PCR-EIA negatives showed the presence of inhibitors of DNA amplification, although 27 of them became positive after diluting (1/10) the specimens in PCR buffer or after phenol-chloroform extraction and subsequent ethanol precipitation.ConclusionThe present study stresses the need for an internal control to confirm PCR true-negatives and demonstrates the high prevalence of chlamydiosis in Belgian turkeys and its potential zoonotic risk. The ompA nested PCR-EIA described here is a rapid, highly sensitive and specific diagnostic assay and will help to facilitate the diagnosis of Cp. psittaci infections in both poultry and man.

Specific-pathogen-free pigs as an animal model for studying Chlamydia trachomatis genital infection

ABSTRACT The purpose of the present study was to evaluate pigs as a large-animal model for female genital infection with two Chlamydia trachomatis human serovar E strains. Sixteen-week-old specific-pathogen-free female pigs (gilts) were intravaginally infected with the trachoma type E reference strain Bour or the urogenital serovar E strain 468. Several conclusions can be drawn from our findings on the pathogenicity of a primary C. trachomatis genital infection in gilts. First of all, we demonstrated that the serovar E strains Bour and 468 could ascend in the genital tract of gilts. The serovar E strains could replicate in the superficial columnar cervical epithelium and in the superficial epithelial layer of the uterus, which are known to be the specific target sites for a C. trachomatis genital infection in women. Second, inflammation and pathology occurred at the replication sites. Third, the organisms could trigger a humoral immune response, as demonstrated by the presence of immunoglobulin M (IgM), IgG, and IgA in both serum and genital secretion samples. Our findings imply that the pig model might be useful for studying the pathology, pathogenesis, and immune response to a C. trachomatis infection of the genital system.

Simultaneous zoonotic transmission of Chlamydophila psittaci genotypes D, F and E/B to a veterinary scientist.

Two groups of five 1-day-old conventional turkeys were housed in negative pressure stables to become experimentally infected with Avian Metapneumovirus (aMPV) and Ornithobacterium rhinotracheale (ORT) at the age of 3 weeks. However, during the first 2 weeks, turkeys started to show respiratory disease characterized by rhinitis and dyspnoea. Routine bacterial and viral diagnoses remained negative. Therefore, pharyngeal swabs from the turkeys and from the veterinary scientist handling the animals were examined for the presence of Chlamydophila (C.) psittaci by using a combination of cell culture, nested PCR and ompA genotype-specific quantitative real-time PCR, as well as by serology. Results revealed simultaneous transmission of C. psittaci outer membrane protein A (ompA) genotypes D, F and E/B from infected turkeys to the veterinary scientist.

Vaccination of turkeys against Chlamydophila psittaci through optimised DNA formulation and administration.

We have demonstrated that vaccination of turkeys with an unformulated DNA vaccine induces significant protection against Chlamydophila (Cp.) psittaci infections. Nevertheless, the immunogenicity of the DNA vaccine can still be improved by increasing translation and transfection efficiency. Therefore, the ompA codon was adapted to the codon usage in birds, resulting in pcDNA1/MOMP(opt). To increase gene transfer, polyplexes of pcDNA1/MOMP(opt)-EGFP with different cationic polymers, such as linear and branched polyethyleneimine (lPEI and brPEI) and starburst PAMAM dendrimers, and lipoplexes with cationic DOTAP/DOPE liposomes were created. Transfection of lPEI and brPEI polyplexes with an N/P ratio of 8 resulted in the highest transfection efficiencies, but lPEI polyplexes were completely destroyed following nebulisation. Secondly, we examined the capacity of nebulised or intramuscularly (IM) administered brPEI-pcDNA1/MOMP(opt) to induce a significant protective immune response in SPF turkeys experimentally infected with 10(8) TCID(50) of a virulent Cp. psittaci strain. Results were compared to IM administration of naked plasmid DNA and to results of non-vaccinated animals. Intramuscular administration of brPEI-pcDNA1/MOMP(opt) increased the immunogenicity of the Cp. psittaci DNA vaccine as compared to IM administration of pcDNA1/MOMP(opt) or aerosol delivery of brPEI-pcDNA1/MOMP(opt). Improved immunogenicity was correlated with increased protection. Vaccinated groups were significantly protected against Cp. psittaci challenge.