Development of a Chlamydophila psittaci species-specific and genotype-specific real-time PCR

A Chlamydophila psittaci species-specific real-time PCR targeting the rDNA ribosomal spacer was developed as well as a genotype-specific real-time PCR targeting the Cp. psittaci outer membrane protein A (ompA) gene. The SYBR Green-based species-specific real-time PCR detected Cp. psittaci genotypes A to F, and the recently discovered E/B genotype. The genotype-specific real-time PCR could easily distinguish genotypes C, D, F by use of TaqMan probes. Genotypes A, B and E could not be distinguished from each other by simply using TaqMan probes. For this purpose, non-fluorescent competitor oligonucleotides, had to be used next to the TaqMan probes. Genotype E/B could only be detected by use of a minor groove binder (MGB) probe. Both real-time PCR assays allowed reproducible, sensitive (10 rDNA or ompA copies/microL DNA extract) and specific detection of Cp. psittaci DNA. The genotype-specific real-time PCR was compared to ompA sequencing and ompA restriction fragment length polymorphism (RFLP) analysis using five Cp. psittaci field isolates (99, 61/8, 7344/2, 8615/1 and 7778B15) each consisting of two different genotypes. The currently developed real-time PCR assays were used in a case study on a veterinary school and a turkey farm. In the veterinary school, Cp. psittaci genotypes D, E/B and F infection were detected in all five groups of turkeys, and one veterinarian who was taking care of all these turkeys. On the turkey farm, the presence of two Cp. psittaci genotype B infection waves was demonstrated in one randomly selected turkey, the first wave at the age of 6 weeks, and the second at the age of 12 weeks.     

Pathogenic interactions between Chlamydophila psittaci and avian pneumovirus infections in turkeys

Both Chlamydophila psittaci and avian pneumovirus (APV) are highly prevalent in Belgian turkeys and might contribute to the respiratory disease complex observed in turkeys. Initial outbreaks of chlamydiosis occur mostly at the age of 4-8 weeks, often accompanied by an APV infection in APV non-vaccinated farms. Regardless APV vaccination, breakthroughs of APV infection from 8 weeks on do occur, a period when also a second C. psittaci infection appears. Therefore, this study examined the pathogenicity of an APV superinfection in C. psittaci predisposed turkeys. Turkeys were infected with C. psittaci, APV or with C. psittaci followed by APV. Simulating the impact of an APV infection during the acute phase or latent phase of a C. psittaci infection, turkeys have been infected with APV at 1 and 5 weeks post C. psittaci infection, respectively. APV infection during the acute phase of a C. psittaci infection aggravates the severity of clinical signs, macroscopic lesions, pharyngeal APV excretion and histological tracheae lesions. In contrast, no clear interaction could be established after APV infection in latently C. psittaci infected specific pathogen-free (SPF) turkeys. This study clearly demonstrates the exacerbating role of APV during acute C. psittaci infection, which can play an important role in the respiratory disease complex of turkeys.     

Exacerbation of Chlamydophila psittaci pathogenicity in turkeys superinfected by Escherichia coli

Both Chlamydophila psittaci and Escherichia coli infections are highly prevalent in Belgian turkeys and therefore they both might contribute to the respiratory disease complex observed in turkeys. C. psittaci can infect turkeys within the first week of age, even in the presence of maternal antibodies. However, the first C. psittaci outbreaks occur mostly at the age of 3 to 6 weeks, the period when also E. coli infections appear on the farms. Therefore, we examined in this study the pathogenicity of an E. coli superinfection on C. psittaci predisposed turkeys. Turkeys were infected with C. psittaci, E. coli or with C. psittaci followed by E. coli. Simulating the impact of an E. coli infection during the acute phase or the latent phase of a C. psittaci infection, turkeys received E. coli at 1 or 5 weeks post C. psittaci infection, respectively. E. coli superinfection during the acute phase of C. psittaci infection increased C. psittaci excretion and stimulated chlamydial replication in the respiratory tract resulting in exacerbated clinical disease. Interestingly, E. coli superinfection during the latent phase of C. psittaci infection induced chlamydial replication, leading to increased C. psittaci-specific antibody titres. In addition, chlamydial predisposition gave higher E. coli excretion compared with turkeys that had only been infected with E. coli. Overall, the present study clearly demonstrates the pathogenic interplay between C. psittaci and E. coli resulting in more severe respiratory disease.     

Detection of Chlamydophila psittaci by using SYBR green real-time PCR

Chlamydophila psittaci is the causative agent of human psittacosis and avian chlamydiosis. This zoonotic pathogen is frequently transmitted from infected birds to humans. Therefore proper and rapid detection of C. psittaci in birds is important to control this disease. We developed a method for detecting C. psittaci by using SYBR Green Real-time PCR based on targeting the cysteine-rich protein gene (envB) of C. psittaci. This one step procedure was highly sensitive and rapid for detection and quantification of C. psittaci from fecal samples. This assay was also able to detect other zoonotic Chlamydophila species such as C. abortus and C. felis. The assay is well suited for use as a routine detection method in veterinary medicine.     

Key role of Chlamydophila psittaci on Belgian turkey farms in association with other respiratory pathogens

Two hundred turkey sera from eight Belgian and two French farms were tested for the presence of antibodies against avian pneumovirus (APV), Ornithobacterium rhinotracheale (ORT), Mycoplasma gallisepticum, Mycoplasma meleagridis and Chlamydophila psittaci. At slaughter, C. psittaci, APV and ORT antibodies were detected in 94, 34 and 6.5% of the turkeys, respectively. No antibodies against M. gallisepticum or M. meleagridis were present. Additionally, turkeys on three Belgian farms were examined from production onset until slaughter using both serology and antigen or gene detection. All farms experienced two C. psittaci infection waves, at 3-6 and 8-12 weeks of age. Each first infection wave was closely followed by an ORT infection starting at the age of 6-8 weeks, which was still detectable when the second C. psittaci infection waves started. Animals on farm A were not vaccinated against APV leading to an APV subtype B outbreak accompanying the first C. psittaci infection wave. Despite subtype A APV vaccination on farms B and C, the second C. psittaci infection waves were accompanied (farm B) or followed (farm C) by a subtype B APV infection. On all farms respiratory signs always appeared together with a proven C. psittaci, APV and/or ORT infection. This study suggests an association between C. psittaci, APV and ORT, and indicates the multi-factorial aetiology of respiratory infections in commercial turkeys. All three pathogens should be considered when developing prevention strategies for respiratory disease.     

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.     

Isolation and characterization of peacock Chlamydophila psittaci infection in China

The objective of this study was to isolate and identify suspected pathogens from peacocks and peacock farmers with severe pneumonia and to investigate its potential association with peacocks' pneumonia, caused by Chlamydophila psittaci infection. A clinical examination of infected peacocks identified birds with symptoms of anorexia, weight loss, yellowish droppings, airsacculitis, sinusitis, and conjunctivitis, whereas the infected farmers showed high fever and respiratory distress. Immunofluorescence tests detected chlamydial antigens in pharyngeal swabs (12 of 20) and lung tissue samples (four of five) from peacocks. One of four swabs taken from farmers was also positive by the same test. Specific anti-chlamydia immunoglobulin G was detected in 16 of 20 peacocks and four of four peacock farmers. The isolated pathogen was able to grow in specific-pathogen-free (SPF) chicken embryos and McCoy cell lines and was identified as Chlamydiae by immunofluorescence assay and PCR. Avian influenza virus, Newcastle disease virus, and infectious bronchitis virus were eliminated as potential causative agents after pharyngeal swabs inoculated onto the chorioallantoic membrane of embryonate eggs failed to recover viable virus. PCR and restriction fragment length polymorphism indicated the ompA gene from the isolate was similar to that of avian C. psittaci type B. Three-week-old SPF chickens challenged with the peacock isolate via intraperitoneal injection showed a typical pneumonia, airsacculitis, and splenitis. Subsequently, the inoculating strain was recovered from the lungs of challenged birds. This is the first report of C. psittaci infection in peacocks and peacock farmers.     

Evaluation of the prophylactic use of ovotransferrin against chlamydiosis in SPF turkeys

Chlamydophila (C.) psittaci infections are highly prevalent in turkeys and the economical and public health importance of these infections has been recognized since 1950. As there are no vaccines, antibiotic treatment (tetracylines, enrofloxacine) is often needed to allow marketing of poultry. In this study, we explored the use of ovotransferrin (ovoTF), a natural anti-microbial protein, in preventing an experimental C. psittaci infection in specific pathogen free (SPF) turkeys. Turkeys were treated with aerosolized ovoTF prior to the infection. Groups 1 and 2 received a single dose of 10 and 5 mg ovoTF per turkey, respectively. Group 3 received a daily dose of 5mg ovoTF per turkey during 12 days. Group 4 served as untreated, infected control group. Turkeys were aerosol infected using 10(6) TCID(50) of the virulent C. psittaci serovar/genotype D strain 92/1293. Birds were monitored (clinical signs, bacterial excretion) during 12 subsequent days before being necropsied. At necropsy, pathology and C. psittaci replication in various tissues was examined. A single dose of 10mg ovoTF and a repeated daily dose of 5mg ovoTF could not prevent the birds from becoming infected with C. psittaci, but they significantly reduced the outcome of the infection. A single dose of 5mg ovoTF had no influence on the outcome of the infection as compared to the non-treated infected controls. Our results demonstrate the anti-chlamydial effect of ovoTF in vivo and present a base for further research on practical applications of ovoTF on turkey farms.     

More than classical Chlamydia psittaci in urban pigeons

In the literature, studies of Chlamydia infection in birds have usually been confined to the search for Chlamydia (C., formerly Chlamydophila) psittaci, so that little is known about the presence of other chlamydial agents. In the present study, cloacal swabs and faeces samples of urban pigeons have been examined by real-time PCR, DNA microarray assays and partial ompA sequencing. Whilst C. psittaci was the predominant chlamydial agent in this pigeon population (75.8% of all Chlamydiaceae positives), the combined use of highly specific and sensitive molecular assays facilitated the detection of atypical serovars of C. psittaci, as well as other species of Chlamydia, such as C. abortus. Detection of C. pecorum and C. trachomatis from an avian host is reported here for the first time. Rather unexpectedly, 19.5% of all Chlamydiaceae-positive cases turned out to be infected with non-classified organisms. The considerable prevalence of these novel agents raises the question of their epidemiological importance and possible role as pathogens. Future surveys in domestic and wild birds will have to take the extended variety of chlamydial organisms into account.     

Improved sensitivity of PCR for Chlamydophila using pmp genes.

Primers targeting the conserved pmp gene family of Chlamydophila abortus were evaluated for their ability to improve the polymerase chain reaction (PCR) sensitivity. In purified DNA, specific pmp primers (named CpsiA and CpsiB) allowed at least a 10-fold increase of the PCR sensitivity compared to the specific ompA primers for C. abortus, but also for C. psittaci and C. caviae strains. No amplification was observed on C. felis, C. pecorum, C. pneumoniae and Chlamydia trachomatis strains. Tested on contaminated specimens such as genital swabs, the PCR sensitivity observed with CpsiA/CpsiB was also better than with the ompA primers. This study demonstrated that these specific pmp primers could serve as valuable, sensitive and common tools for a specific Chlamydophila diagnosis in ruminant, avian and human diseases. Digestion by AluI of the CpsiA/CpsiB fragments allowed a specific discrimination of the strains in function of their hosts and/or their serotypes.     

DNA microarray-based genotyping of Chlamydophila psittaci strains from culture and clinical samples

The avian and human pathogen Chlamydophila (C.) psittaci represents a genetically heterogeneous species. To facilitate epidemiological surveys, more rapid yet highly specific molecular tests are needed. Currently used typing methods, i.e. serotyping and PCR-RFLP, have only limited sensitivity and are incapable of covering the wide spectrum of naturally occurring types of C. psittaci strains. In the present study, a new DNA microarray assay based on the ArrayTube (AT) technology was used to genotype C. psittaci in 98 isolates and 23 clinical tissue samples. The present array carries 35 oligonucleotide probes derived from variable domains 2 and 4 of the ompA gene. The assay proved highly sensitive, allowing correct genotyping of DNA from 2 inclusion-forming units. The results of DNA microarray genotyping of cultured strains proved highly concordant with the data from PCR-RFLP typing and serotyping. Sequencing of the ompA gene served as the reference test to verify the accuracy of AT genotyping results. In 15 instances (15.3%), strains were successfully typed by the AT assay, while serotyping and/or PCR-RFLP genotyping failed to produce unambiguous results. Eleven of these samples were ompA sequenced to confirm the AT findings. In addition to the currently accepted nine ompA genotypes, the microarray test was shown to recognise new provisional genotypes, such as Mat116 and YP84. In conclusion, the new AT assay proved to be suitable for rapid, sensitive and reproducible genotyping of C. psittaci strains and can be recommended for routine diagnosis.     

Chlamydophila psittaci infections in humans during an outbreak of psittacosis from poultry in Germany

In 2005, an outbreak of severe respiratory disease in a mixed poultry flock that was infected with Chlamydophila (C.) psittaci led to dissemination of the infection to at least 100 small poultry farms in 11 districts of Central Germany. At the same time, a total of 24 persons in contact with poultry from one of the flocks reported flu-like symptoms to their physician, thus suggesting zoonotic transmission. Within 3 weeks, seven individuals had to be hospitalized, with three of them requiring intensive care. Analysis of ompA sequences from chlamydial isolates and directly from clinical samples revealed the presence of both genotype A and E/B of C. psittaci at the source of the outbreak and in contact flocks. Genotype A was also detected in the three severely ill patients. The findings of the present study demonstrate the high zoonotic potential of avian chlamydiae. To ensure speedy eradication of psittacosis in poultry flocks and effective treatment of infected humans, fast, sensitive and species-specific detection of the causative agent is essential, as well as close collaboration between regional public health services, attending physicians and the diagnostic laboratories involved.     

A pmp genes-based PCR as a valuable tool for the diagnosis of avian chlamydiosis

In a previous study we described the use of a new set of PCR primers (CpsiA/CpsiB) specific of the conserved pmp-family genes of Chlamydophila abortus as an efficient tool for the detection of these bacteria in ruminants including also preliminary results on avian strains. In this work, the use of this set of primers was extended to representative strains of the six major avian serovars (serovars A-F) and to field isolates of C. psittaci. For all the studied representative strains, using purified genomic DNA as a template, CpsiA/CpsiB primers allowed, as observed for C. abortus, a minimal 10-fold PCR signal increase compared to the one observed with ompA specific primers. In comparison to primers targeting the 16S-23S rRNA intergenic spacer, similar or increased sensitivity was observed depending on the strain. All the field isolates were amplified with CpsiA/CpsiB primers. On clinical samples, our primers are the best among those tested for detection of C. psittaci by simple conventional PCR. RFLP experiments performed using PCR fragments amplified with the CpsiA/CpsiB primers gave promising results demonstrating that these primers may provide an interesting tool for molecular typing when the bacterium cannot be grown from pathological samples.     

Isolation of chlamydia psittaci from domestic cats with oculonasal discharge in Japan

Eight strains of Chlamydia psittaci were isolated in Japan from the nasal and conjunctival swabs of six household cats using the L929 cell line of mouse fibroblast origin. The isolates were identified as C. psittaci on the basis of the formation of characteristic inclusion bodies in the cell culture detected by Giemsa stain and immunofluorescence. Comparison of nucleotide sequences of the ompA gene amplified from the three isolates with the published sequence of feline FEPN strain of C. psittaci showed almost 100% homology.     

Respiratory and pericardial lesions in turkeys infected with avian or mammalian strains of Chlamydia psittaci

Three groups of turkeys were inoculated with strains of C. psittaci (B577, VS1, TT3) from different restriction endonuclease groups. Turkeys were necropsied at 15 times through post-inoculation day 70. Birds infected with the TT3 strain were lethargic and had decreased body weight. After forced exercise, dyspnea was seen in VS1-infected turkeys. Pericarditis was the most severe lesion in TT3-infected birds. Airsacculitis and bronchopneumonia were the most severe lesions in VS1-infected turkeys. Lateral nasal adenitis was in both VS1- and TT3-infected birds. Only mild peribronchial pneumonia was in B577-infected turkeys. Chlamydial antigen, identified by light microscopy using an immunoperoxidase technique, was seen from post-inoculation days 9 through 50 in the lateral nasal gland and at earlier times in other tissue from VS1- and TT3-infected turkeys. No chlamydial antigen was detected in tissue from B577-infected birds. These studies showed that chlamydial strains from different restriction endonuclease groups are associated with distinct disease syndromes in turkeys.     

New real-time PCR tests for species-specific detection of Chlamydophila psittaci and Chlamydophila abortus from tissue samples

Chlamydophila psittaci and Chlamydophila abortus are the causative agents of avian chlamydiosis (psittacosis) and ovine enzootic abortion, respectively. Both pathogens are known to possess zoonotic potential. Due to their close genetic relatedness, direct and rapid species identification is difficult. In the present study, new real-time PCR assays are reported for both species. The tests are based on highly specific probes targeting the ompA gene region and were conducted as duplex PCRs including an internal amplification control. The Cp. psittaci assay successfully passed a proficiency test at national level. Examination of field samples revealed Cp. psittaci as the dominating species in birds, but also Cp. abortus in a few psittacines. Real-time PCR assays for species-specific detection of Cp. psittaci and Cp. abortus are suited for routine diagnosis, which renders them important tools for the recognition of outbreaks of psittacosis and ovine enzootic abortion.     

Chlamydial infections in feral pigeons in Europe: Review of data and focus on public health implications

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 over the last thirty years across Europe have detected high seropositivity values and high percentages of infection in feral pigeon populations. Overall, when 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, the complement fixation test was used, and antibodies were detected in 19.4-66.3% of the samples, with a median of 46.1%. Indirect immunofluorescence and ELISA tests were employed less frequently, but led to the detection of higher percentages of seropositivity (23.7-67.7% and 35.9-95.6%, respectively). Attempts to grow C. psittaci in cell culture or 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 using different genomic targets detected the organism in 3.4-50% of samples. Twenty-five C. psittaci isolates from pigeons were typed as ompA 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 on 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 of the human cases, while loose or transient contacts with feral pigeons have been mentioned in about 40% of the cases. Education initiatives as to the communication of a health risk resulting from contact with pigeons and pigeon excreta should primarily be targeted at individuals who may be exposed to C. psittaci-contaminated dust, such as demolition/construction workers. Recommendations to this category of workers include wearing protective clothes with hoods, boots, gloves and air filter face masks when removing pigeon faeces from roofs, garrets and buildings, especially if working indoors. Monitoring for C. psittaci infections in these workers over time should also be considered. Children should be warned not to handle sick or dead pigeons, and immunocompromised individuals should be advised to carefully limit their contact to feral pigeons. Culling of pigeons by shooting or poisoning is both unethical and ineffective as the place of the killed birds in the population is quickly filled by new juveniles or immigrating birds 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 size regulation of the pigeon populations. Nevertheless, the measure that will ultimately lead to permanent reduction and will establish healthy sustainable populations is the restriction of indiscriminate feeding by pigeon lovers. The erection 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.     

Abortion in cattle caused by Chlamydia psittaci

A case of bovine abortion was for the first time in Switzerland diagnosed by immunohistochemistry PCR (16S rRNA, ompA) and gene sequence analysis to be caused by Chlamydia psittaci. A possible occurence of further cases has to be investigated. Based on the existence of several methods with very different sensitivity and specificity to diagnose chlamydia it is suggested that at least two verified diagnostic tools should be used to diagnose an involvement of chlamydia in bovine abortion.     

Evaluation of a recombinant enzyme-linked immunosorbent assay for detecting Chlamydophila psittaci antibodies in turkey sera

Chlamydophila psittaci (formerly Chlamydia psittaci) is one of the major pathogens associated with turkey respiratory disease. Devastating outbreaks with high mortality rates, similar to those of 1950 to 1970 in the USA occasionally occur, but respiratory signs without or with low mortality mostly characterize outbreaks now a day. Accurate diagnostic methods should be made available. The present study examined the sensitivity and specificity of a recombinant ELISA (rMOMP ELISA) for detecting Cp. psittaci major outer membrane specific antibodies in turkey sera. Test results were compared to those of immunoblotting and of a competitive ELISA (Chlamydia-psittaci-AK-EIA, R?hm Pharma, Germany) and an indirect ELISA (LPS/LGP) detecting antibodies to the lipopolysaccharide/lipoglycoprotein complex. The rMOMP ELISA was most sensitive as determined on serial dilutions of positive control sera originating from experimentally infected SPF turkeys. The competitive ELISA gave false positives since three negative controls reacted positive. For conventional sera, the sensitivities of the competitive ELISA, immunoblotting and the indirect ELISA were found to be 99.4, 93.1 and 82.2%, respectively, as compared to the rMOMP ELISA (100%). The specificities of the rMOMP ELISA, immunoblotting and the indirect ELISA were found to be 100% while the specificity of the competitive ELISA was only 2.7%. The rMOMP ELISA was chosen to compare the prevalence of chlamydiosis in 2002 with the one from 1992. In 2002, 188 on 200 (94%) turkey sera reacted positive compared to 175 on 200 (87.5%) in 1992 and like 10 years ago all examined farms were seropositive at slaughter. Interestingly, Belgian as well as French farms were seropositive.