Methods of detection of Chlamydia psittaci in domesticated and wild birds

OBJECTIVE: To study the occurrence of Chlamydia psittaci in domesticated and wild birds and compare the sensitivity of molecular detection with cell culture isolation. DESIGN: Study of cell culture isolation and PCR detection of C psittaci in avian samples. PROCEDURE: Samples were obtained from 485 birds. Domesticated birds were selected at random from pet shops, private aviaries and zoos, while wild birds were captured locally, sampled, and immediately released. Swabs were collected from choanal slit, conjunctiva and cloaca of each bird and pooled. Samples were divided into equal portions for use in PCR dot-blot and cell culture detection. PCR and dot-blot detection was based on the ompB gene. RESULTS: Prevalence of infection varied markedly between flocks of captive birds. It was highest where there were frequent changes in the flock members or where there were many birds confined in small areas. C psittaci was not detected in wild birds or water birds. The sensitivity of cell culture compared to PCR dot-blot detection was 68%. All samples positive by cell culture were also positive by PCR. CONCLUSIONS: PCR-dot blot detection of C psittaci in birds appears to be more sensitive than cell culture isolation in this study. C psittaci infection of birds may occur in clinically normal captive birds.     

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.     

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.     

Use of ovotransferrin as an antimicrobial in turkeys naturally infected with Chlamydia psittaci, avian metapneumovirus and Ornithobacterium rhinotracheale

Respiratory pathogens are difficult to control in large-scale turkey production. This report describes a clinical trial of antimicrobial ovoTF aerosol on a large Belgian turkey farm. ovoTF was administered to reduce Chlamydia psittaci (C. psittaci) infections and to study the impact of this action on the occurrence of Ornithobacterium rhinotracheale (O. rhinotracheale) and avian metapneumovirus (aMPV) infections. Two subsequent broods were included; (i) a control brood receiving no ovoTF and (ii) an ovoTF brood receiving ovoTF aerosol (5mg/animal) at the age of 2 weeks, continuing daily for 12 days. Twenty-four one-day-old toms of the control and ovoTF brood were tagged and monitored for 15 weeks. The control brood experienced two periods of respiratory disease, the first (2-3 weeks of age) due to C. psittaci and the second (8-17 weeks of age) in the presence of C. psittaci, O. rhinotracheale and maybe aMPV. Extensive antibiotic treatment was needed in 2, 8 and 9 week-old toms. In the ovoTF brood, toms stayed healthy until the age of 9 weeks, whereafter respiratory disease occurred in the presence of C. psittaci, O rhinotracheale and aMPV. OvoTF administration: (i) reduced the amount of C. psittaci in the air as demonstrated by bioaerosol monitoring, (ii) prevented respiratory disease during the first half of the brood period, (iii) was associated with 46% reduction of mortality, and (iv) reduced the antibiotic cost. Our results justify additional clinical trials to explore the use of this innovative antimicrobial strategy for poultry.     

Shedding and transmission of Chlamydia psittaci in experimentally infected chickens

Three avian strains of Chlamydia psittaci were inoculated into 8-day-old chickens by the intra-air-sac or peroral route. Uninoculated chickens were kept as cagemates with the air-sac-inoculated birds. The air-sac-inoculated birds had systemic infection, and chlamydiae were detected frequently in the livers, lungs, jejunums, and colo-rectums at high titers (greater than or equal to 10(5.0) ELD50). All three groups of birds had intermittent and persistent shedding of chlamydiae into feces during the 28-day observation period. In the cagemates, organisms were detected first in the colo-rectum 3 days postexposure and later in the liver, but not in the lung. Limited infection was seen particularly in the colo-rectum of the cagemates and perorally inoculated birds. Antibody response was markedly higher in the air-sac-inoculated chickens than in their cagemates and the perorally inoculated birds. These findings suggest that the colorectum is an important target organ for C. psittaci infection in chickens and that it may be the main site from which the organisms are shed into feces of chickens.     

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.     

Diagnosis of avian mycobacteriosis: comparison of culture,acid-fast stains,and polymerase chain reaction for the identification of Mycobacterium avium in experimentally inoculated Japanese quail (Coturnix coturnix japonica)

In this study we compared culture, acid-fast stains, and polymerase chain reaction (PCR) for the detection of acid-fast organisms in fecal and tissue samples from Japanese quail (Coturnix coturnix japonica) that were experimentally inoculated intravenously with Mycobacterium avium. For culture, three different culture media (modified Herrold egg yolk with mycobactin; Lowenstein-Jensen [L-J]; and L-J with cyclohexamide, naladixic acid, and lincomycin) were tested to determine which medium had the greatest success in isolating mycobacteria. Acid-fast staining methods included Zichl-Neelsen (Z-N) and Truant. The PCR assay detected mycobacterial DNA with primers specific for the 65-kD heat shock protein gene. Culture was considered the "gold standard." Compared with other culture media, L-J yielded more positive cultures and greater numbers of colonies on positive tubes, and incubation times were shorter. Mycobacterium avium was isolated from all of the harvested tissue samples (liver, spleen, and intestine) of inoculated birds. Mycobacteria were isolated from 53% (69/130) of fecal samples from inoculated birds. As the disease advanced, fecal culture was positive on more culture days, indicating that the culture-positive rate was higher later in the course of the disease. Compared with culture, all of the laboratory methods had 100% specificity for the tissue samples. Sensitivities for the tissue samples were 82.6% (Z-N), 95.7% (Truant), and 100% (PCR). For the fecal samples, the specificity was >95% for all methods. Sensitivities compared with fecal culture were 7.2% (Z-N), 30.4% (Truant), and 20.3% (PCR). Tissue and fecal samples from the two control birds were negative for acid-fast organisms by any method. These results were comparable with clinical cases of avian mycobacteriosis where culture and PCR of tissue samples seem to be the most sensitive and specific laboratory tests and evaluation of fecal samples still remains challenging. On the basis of the results of this study, identification of mycobacteria in fecal samples from Japanese quail can be optimized by repeated cultures and Truant acid-fast staining of fecal smears.     

Chlamydophila psittaci DNA detection in the faeces of cage birds

In this study, we investigated the shedding of Chlamydophila psittaci in faecal samples from cage birds using PCR testing. A total of 47 faeces samples were collected from four different aviaries. Main symptoms determined after clinical investigation and owner histories of the birds showed that the birds had respiratory system problems changing from mild to severe. They also showed conjunctivitis, diarrhoea or no symptoms at all. DNA extractions from faeces were performed with the QIAamp DNA Stool Mini Kit. Following PCR with Cp. psittaci specific primers, 43 (91.5%) samples were determined to harbour-specific DNA. Only one bird from each aviary was found to be negative by PCR. As all the samples from birds showing clinical signs were PCR positive, these signs could be correlated to psittacosis in these birds. Cp. psittaci shedding in faeces was detected in all the aviaries. After restriction analysis of PCR amplicons with AluI enzyme, all the isolates showed the same RFLP (Restriction Fragment Length Polymorphism) patterns with the control Cp. psittaci DNA. PCR following QIAamp DNA stool mini kit extraction of faecal samples was found to be a rapid, specific, sensitive, reproducible test, which did not need additional nested PCR of samples.     

Evidence of Chlamydophila psittaci infection in captive Amazon parrots in Brazil.

The prevalence of Chlamydophila psittaci (formerly Chlamydia psittaci) infection was assessed in 95 apparently healthy, captive Amazon parrots from three breeder collections in southeastern and west-central Brazil. Cloacal swabs from 95 birds were tested for chlamydial antigen, which was detected by direct immunofluorescence (DIF), and serum samples from 44 of these birds were tested for antibodies to C. psittaci using an enzyme-linked immunosorbent assay. The prevalences of active infection as detected by DIF were 16.7%, 22.2%, and 56.1%, and seroprevalences were 100%, 87.5%, and 60% in flocks A, B, and C, respectively. We can therefore infer that C. psittaci may be widespread in captive parrot populations in Brazil.     

Chlamydiosis in captive raptors

Chlamydia psittaci was isolated from four red-tailed hawks (Buteo jamaicensis) that died suddenly and from seven birds that survived at a raptor rehabilitation center in California in 1983. One hundred captive raptors representing 14 species in five families were subsequently tested serologically and by direct cloacal culture. C. psittaci was isolated from seven clinically normal birds. Forty-four percent of the raptors were considered positive using an enzyme-linked immunosorbent assay (ELISA), and 19% were suspects. The ELISA was repeated on 54 raptors in 1986. Forty-one percent of the birds were considered positive, and 35% were suspect, indicating that C. psittaci is endemic in the population.     

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.     

A comparative study of detecting Chlamydophila psittaci in pet birds using isolation in embryonated egg and polymerase chain reaction

This study, for the first time in Turkey, investigated the existence of Chlamydophila psittaci and determined the prevalence of its disease, chlamydiosis, in pet birds. Polymerase chain reaction (PCR) was compared with other testing methods that have been typically used in the diagnosis of C. psittaci. Fecal specimens (n =96) of avian origin were tested by PCR and two identification methods, modified Gimenez staining (mGS) and direct fluorescein-conjugated monoclonal antibody staining (FA). The identification methods were implemented by staining the yolk sacs of embryonated chicken eggs inoculated at 6 days of age and harvested between 3 and 10 days after inoculation. Fecal specimens from pet birds were randomly collected from pet shops and homes. These specimens were then used to isolate C. psittaci and to detect its specific DNA. The inocula that were prepared from fecal specimens were then inoculated into yolks of 6-day-old embryonated chicken eggs. The preparations from egg yolk sacs were examined with mGS and direct FA after three blind passages. The PCR method was used to detect specific DNA in feces. In 96 fecal specimens, 33 (34.4%) were positive with PCR, 21 (21.9%) were positive with mGS, and 29 (30.2%) were positive with FA. Among 33 positive specimens with PCR, 28 specimens were positive with FA, and 20 specimens were positive with mGS. The sensitivity and specificity were 59% and 94% between FA and mGS, and 97% and 93% between FA and PCR, respectively.     

Chlamydiae and atherosclerosis: can psittacine cases support the link?

Atherosclerosis is a common disease in pet birds, particularly in psittacines. Little is known about the role of risk factors predisposing birds to this disease. In our study, we tried to detect chlamydiae in formalin-fixed and paraffin-embedded atherosclerotic tissue from 103 pet birds to clarify their role in atherosclerosis. Methods used were polymerase chain reaction (PCR), sequencing, and immunohistochemistry. Histopathologic examination served to classify the extent of atherosclerotic lesions. In the PCR, 4 (3.9%) of 103 cases, all of them with advanced stages of atherosclerosis, were positive. Subsequent sequence analysis revealed high identities (94%-100%) with Chlamydophila psittaci in three cases. Interestingly, two of these birds came from C. psittaci-infected populations. Because of the low incidence (3.9%), the occurrence only in advanced stages, and the association with C psittaci-infected avian populations, a causal relationship between chlamydiae and atherosclerosis in pet birds is rather improbable.     

Chlamydophila psittaci in free-living Blue-fronted Amazon parrots (Amazona aestiva) and Hyacinth macaws (Anodorhynchus hyacinthinus) in the Pantanal of Mato Grosso do Sul, Brazil

Chlamydophila psittaci (C. psittaci) infection was evaluated in 77 free-living nestlings of Blue-fronted Amazon parrots (Amazona aestiva) and Hyacinth macaws (Anodorhynchus hyacinthinus) in the Pantanal of Mato Grosso do Sul, Brazil. Tracheal and cloacal swab samples from 32 wild parrot and 45 macaw nestlings were submitted to semi-nested PCR, while serum samples were submitted to complement fixation test (CFT). Although all 32 Amazon parrot serum samples were negative by CFT, cloacal swabs from two birds were positive for Chlamydophila DNA by semi-nested PCR (6.3%); these positive birds were 32 and 45 days old. In macaws, tracheal and cloacal swabs were positive in 8.9% and 26.7% of the samples, respectively. Complement-fixing antibodies were detected in 4.8% of the macaw nestlings; macaw nestlings with positive findings were between 33 and 88 days old. These results indicate widespread dissemination of this pathogen in the two evaluated psittacine populations. No birds had clinical signs suggestive of chlamydiosis. To the best of our knowledge, this is the first report on C. psittaci in free-living Blue-fronted Amazon parrots and Hyacinth macaws in Brazil.     

Evaluation of the polymerase chain reaction in comparison with other diagnostic methods for the detection of Chlamydia psittaci.

Various diagnostic methods exist for the detection of Chlamydia psittaci. In the current study, the test performance of polymerase chain reaction (PCR) was compared with other testing methods used in the diagnosis of C. psittaci. Tissue and fecal specimens (n = 119) of avian and mammalian origin were tested by PCR and one or more of the following methods: cell culture, enzyme-linked immunosorbent assay, and direct fluorescein-conjugated monoclonal antibody staining. Several gold standards, based on results of testing methods other than PCR, were used to calculate the following test performance characteristics of PCR: sensitivity and specificity, with their 95% confidence intervals; kappa statistics, a measure of intertest agreement; and lambda statistics, a chance-corrected estimate of the sensitivity and specificity. Overall, the test performance characteristics of PCR were low compared with the other testing methods. Possible reasons for the poor test performance of PCR in the current study include destruction of the organisms during storage, interference with the PCR by other reagents, or technical errors.     

Optimization of a culture technique for the isolation of Listeria monocytogenes from faecal samples

A culture technique employing cold enrichment at 4 degrees C followed by selective enrichment and plating at higher temperatures (30 degrees C) was used to isolate Listeria monocytogenes from faecal samples. The samples were held at 4 degrees C for 15 weeks and cultured weekly to assess the sensitivity of the culture after cold storage for different lengths of time. No media, Listeria selective enrichment broth (LSEB), nutrient broth (NB) and saline were used as cold storage medium. Cold storage increased the frequency of Listeria positive samples. The sensitivity of the culture for Listeria spp. and L. monocytogenes was 72 and 94%, and 56 and 61% after third and seventh week of cold storage, respectively. When the results of third and seventh week of cold storage were combined, the sensitivity was 100% for Listeria spp. and 94% for L. monocytogenes. LSEB and NB as storage medium increased Listeria positive samples after the first week of cold storage but did not maintain the increase thereafter while saline had an adverse effect on the growth of the bacteria. However, samples held in no media in a pilot study involving monthly sampling of a herd revealed better results. Detection limit of the culture media was also investigated. The lowest concentration detected by culture media was 3.17 organisms/ml. This was seven organisms/g for known Listeria positive sample. The faecal samples spiked with 10-fold dilutions of L. monocytogenes and held at 4 degrees C revealed that the sample spiked with 3.17 x 10-1 cfu/ml organisms resulted in growth after the second week of cold storage. The results suggest that the culture technique employing cold enrichment followed by selective enrichment and plating is more sensitive, the storage of faecal samples in no media when compared with the samples in storage medium, LSEB, NB and saline, during cold enrichment is a better application and culture of faeces, immediately after collection, at third and seventh week of cold enrichment produce more satisfactory results.     

动物衣原体病诊断方法及疫苗研究进展

文章叙述了鹦鹉热衣原体的特性以及感染的宿主范围;国外通过过氧化物酶-抗过氧化物酶法对鹦鹉热衣原体在鸟体内的分布进行定位;为提高诊断的准确性,建立了单克隆抗体技术以及PCR诊断技术.国内通过对羊衣原体和猪衣原体的分离,建立了间接血凝试验(IHA),并进行了大面积的推广应用.同时为提高诊断的准确性,ELISA诊断方法以及PCR诊断方法相应被建立.国外对于鹦鹉热衣原体弱毒疫苗、灭活疫苗以及基因工程亚单位疫苗进行了详细的研究.我国在20世纪已完成对鹦鹉热衣原体灭活疫苗的研究,但对于基因工程疫苗的研究尚处于早期阶段.     

Isolation and identification of Chlamydia psittaci from pet birds

Culturing of Chlamydia psittaci from pet birds requires the inoculation of a susceptible living host system with suspensions of various tissues from dead birds or with tracheal and/or cloacal swabs and fresh feces from live birds. Cell cultures have been used as the host system. The most commonly used cell cultures for isolation of C psittaci from pet birds are McCoy and mouse L cells. The sensitivity and specificity of cell culture equals or surpasses embryonating chicken eggs and mice, and results can be obtained in less than 7 days. To obtain satisfactory results, the inoculum must be centrifuged onto the cell cultures at 37 C, and the cells must be treated with a metabolic inhibitor such as colchicine or cycloheximide. Chlamydia psitaci can be detected in infected cells by use of fluorescent antibody, Giemsa, or Gimenez staining.     

Prevalence of chlamydia, toxoplasma, toxocara and ringworm in farm cats in south-west England

The prevalence of infection with Chlamydia psittaci, Toxoplasma gondii, Toxocara cati and Microsporum canis was examined in 51 cats on 22 sheep farms in the Bristol area. Serum antibody to C psittaci and T gondii was present in 45 per cent and 47 per cent of cats, respectively. At the time of sampling C psittaci was isolated from 6 per cent of the cats, T cati was identified in 63 per cent of faecal samples but neither T gondii nor M canis was isolated. When examined according to the farm of origin, 22.7 per cent of farms had cat populations with no evidence of infection with C psittaci or T gondii. Of the remainder, 45.5 per cent supported cat populations with evidence of both infections and 31.8 per cent had evidence of T gondii infection alone. None of the farms had cat populations with evidence of C psittaci infection alone. Two of the cats infected with C psittaci were excreting viable organisms in the faeces. The possible significance of this to the epidemiology of ovine enzootic abortion is discussed.     

Comparison of DNA-spot hybridization, cell culture and direct immunofluorescence staining for the diagnosis of avian chlamydiae

DNA-spot hybridization, cell culture and direct immunofluorescence staining were compared for the detection of avian Chlamydia psittaci strains in cell culture dilutions and in routine samples submitted for diagnosis. With dilutions of infected cell culture material, growth in BGM cells was by far the most sensitive technique, detecting 0.01 infected cells (20 elementary bodies) ml-1. DNA-spot hybridization and direct immunofluorescence staining were of approximately equal sensitivity, both detecting 16 infected cells (3.2 x 10(4) elementary bodies) per ml-1. When 27 avian liver and spleen samples were assayed, all 3 tests performed similarly (13 positive and 12 negative by all 3 tests). This suggests that in most avian samples presented for diagnosis, sufficient numbers of chlamydiae are present to allow any of the test to the be used. Thus, the direct immunofluorescence staining method is currently the test of choice for routine diagnosis since it is available in kit form, is relatively simple and quick to perform, and like DNA-spot hybridization, detects non-viable as well as viable organisms. However, if low levels of chlamydiae are to be effectively detected, such as in carrier birds or birds with recently acquired infections, then cell culture should be used.