Evaluation of an enzyme immunoassay for detection of Chlamydia psittaci in vaginal secretions, placentas, and fetal tissues from aborting ewes

A commercially available enzyme immunoassay (EIA) for the detection of Chlamydia trachomatis in human urogenital and conjunctival specimens was compared with isolation in cell culture for the detection of Chlamydia psittaci in vaginal and placental swabs from aborting ewes and swabs of aborted fetal tissues. The EIA on vaginal swabs collected from 10 ewes experimentally infected with C. psittaci had a sensitivity of 85.7% and a specificity of 85.7%. Vaginal swabs collected at the time of abortion or within 3 days were the best samples for detection of chlamydial infection. The 29 vaginal swabs collected during this period from experimentally infected ewes were all strongly EIA-positive, and chlamydia were isolated from 28. The EIA on vaginal swabs from 78 field cases of abortion had a sensitivity of 78.0% and a specificity of 76.8%. The EIA on swabs of cotyledons from 65 placentas had a sensitivity of 100% and a specificity of 75.0% compared with isolation in cell culture. The EIA on 57 swabs of fetal tissues or body fluids from 10 aborted fetuses or weak lambs from experimentally infected ewes had a sensitivity of 26.6% and a specificity of 88.1% compared with isolation in cell culture. Limitations of the EIA are discussed.     

Rapid detection of Chlamydia psittaci in vaginal swabs of aborted ewes and goats by enzyme linked immunosorbent assay (ELISA)

An enzyme linked immunosorbent assay (ELISA) for the detection of Chlamydia psittaci in vaginal swabs of aborted ewes and goats has been developed using microtiter plates coated with sheep anti-Chlamydia immunoglobulin G. This technique was compared to the direct isolation of the agent by plaque assay on McCoy cells. Among 89 specimens from animals in infected flocks, 58 were positive by both methods, seven were only positive by ELISA, and nine others were only positive by direct isolation (plaque assay). None of the 75 specimens from animals in healthy flocks gave a positive response in ELISA or the plaque assay. Unlike direct isolation in cell culture, the ELISA technique permitted the detection of Chlamydia even in the absence of special care in sampling and conservation of specimens.     

Direct immunofluorescence tests with counterstains for detection of Chlamydia psittaci in infected avian tissues.

Different methods of preparation and serological evaluation of rabbit globulins for use in fluorescent antibody conjugate and different methods of counterstaining with fluorescent antibody tests were evaluated for detection of Chlamydia psittaci in infected turkey tissues. The agar gel precipitin reaction was that chosen for testing and selecting antiserums to be used for fluorescein isothiocyanate conjugation. The fluorescent antibody staining was most pronounced with conjugate made from globulins precipitated with ammonium sulfate. A direct fluorescent antibody method with Evans blue counterstain correctly identified "coded" specimens of C. psittaci-infected and noninfected turkey air sacs. However, naphthalene black was superior to Evans blue as a counterstain when infected pericardial sacs were tested.     

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.     

Serological detection of phage infection in Chlamydia psittaci recovered from ducks

Antiserum prepared against a phage which infects a Chlamydia psittaci isolate recovered from domestic ducks was used to screen other recent avian C psittaci isolates by indirect immunofluorescence. Two more phage infected strains from ducks were discovered. However, phage was not detected in every isolate examined from common source ducks, although such birds are likely to be infected with the same C psittaci strain. Moreover, phage could not always be demonstrated by indirect immunofluorescence in McCoy cell monolayers infected with the phage-containing strain. The results suggest that phage infection is probably an integral part of duck chlamydiosis in the United Kingdom at present, but that the infection is often cryptic.     

Variations in the virulence of strains of Chlamydia psittaci for pregnant ewes

Invasive and non-invasive strains of Chlamydia psittaci isolated from faeces of clinically healthy ewes and from vaginal swabs of ewes which had aborted were injected intravenously or intradermally into pregnant ewes. The results were studied by recording the ewes' thermal and serological responses, lambing performance and the excretion of chlamydia from the vagina. The differences between the effects of different invasive strains were greater after intradermal inoculation than after intravenous inoculation. After intradermal inoculation non-invasive strains did not disturb pregnancy (11 of 13 ewes lambed normally) whereas invasive strains induced abortion in 23 of 25 ewes, 24 of which excreted chlamydia in vaginal secretions.     

Efficacy of a bacterin prepared from Chlamydia psittaci grown in cell culture for experimental immunization of ewes

The efficacy of a bacterin prepared from Chlamydia psittaci grown in mouse L-cells was compared to a similar bacterin prepared from C. psittaci grown in chicken embryos (CE). Both bacterins significantly reduced (P < 0.03) incidence of abortion and weak lambs compared to non-vaccinated control ewes. The L-cell bacterin elicited a greater antibody response than the CE bacterin (P < 0.03).     

鹦鹉热衣原体PCR检测方法的建立

针对16S-23S rRNA和主要外膜蛋白(MOMP)基因序列,分别设计引物,并建立了2种TaqMan荧光PCR、2种常规PCR和2种衣原体科通用PCR方法。经条件优化后,建立的6种PCR方法均具有良好的特异性,其中建立的2种荧光PCR分别能检测出7拷贝和2拷贝的目标双链DNA,其灵敏度明显优于常规PCR;建立的2种衣原体通用PCR,其扩增的目标片段中包含的可变区能有效区分鹦鹉热衣原体和其他衣原体。本试验建立的多种PCR方法敏感特异,具有良好的应用前景。     

Isolation of Chlamydia psittaci from bull ejaculate

During the repeated serological examination (RVK) in five breeding bulls the positive levels of antibodies to Chlamydia psittaci in titre 1 : 128 were found. In the isolation experiments the pelleted ejaculates deposited in liquid nitrogen were used. The isolation of Chlamydia psittaci on yolk sacs of chicken embryos was positive in two breeding bulls. The isolated strains are labelled GN-33 and OK-107. The serological examination of blood samples was in all five breeding bulls negative on brucellosis (BAB), infectious bovine rhinotracheitis and coxiellosis and positive on PI-3. Bacteriological examination of spermatic fluid proved only sporadic contamination with moulds and saprophytic bacteria.     

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.     

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.     

First detection of Chlamydia psittaci from a wild native passerine bird in New Zealand

AIMS: To undertake disease surveillance for Chlamydia psittaci in native birds as part of a pilot study to examine pathogen diversity on Hauturu-o-Toi/Little Barrier Island. To retrospectively review the Massey University post-mortem database to determine previous cases of avian chlamydiosis in New Zealand.

METHODS:Mistnetting of forest birds was conducted across an elevational gradient on Hauturu-o-Toi/Little Barrier Island. Minitip culture swabs were used to collect cloacal samples from native birds. These swabs were screened for Chlamydia family DNA using two PCR methods. Positive results were sequenced. A retrospective review of the Massey University post-mortem database of all avian cases from 1990 to 2011 was conducted.

RESULTS:Ten native birds including four bellbirds (Anthornis melanura), three rifleman (Acanthisitta chloris), two hihi (Notiomyces cincta), and one whitehead (Mohoua albicilla) were sampled and one otherwise healthy female hihi was positive by both PCR screening methods for Chlamydophila. Sequencing confirmed 99–100% genetic similarity to C. psittaci. A retrospective review of the Massey University post-mortem database revealed no previous diagnoses of avian chlamydiosis in wild native New Zealand birds although it has been detected in captive parrots, and wild and captive exotic pigeons.

CONCLUSIONS:This is the first report of the detection of C. psittaci from a wild native bird in New Zealand. The bird was a Passeriforme from an endangered species that was captured free-living on Little Barrier Island. The incidence of avian chlamydiosis in native birds in New Zealand appears to be very low, based on the retrospective review of the post-mortem database.

CLINICAL RELEVANCE: It is unlikely that avian chlamydiosis is a significant problem for hihi population health. The detection of this organism has greater significance for other more susceptible species on Little Barrier Island and for human health, particularly for conservation workers involved in wildlife translocations. It further suggests that passerine birds may be a reservoir for C. psittaci in New Zealand ecosystems.     

Characterisation of Chlamydia psittaci isolated from a horse

This paper describes the isolation and characterisation of a strain of Chlamydia psittaci obtained from a nasal swab taken from a horse with serous nasal discharge. Initial isolation was achieved in cycloheximide-treated McCoy cell monolayers. Chlamydial inclusions stained by immunofluorescence either with a rabbit antiserum raised against C. psittaci or with a monoclonal antibody directed against the genus-specific lipopolysaccharide antigen were single and compact. They did not stain with iodine or with a monoclonal antibody reactive against Chlamydia trachomatis. The agent was re-isolated in the yolk sacs of embryonated hens eggs and designated N16. Identification of the agent was confirmed by electron microscopy. Unique plasmid DNA was prepared from a purified suspension of chlamydial elementary bodies (EBs), and analysed by electrophoresis through 1.0% agarose gels stained by ethidium bromide. This strain of C. psittaci grew relatively slowly in cycloheximide-treated McCoy cells, and the yield of elementary bodies during the course of one growth cycle was relatively low.     

Detection of Chlamydia psittaci DNA in avian clinical samples by polymerase chain reaction

A polymerase chain reaction (PCR) assay was developed to detect Chlamydia psittaci DNA in faeces and tissue samples from avian species. Primers were designed to amplify a 264 bp product derived from part of the 5′ non-translated region and part of the coding region of the ompA gene which encodes the major outer membrane protein. Amplified sequences were confirmed by Southern hybridization using an internal probe. The sensitivity of the combined assay was found to be between 60 to 600 fg of chlamydial DNA (approximately 6 to 60 genome copies). The specificity of the assay was confirmed since PCR product was not obtained from samples containing several serotypes of C. trachomatis, strains of C. pneumoniae, the type strain of C. pecorum, nor from samples containing microorganisms commonly found in the avian gut flora. In this study, 404 avian faeces and 141 avian tissue samples received by the Central Veterinary Laboratory over a 6 month period were analysed by PCR, antigen detection ELISA and where possible, cell culture isolation. PCR performed favourably compared with ELISA and cell culture, or with ELISA alone. The PCR assay was especially suited to the detection of C. psittaci DNA in avian faeces samples. The test was also useful when applied to tissue samples from small contact birds associated with a case of human psittacosis where ELISA results were negative and chlamydial isolation was a less favourable method due to the need for rapid diagnosis.