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.     

Relationship between infectivity and cytopathology for L-929 cells, membrane proteins, and antigenicity of avian isolates of Chlamydia psittaci

Seventeen isolates of Chlamydia psittaci from various avian species were examined. Based on their infectivity and cytopathology for L-929 cells, these isolates were separable into a high-infectivity group (HIG) and a low-infectivity group (LIG). Differences in the molecular weight of the major outer membrane proteins (MOMPs) of the isolates correlated with differences in infectivity. The HIG MOMPs had a molecular weight of 43,500, and the LIG MOMPs had a molecular weight of 45,500. The MOMP of one mammalian isolate of C. psittaci examined had a molecular weight of 43,500. Antisera raised against some of the isolates reacted with only the MOMP from isolates of their respective groups. The MOMPs of a mammalian C. psittaci isolate and of the C. trachomatis LGV 440 isolate did not react with HIG or LIG antisera. The MOMPs of some avian C. psittaci did react weakly with antiserum against the LGV 440 isolate of C. trachomatis.     

Chlamydia psittaci infection in horses: results of a prevalence survey and experimental challenge.

Nasal and conjunctival swabs were obtained from 300 horses and Chlamydia psittaci was isolated from 15 of them (5 per cent). Eleven nasal swabs and six conjunctival swabs were positive on culture, but there was no association between the isolation of the organism and the presence of clinical ocular or respiratory disease. Six ponies were challenged with an equine isolate of C psittaci into the eye, nasal cavity or bronchial tree. The organism could be isolated from nasal and conjunctival swabs taken from the ponies for up to 17 days after challenge, but there was no clinical evidence of disease.     

Isolation and characterization of Chlamydophila psittaci isolated from laying hens with cystic oviducts

The objective of this study was to isolate and identify a hypothetical Chlamydiaceae pathogen from laying hens with an oviduct cyst, and to characterize its potential causal relation with decreased egg production. Our clinical survey showed that cystic oviducts were prevalent at rates of 10% and 15.1% in breeder and commercial hen flocks, respectively. Chlamydial antigens were detected in 20 of 50 pharyngeal swabs (40%) and in 17 of 20 oviduct tissues (85%) using enzyme-linked immunosorbent assay (ELISA) antigen detection kits. The isolated pathogen was identified as Chlamydophila psittaci via complement fixation test, PCE-ELISA, and immunofluorescence assay. Avian influenza virus, Newcastle disease virus, and infectious bronchitis virus were excluded after oviduct tissues were inoculated onto the chorioallantoic membrane of embryonating eggs. The nucleotide sequence of the omp1 gene (accession no. EF202608) from the isolate was similar to that of C. psittaci avian type C (accession no. L25436). Typical cystic oviducts were observed in specific-pathogen-free hens inoculated intraperitoneally with the isolate. The high presence of chlamydial antigen is consistent with the cystic oviducts and poor egg production. We conclude that the isolated C psittaci is most likely associated with cystic oviducts in laying hens.     

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.     

Preparation and use of a monoclonal antibody to detect Chlamydia psittaci antigen in paraffin-embedded tissue sections

A murine monoclonal antibody prepared against an ovine abortion isolate of Chlamydia psittaci (A22/Teramo) revealed specific binding to a 57 kDa chlamydial antigen in immunoblotting studies. The monoclonal antibody was able to detect intracytoplasmic chlamydial inclusions and scattered elementary bodies in infected McCoy cell culture, and on formalin-fixed paraffin-embedded tissue sections both from experimentally infected mice and from fetal membranes of cases of ovine enzootic abortion.     

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.     

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.     

Evaluation of a monoclonal antibody based ELISA for detection of feline Chlamydia psittaci

A commercially available ELISA designed for the detection of C trachomatis in human urogenital specimens was compared with cell culture for the detection of Chlamydia psittaci in cat conjunctival swabs and in twofold dilutions of a cell culture pool of a feline strain of C psittaci. Cell culture was more sensitive than the ELISA test for detection of C psittaci.     

A simple staining method for the identification of chlamydial elementary bodies in the fetal membranes of sheep affected by ovine enzootic abortion.

A dark-ground methylene blue (DGMB) staining method was used to demonstrate chlamydial elementary bodies in fetal membranes of sheep affected by Chlamydia psittaci. Before evaluation on material from clinically affected animals, the DGMB method was compared with modified Ziehl-Neelsen (MZN) and dark-ground Giemsa (DGG) staining methods for its ability to demonstrate chlamydial elementary bodies in hens' eggs which had been experimentally infected with C. psittaci. DGMB was more specific in its staining of chlamydial elementary bodies than DGG or MZN. The DGMB method was found to be a more reliable technique for the examination of fetal membranes from sheep affected with C. psittaci than DGG or MZN. Those samples diagnosed as positive using the DGMB showed a good correlation with those diagnosed as positive on macroscopic examination.     

Evaluation of bioaerosol sampling techniques for the detection of Chlamydophila psittaci in contaminated air

Chlamydophila (C.) psittaci, a category B bioterrorism agent, causes respiratory disease in birds and psittacosis or parrot fever in man. The disease spreads aerogenically and no vaccines are available for either birds or man. Highly sensitive C. psittaci bioaerosol monitoring methods are unavailable. We evaluated: (1) dry filtration for collecting C. psittaci from contaminated air using different samplers and membrane filters, (2) impingement into different liquid collection media by use of the AGI-30 impinger and the BioSampler and (3) impaction into newly designed C. psittaci media utilizing the MAS-100 aerosol impactor. For personal bioaerosol sampling, we recommend the use of a gelatin filter in combination with the IOM inhalable dust sampler at an airflow rate of 2L/min. This allowed the detection of 10 organisms of C. psittaci by both PCR and culture. For stationary bioaerosol monitoring, sampling 1000L of air in 10min with the MAS-100 impactor and ChlamyTrap 1 impaction medium was most efficient and made it possible to detect 1 and 10 C. psittaci organisms by PCR and culture, respectively. ChlamyTrap 1 in combination with the MAS-100 impactor might also be applicable for bioaerosol monitoring of viruses.     

Experimental conjunctival infection of lambs with a strain of Chlamydia psittaci isolated from the eyes of a sheep naturally affected with keratoconjunctivitis

Five ram-lambs were inoculated into the left conjunctival sac with the 15R isolate of Chlamydia psittaci, recovered from a sheep with keratoconjunctivitis. A sixth ram-lamb was kept in contact with them. The five lambs developed varying degrees of acute conjunctivitis and 14 days later C psittaci could be recovered from the inoculated eyes, from which Branhamella ovis was also isolated. The eyes were examined regularly for four months; C psittaci could not be re-isolated but the eyes developed varying degrees of follicular conjunctivitis. After four months the sheep were treated with corticosteroids in an attempt to reactivate a latent chlamydial infection but no chlamydiae could be isolated. Five months after the start of the experiment the six lambs were inoculated with 15R into the left conjunctival sacs. Acute conjunctivitis developed which was not as severe as after the first inoculation, but C psittaci could only be recovered from the left eyes of three sheep three days after inoculation. The eyes remained chronically affected by follicular conjunctivitis. Six months after the start of the experiment the left eyes were again inoculated with 15R; on this occasion acute conjunctivitis did not develop and chlamydiae could not be isolated. Chronic follicular conjunctivitis persisted until the experiment was terminated three months later.     

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.     

Protection of ewes vaccinated with A22 strain Chlamydia psittaci (ovis) against challenge in pregnancy with homologous and heterologous strains of the organism

Fifty ewes were randomly divided into four groups. Groups A and B were vaccinated with an experimental vaccine derived from the A22 isolate of Chlamydia psittaci (ovis), an isolate known to cause ovine enzootic abortion (OEA). Groups C and D were unvaccinated controls. In mid-pregnancy, animals in group A and C were challenged with live A22 C. psittaci (ovis) and those in B and D were challenged with a field isolate of the organism (BS) against which the commercially available A22 vaccine appeared to offer poor protection. In group A, three animals showed clinical signs of OEA and six excreted chlamydiae. In group B, five ewes showed clinical signs of OEA and excreted chlamydiae. In group C, three ewes had clinical signs of OEA but seven excreted chlamydiae. In group D, all 11 ewes showed clinical signs of OEA and excreted chlamydiae in the products of parturition. This group produced only four live lambs with an average weight of viable lamb per ewe of 1.4 kg, whereas the other groups each produced 12 or 13 lambs with an average weight of viable lamb per ewe of more than 4 kg. The BS isolate was much more virulent than the A22 isolate for unvaccinated, pregnant ewes. However, the A22 vaccine offered significant protection against the heterologous BS isolate although on this occasion it did not appear to alter the course of disease produced by the less pathogenic A22 isolate.     

The clinical efficacy of topical and systemic therapy for the treatment of feline ocular chlamydiosis

Twenty-four specific-pathogen-free-derived cats aged four to 11 months were challenged by ocular application of a field isolate of Chlamydia psittaci to evaluate the effect of topical and systemic therapy on the course of disease. The cats were monitored for 35 days post-challenge, with severity of clinical signs being measured using a scoring system, and ocular shedding of the organism monitored by culture of conjunctival swabs. All cats developed active C psittaci infection, and after 7 days the cats were randomly assigned to one of four treatment groups: Group P (placebo) was given twice-daily ophthalmic tear-replacement ointment; group F was given twice-daily topical 1% fusidic acid ophthalmic viscous drops; group C was given twice-daily topical 1% chlortetracycline ophthalmic ointment; and group D was given doxycycline at 10 mg/kg daily per os in addition to twice-daily topical 1% fusidic acid ophthalmic ointment. Within 24 h of commencement of therapy, group D had significantly lower median clinical scores than group P, and with the exception of day 16, this trend was maintained throughout the observation period. Median clinical scores of cats in group F were not appreciably different to those in group P, whereas the median scores of cats in group C generally fell between those of groups P and D. The median duration of C psittaci shedding was 10 and 15 days for groups D and C respectively, but four of the six cats in groups F and P were still shedding organisms at the end of the study (day 35). In this study, systemic therapy with doxycycline proved superior to topical therapy in the treatment of feline chlamydiosis.     

Distinguishing between ovine abortion and ovine arthritis Chlamydia psittaci isolates with specific monoclonal antibodies

Monoclonal antibodies to Chlamydia psittaci were prepared by both in vivo and in vitro immunization methods, using an abortion strain of C psittaci as the immunizing antigen. Seven of the 8 monoclonal antibodies produced were genus-specific by the enzyme-linked immunosorbent assay and immunofluorescence test. The genus-specific antibodies were reactive with a protease-resistant, periodate-sensitive antigen of less than 14 kilodaltons. The remaining monoclonal antibody, 10D7, was specific for ovine abortion strains of C psittaci and nonreactive with 2 strains isolated from the joints of lambs with polyarthritis. The type-specific antigen was protease sensitive, but could not be detected in the immunoblot assay.     

Oxidative activity of turkey monocytes, following the inoculation with Chlamydia psittaci

Chemiluminescence (CL) was used to investigate the competence of turkey monocytes to mount a respiratory burst response upon interaction with Chlamydia psittaci. The oxidative activity of purified turkey monocytes, following inoculation with the avian C. psittaci serovar D strain 92/1293, was studied using luminol- and lucigenin-enhanced CL. Purified turkey monocytes were inoculated with C. psittaci at multiplicity of infection (MOI) of approximately 100, 10 and 1. In the presence of luminol, no detectable CL or only a weak CL response was obtained, and if present it increased with increasing MOI. Either sham inoculated monocytes, or monocyte-free control assays supplemented with C. psittaci, gave no detectable luminol-enhanced CL responses. In the lucigenin-enhanced assays, monocytes inoculated with C. psittaci demonstrated an immediate CL peak, the height of which was proportional to the MOI used. Following inoculations at a MOI 1, a faint second peak was observed, when applying high concentrations of lucigenin. Sham inoculated monocytes gave no detectable lucigenin-enhanced CL responses. However, in the presence of lucigenin, the addition of C psittaci to monocyte-free controls also resulted in an immediate CL peak, though no second peak was detected. This immediate lucigenin-dependent CL peak induced by C. psittaci was similar to the one observed in the presence of monocytes, and was not inhibited by superoxide dismutase. We demonstrated that this avian C. psittaci strain induces only a very weak respiratory burst response in turkey monocytes. In contrast, C. psittaci itself elicited an intense non-superoxide mediated lucigenin-dependent CL, indicating that in chlamydial research the detection of superoxide, using lucigenin, should be confirmed with a specific superoxide inhibitor.     

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.     

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.     

Diagnosis of avian chlamydiosis: specificity of the modified Giménez staining on smears and comparison of the sensitivity of isolation in eggs and three different cell cultures.

For the diagnosis of chlamydiosis in dead and live birds different methods were compared for their sensitivity and specificity. The specificity of the modified Giménez staining and the direct immunofluorescence (DIF) test for direct demonstration of Chlamydia psittaci in organ, cloacal and/or conjunctival smears was examined. The sensitivity of the isolation of Chlamydia psittaci in 6 days embryonated specific pathogen free (SPF) chicken eggs, Buffalo Green Monkey (BGM) cell line, McCoy cell line and Vero cell line was compared. On smears, the direct immunofluorescence test was more specific than the modified Giménez staining. The concordance between the results of both detection methods was 80%. The BGM cell culture was the most sensitive artificial host for isolation of Chlamydia psittaci, followed by the embryonated eggs, the Vero cell line and the McCoy cell line. The concordance between the results of isolation in BGM cell culture and eggs was 96.5%, while it was 86% between the results of isolation in BGM cell culture and Vero cell culture and only 65.5% between the results of isolation in BGM cell culture and McCoy cell culture. For dead bird species, chlamydiosis could be diagnosed more often using DIF on smears than with isolation. The concordance between the results of the DIF on smears and isolation followed by DIF was 91%.