Experimental enteric infection of gnotobiotic piglets with a Chlamydia psittaci strain of avian origin

The pathogenicity of a Chlamydia psittaci isolate of pigeon origin was assessed using a litter of gnotobiotic piglets. At 3 days of age, six piglets were inoculated intragastrically with egg-grown chlamydiae, the remaining six pigs were sham-inoculated. The animals were observed for clinical signs, and they were killed and necropsied sequentially between 4 and 15 days of age. Clinical manifestations consisted of slight softening of the faeces between 6 and 10 days post-inoculation (DPI). Immunohistochemistry revealed chlamydial replication predominantly in the small intestine, initially within villous enterocytes, after 4 DPI mostly in the lamina propria. Histopathology showed villous atrophy and increased numbers of inflammatory cells in the gut up to 6 DPI. Chlamydial stages of normal morphology were identified within enterocytes using transmission electron microscopy. An enzyme-linked immunosorbent assay (ELISA) run on faecal samples revealed shedding of chlamydial antigen from 3 until 11 DPI. Systemic dissemination of Chlamydia occurred to a limited extent according to polymerase chain reaction and immunohistochemistry results of several extraintestinal organs. Corresponding histopathological changes were minimal. Sera of all pigs were negative for anti-chlamydial antibodies using a complement fixation test. In conclusion, inoculation of this isolate in gnotobiotic piglets resulted in a productive enteric infection with mild lesions, weak systemic dissemination, and faecal shedding, indicating the pig as a potential host for avian chlamydiae.     

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.     

Characterization of circulating antibodies against Chlamydia psittaci in turkeys.

Plasma and joint fluids from turkeys experimentally inoculated with Chlamydia psittaci strain TT3 were evaluated by immunoblotting to identify antibodies elicited by chlamydial antigens during the course of infection. Protein antigens from elementary bodies of TT3 were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred electrophoretically to nitrocellulose before being probed with plasma or synovial fluid from TT3-inoculated birds. The major outer-membrane protein (MOMP), the 60,000-molecular-weight proteins, and a 97,400-molecular-weight protein were the predominant antigens recognized by IgG in the plasma and joint fluids. Plasma IgG specific for the 97,400 protein band was first detectable at day 10 postinoculation (PI). Antibodies to the 60,000-molecular-weight protein and MOMP were first detected at days 14-17 PI and at days 7-10 PI, respectively, in some birds, and as late as days 36-42 PI and days 42-70 PI in others. The antibodies were still present at day 142 PI. Immunoblotting techniques indicated that the antigens to which these antibodies were reacting were protein. These observations may have implications for the development of serodiagnostic assays as well as the identification of potential proteins for subunit immunogens in birds.     

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.     

Restriction endonuclease analysis of DNA from ruminant Chlamydia psittaci and its relation to mouse virulence.

DNA from 20 pathogenic or non-pathogenic ruminant strains of Chlamydia psittaci was compared by restriction endonuclease analysis. The strains could be easily differentiated according to their invasiveness for mouse, whatever their pathological origin. DNA patterns of invasive strains were similar, whereas those of non-invasive strains were distributed in two groups.     

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.     

山东省部分地区家禽鹦鹉热衣原体感染情况调查

为了解山东地区家禽鹦鹉热衣原体(Cps)感染现状,本研究采用间接血凝法(IHA)对2013年~2014年采集自山东潍坊、淄博、济南、临沂、烟台等地区的1 020份鸡、鸭血清样品进行Cps抗体的检测,并对检测数据进行了统计分析;结果显示:IHA测得总阳性率为29.51%(301/1 020);鸡阳性率为25.26%(197/780);鸭阳性率为43.33%(104/240);各地区间阳性率存在一定差异。本调查结果表明,家禽Cps感染在山东地区具有较高的感染率。     

Antigenic analysis of avian Chlamydia psittaci using monoclonal antibodies to the major outer membrane protein.

Monoclonal antibodies to the major outer membrane protein (MOMP) of Chlamydia psittaci derived from a parrot were established for antigenic analysis of avian C. psittaci. With 17 monoclonal antibodies to MOMP, 17 reactivity patterns were identified on 112 strains of C. psittaci, C. pneumoniae and C. trachomatis, which were isolated from birds, mammals and humans in Japan, U.S.A., Canada and Taiwan, from 1938 to 1987. Immunological reactivity of budgerigar-derived strains to the monoclonal antibodies was different from that of pigeon-derived strains. Imported bird-derived strains were distinguishable from domestic bird-derived strains by the reactivity to the monoclonal antibodies. A close relationship between the subtypes and geographic origins was indicated on budgerigar-derived strains. On the contrary, various reactivity patterns were shown in pigeon-derived strains isolated in a narrow area. The monoclonal antibodies established in the present work may be useful probes for ecological study of avian C. psittaci.     

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.     

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.     

Nucleotide sequence of a gene encoding a new genus specific protein of Chlamydia psittaci.

DNA fragment No. 13 from the C. psittaci pigeon strain, has been cloned in the plasmid pUC19. Hybridization analysis revealed that the fragment maintained a chlamydial common sequence. Furthermore, nucleotide sequencing identified two partial open reading frames (ORF), 675b. p. and 530b. p. Expression of ORFs revealed that the second ORF encoded 25KD polypeptide, whereas the first ORF did not produce any antigenic product. The 25KD beta-galactosidase fusion protein reacted strongly with chlamydia-specific antibodies elicited against a number of different chlamydial strains. Gene Bank analysis showed that this cloned gene is not highly homologous with chlamydia or other organisms for which nucleotide sequences have already been published. This 25KD polypeptide may be an additional genus-specific antigen of Chlamydiae.     

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.     

Antigenic analysis of feline and bovine Chlamydia psittaci with monoclonal antibodies.

Monoclonal antibodies were established for antigenic analysis of feline and bovine Chlamydia psittaci. The monoclonal antibodies recognized lipopolysaccharide (LPS), 56-64, 84 or 86 kDa antigens. At least 5 antibody-binding sites were detected on LPS with the monoclonal antibodies. The 56-64 kDa antigen was suggested to have both polypeptide and carbohydrate antibody binding sites. Immunoblotting analysis of cat and cattle sera indicated that the 56-64 kDa antigen is an important antigen in host immune response. The monoclonal antibodies are extremely useful tools to analyse the structure and function of chlamydial antigens.     

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.