Characterization of DNA fragment from Chlamydia psittaci avian strain which shows high homology with hypB gene of Chlamydia.

A study was performed to characterize DNA fragment No. 17 of C. psittaci strain P-1041 which encoded 42 KD beta-galactosidase fusion protein with type-specific antigenicity. Sequence determination identified a partial open reading frame that spanned about 1,200b. p. nucleotides. Screening the literatures for the nucleotide and deduced amino acid sequences revealed extensive similarity between the DNA fragment of P-1041 and two chlamydial hypB genes. This DNA showed 91.5% homology with C. psittaci GPIC hypB gene in nucleotide sequence and 96.4% homology in deduced amino acid sequence. The hypB gene of C. trachomatis serovar A and the P-1041 DNA fragment showed 81.2% and 91.3% homology in nucleotide and amino acid sequences, respectively. Dot enzyme-linked immunosorbent assay, for the products of deleted DNA fragments defined the coding region for type-specific antigenic polypeptide. In addition, the P-1041 DNA fragment carried a sequence highly homologous (greater than 49%) with other bacterial and plant genes called chaperonin which responds to various stress in cells. From these results, the P-1041 DNA fragment was found to be a part of hypB gene and to encode the region critical for type-specific antigenicity.     

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.     

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.     

Immunocytologic detection of Chlamydia psittaci from cervical and vaginal samples of chronically infected ewes.

An immunocytologic method was developed for the detection of chronic Chlamydia psittaci infection from the reproductive tract of ewes. Vaginal and cervical samples from 8 infected and 2 non-infected ewes were stained with a C. psittaci-specific monoclonal antibody. Cells containing C. psittaci were only detected from the 8 infected ewes and the level of detection varied with respect to the estrus cycle. An increased number of infected cells were observed during the periovulation period, thus indicating an optimal window for detection.     

Production and characterization of monoclonal antibodies against Actinobacillus (Haemophilus) pleuropneumoniae serotype 2

Four hybridoma cell lines producing monoclonal antibodies (MAbs) against Actinobacillus (Haemophilus) pleuropneumoniae were established by fusion of mouse myeloma and spleen cells obtained from mice immunized with a serotype 2, strain SH-15. Enzyme-linked immunosorbent assay-inhibition tests with antigens obtained from 12 serotype strains of A. pleuropneumoniae and 9 other gram-negative bacteria showed that all the MAbs bound to only serotype 2 strains of A. pleuropneumoniae. The epitopes recognized by the MAbs were located on a carbohydrate moiety of lipopolysaccharide (LPS) of the organism, which was sensitive to periodate oxidation. In immunoblotting analyses of LPS obtained from A. pleuropneumoniae serotype 2, all the four MAbs reacted specifically with the characteristic ladder bands of LPS detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results suggest that O-antigen side chains of the LPS are one of the antigenic determinants responsible for the serotype-specificity of A. pleuropneumoniae.     

Characterization of the systemic disease and ocular signs induced by experimental infection with Chlamydia psittaci in cats

In addition to the commonly reported ocular signs, Chlamydia psittaci infection of kittens resulted in fever, lethargy, lameness and reduction in weight gain following ocular instillation of virulent organisms. The appearance of these systemic signs was late with respect to the appearance of ocular symptoms and occurred simultaneously with increasing levels of chlamydia-specific IgG. Measurement of acute phase reactants and IL-6 in plasma indicated that both became elevated concurrent with or slightly after the appearance of fever and remained elevated after the fever began to resolve. Preliminary data also indicated that infectious C. psittaci was present in the blood stream during this time period. The results of ocular instillation of three different levels of C. psittaci (10^3.8, 10^2.8 and 10^1.5 TCID₅₀) indicated that the frequency of infection and the severity of ocular signs were diminished in the group receiving the lowest dose. However, the magnitude of systemic disease was similar in all animals which exhibited clinical signs, irrespective of the dose administered. The immune response to infection included elementary body (EB)-specific lymphocyte proliferation as well as the development of EB-specific IgG and IgM antibodies. The predominant antibody response was to a 45 kDa protein, the major outer membrane protein (MOMP), lipopolysaccharide (LPS), a 58 kDa doublet and 32 and 16–19 kDa proteins.     

应用核酸杂交技术检测畜禽衣原体病的研究

从纯化山羊流产衣原体颗粒提取ＤＮＡ，用ＤＮＡ限制性内切酶切割，进行酶切图谱分析，与文献报道的图谱对比，证实确系纯的衣原体ＤＮＡ制品。将衣原体ＤＮＡ用光敏生物素标记，制成核酸探针，用斑点杂交法检测衣原体核酸，灵敏度可达１０ｐｇ。又用重组克隆技术将衣原体ＤＮＡ片段克隆于大肠杆菌质粒载体上，筛选出衣原体特异的ＤＮＡ片段制成光生物素探针，可以同样有交效地检测衣原体核酸。用光生物素衣原体探针检测了山羊、绵羊、豚鼠、小白鼠、鸡胚等的衣原体感染病料，结果准确。与间接血凝法检测衣原体感染做了对比试验，证明核酸杂交法检测衣原体病更为灵敏和特异     

Serotyping of chlamydial clinical isolates from birds with monoclonal antibodies

Forty-nine avian chlamydial strains, isolated mainly from various regions in France and from different species of birds, were analyzed and tested with a panel of nine monoclonal antibodies (MAbs) by the indirect microimmunofluorescence test (MIF). The MAbs included five serovar-specific MAbs, three MAbs raised against Chlamydia psittaci and Chlamydia pecorum ovine strains, and one genus-specific MAb. Of the 49 isolates, 41 came from parrots or budgerigars; the rest were from pigeons, a canary, a duck, and a dove. Two additional strains were from unknown hosts. Most of these avian strains were successfully serotyped according to their reactions with five serovar-specific MAbs by the MIF test. The serovars of 44 strains were determined: 39 were of serovar A, 3 of serovar B, and 2 of serovar E. The remaining five isolates were unclassified because they did not react with any of five serovar-specific MAbs but did react with genus MAb or the MAbs produced with ovine strains. The five unclassified isolates (two from budgerigars, two from Gabon gray parrots, and one from a duck) indicate that one or more additional serovars of C. psittaci exist in birds. The heterogeneity within each subgroup was evident because the 49 avian isolates gave 10 subgroups when the results of the five serovar-specific MAbs were combined with results from the three MAbs produced with ovine strains. This heterogeneity of the serovar isolates, as shown by the combination of MAbs, could provide strain markers very useful for epidemiologic studies.     

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.     

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.     

The tick-borne rickettsia Cowdria ruminantium has a Chlamydia-like developmental cycle.

The development of the tick-borne rickettsial pathogen Cowdria ruminantium (S stock) was studied in bovine umbilical endothelial (BUE) cell cultures and in goat choroid plexus, by light- and electron microscopy. Cowdria divided by binary fission within intracytoplasmic vacuoles resulting in large colonies of reticulate bodies. After three to four days in culture, reticulate bodies developed into smaller intermediate bodies characterized by an electron-dense core. Shortly before disruption of the host cells, intermediate bodies condensed further into electron-dense elementary bodies, which were released into the culture medium. Elementary bodies invade other endothelial cells thus initiating a new infectious cycle which lasts between 5 and 6 days. In the infected goat choroid plexus similar reticulate and intermediate bodies were identified within vacuoles of capillary endothelial cells. However, extracellular elementary bodies were not detected. Another stock of Cowdria (W) showed an identical developmental cycle as that of the S stock. The W isolate was also pathogenic for mice, making it possible to test the infectivity of reticulate and elementary bodies in these animals. Reticulate bodies appeared to be less infective than elementary bodies. The developmental cycle of Cowdria resembles the cycle known to occur in Chlamydia. Moreover, Cowdria has other similarities with Chlamydia. It has a Gram-negative envelope, it does not store iodine-stainable carbohydrates and may lack peptidoglycan as does Chlamydia. It is concluded, that Cowdria and Chlamydia are to a certain extent related, confirming a recent report that both organisms have certain antigenic determinants in common. Since Cowdria is also related to Ehrlichia it may well be that Cowdria takes an intermediate position between Chlamydia and Ehrlichia. The phylogenetic relationship between Cowdria and Chlamydia and also with Ehrlichia should be further elucidated by molecular analysis using 16S ribosomal DNA sequences.     

Immunoblot analysis of cyanogen bromide-cleaved Moraxella bovis pilin reveals presence of shared antigenic determinants on pili from heterologous strains

Moraxella bovis pilus proteins, collected and purified from four strains of M. bovis, were cleaved with cyanogen bromide. Two major fragments were produced. Antisera were produced in rabbits to the pilin protein fragments and to whole uncleaved pili from these strains. Immunoblots of whole and cyanogen bromide-cleaved pilin were reacted with the homologous and heterologous antisera to whole pili and cleaved pilin. Antisera to whole pili reacted strongly with homologous pilin. Weaker and inconsistent reactions were detected with heterologous pilin. Antisera produced to cyanogen bromide-cleaved pilin proteins reacted strongly with homologous and heterologous pilin fragments and uncleaved pilin proteins. These findings demonstrate the presence of conserved antigenic determinants on pili from heterologous strains that are non-immunogenic in the intact pilus but are immunogenic after treatment with cyanogen bromide. Cyanogen bromide-treated pilus preparation might have potential as a vaccine because antibodies are induced against heterologous strains of M. bovis, whether these cross-reactive antibodies are protective remains to be determined.     

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.     

Chlamydia psittaci infection and associated infertility in sheep.

Nineteen ewes were injected subcutaneously with the agent of enzootic ovine abortion, Chlamydia psittaci serovar 1, at 50 days gestation. Placental and fetal tissues were examined at 15 days postinfection and thereafter at ten day intervals. Placental infection was detected at 15 days postinfection. Only postinoculation sera collected from postinfected ewes contained antibodies reactive to C. psittaci. Five (26%) chlamydial infected ewes experienced inapparent fetal loss before day 105 of gestation. This finding is significant since C. psittaci infection in sheep is commonly associated with abortion and not infertility.     

Immunoblot analysis of cyanogen bromide-cleaved Moraxella bovis pilin reveals presence of shared antigenic determinants on pili from heterologous strains

Moraxella bovis pilus proteins, collected and purified from four strains of M. bovis, were cleaved with cyanogen bromide. Two major fragments were produced. Antisera were produced in rabbits to the pilin protein fragments and to whole uncleaved pili from these strains. Immunoblots of whole and cyanogen bromide-cleaved pilin were reacted with the homologous and heterologous antisera to whole pili and cleaved pilin. Antisera to whole pili reacted strongly with homologous pilin. Weaker and inconsistent reactions were detected with heterologous pilin. Antisera produced to cyanogen bromide-cleaved pilin proteins reacted strongly with homologous and heterologous pilin fragments and uncleaved pilin proteins. These findings demonstrate the presence of conserved antigenic determinants on pili from heterologous strains that are non-immunogenic in the intact pilus but are immunogenic after treatment with cyanogen bromide. Cyanogen bromide-treated pilus preparation might have potential as a vaccine because antibodies are induced against heterologous strains of M. bovis, whether these cross-reactive antibodies are protective remains to be determined.     

Efficacy against ovine enzootic abortion of an experimental vaccine containing purified elementary bodies of Chlamydia psittaci

A vaccine prepared from purified, inactivated elementary bodies of Chlamydia psittaci protected sheep against abortion after subcutaneous challenge with live chlamydiae. Immunoblot analysis of serum samples revealed a consistently dominant antibody response against the chlamydial major outer membrane protein in all vaccinated sheep. Reactions to other chlamydial antigens were also detected but were less pronounced or inconsistent. Serological responses detected by complement fixation were variable and did not correlate with immunity.     

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.     

Evaluation of relationship among three purified antigens from Pasteurella multocida strain P-1059 and of their protective capacities in turkeys

Three antigens were prepared from Pasteurella multocida strain P-1059, and their immunogenicity and antigenic relationships were investigated. The 3 antigens were a soluble antigen purified from a 2.5% NaCl extract (2.5S), a similar antigen purified from an extract in 0.3% formalin solution containing 0.85% NaCl (FS), and lipopolysaccharide (LPS). The antigens were treated with various chemicals and enzymes to study their antigenic and immunogenic determinants. Antigenic analyses with ELISA inhibition tests indicated that 2.5S and FS were similar LPS-protein complex antigens. The 2.5S and FS antigens induced protective immunity in turkeys with high antibody titers against LPS antigen. Although LPS was a component of 2.5S and FS, LPS itself was poorly immunogenic in turkeys. The antigenicity of protein compounds in 2.5S was deteriorated by protease treatment, which, however, did not significantly diminish the protective immunogenicity. Treatment of 2.5S with sodium periodate, altering its carbohydrate moieties, decreased its immunogenicity. The immunogenicity of 2.5S also was abolished by phenol-water treatment, owing to dissociation of the LPS-protein complex. These findings suggest that a certain form of LPS-protein complex is essential for the induction of immunity against the P multocida infection in turkeys.     

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 response to pgp3 protein in animal and human chlamydial infections

Specific antibodies to plasmid-encoded protein pgp3 are known to be encountered in human Chlamydia (C.) trachomatis infections. In order to verify whether antibodies to this protein could be developed in animals infected with plasmid-carrying chlamydial strains, 454 animal sera were examined using a home-made pgp3 protein ELISA and Western blots (WB) of recombinant pgp3 protein from Chlamydophila (Cp.) psittaci. Likewise, 50 human sera were tested by ELISA and WB of recombinant pgp3 from C. trachomatis. The reactivity against pgp3 protein was compared to the reactivity against chlamydial elementary bodies (EBs) detected by microimmunofluorescence (MIF) test. The presence of pgp3-specific antibodies was demonstrated in most ducks and pigeons with Cp. psittaci infection detected by MIF, as well as in the majority of symptomatic cats and pigs infected with Cp. felis and C. suis, respectively, which reacted at high titres to Cp. felis and C. suis EBs by MIF. Moreover, most of the sera collected from patients with C. trachomatis culture-confirmed infection and seropositive to C. trachomatis by MIF, presented antibodies specific to C. trachomatis pgp3 recombinant protein. Therefore, pgp3 protein could be a useful marker of chlamydial infections in animals, as well as in humans.