Single channel analysis of recombinant major outer membrane protein porins from Chlamydia psittaci and Chlamydia pneumoniae.

We recently demonstrated that the major outer membrane protein of Chlamydia psittaci, the primary vaccine candidate for combating chlamydial infections, functions as a porin-like ion channel. In this study, we have cloned, expressed and functionally reconstituted recombinant major outer membrane proteins from C. psittaci and Chlamydia pneumoniae and analysed them at the single channel level. Both form porin-like ion channels that are functionally similar to those formed by native C. psittaci major outer membrane protein. Also, like the native channels, recombinant C. psittaci channels are modified by a native major outer membrane protein-specific monoclonal antibody. This is the first time that native function has been demonstrated for recombinant chlamydial major outer membrane proteins. Future bilayer reconstitution will provide a strategy for detailed structure/function studies of this new subclass of bacterial porins and the work also has important implications for successful protein refolding and the development of improved subunit vaccines.

Identification of a multigene family coding for the 90 kDa proteins of the ovine abortion subtype of Chlamydia psittaci.

While attempting to identify genes and their corresponding antigens that could be used to improve the current methods of diagnosing Chlamydia psittaci infection which causes enzootic abortion in ewes, two candidate clones were isolated from a lambda gt 11 genomic DNA expression library of ovine abortion subtype (strain S26/3) C. psittaci. These clones contained fragments of a gene coding for a group of three chlamydial proteins of approximately 90 kDa which appeared as major immunogens by immunoblotting experiments, indicating their potential as diagnostic or possibly protective antigens. Southern blotting of S26/3 genomic DNA using the two clones as probes identified a family of three or four genes. These represent the first example of protein gene duplication reported in Chlamydia.

Sequence analysis of the major outer membrane protein gene of an ovine abortion strain of Chlamydia psittaci.

The major outer membrane protein (MOMP) gene from an ovine abortion strain of Chlamydia psittaci (S26/3) has been cloned and sequenced. The gene shows the features of other chlamydial MOMPs but comparison with the previously reported sequence for the ovine abortion isolate A22/M has revealed substantial sequence divergence which is clustered into the same four intramolecular regions as the sequence variation found between C. trachomatis serovars. Subsequent restriction enzyme analysis of A22/M DNA has shown that it has an avian-type genomic profile and thus the comparison is between types rather than between strains.

A liquid-hybridization method for typing the Vp4 and Vp7 genes of bovine rotaviruses.

A simple liquid-hybridization assay was developed which allows assessment of the degree of hybridization between the two serotype-determining genes of the bovine rotavirus strain UK and the homologous genes of the isolate under test. 32P-labelled transcription probes were produced from cloned complementary DNA (cDNA) copies of UK gene segments 4 and 8 and hybridized to double stranded RNA (dsRNA) extracted from rotavirus-positive field samples. Subsequent treatment with ribonuclease A (RNase A), separation of the RNase A-resistant hybrid fragments by polyacrylamide gel electrophoresis (PAGE) and autoradiography yielded a specific, reproducible banding pattern for each isolate. A total of 74 field samples was tested by both the hybridization assay and by an enzyme-linked immunosorbent assay (ELISA) using serotype-specific monoclonal antibodies (Mabs). The results obtained were in excellent agreement and confirmed that serotype G6 rotaviruses predominated. Hybridization of these G6 viruses with the gene 4 probe suggested that viruses with Vp4s related to that of UK rotavirus are also common. The hybridization assay was more sensitive than the ELISA.

Comparison of isolates of Chlamydia psittaci of ovine, avian and feline origin by analysis of polypeptide profiles from purified elementary bodies.

The single species Chlamydia psittaci is a diverse grouping which contains several different types of chlamydial strain for which there is no generally accepted typing method. The results obtained when profiles of polypeptides from purified elementary bodies are compared are consistent with type designations obtained using other criteria. However, the method still requires large scale culture and extensive purification of the chlamydial cells.

Rapid detection of bovine herpesvirus 1 (BHV 1) using the polymerase chain reaction.

A polymerase chain reaction (PCR) assay based on primers from the viral gI glycoprotein gene detected 3 fg pure BHV-1 DNA, 0.1-1.0 TCID50 or a single infected cell. No amplification was observed with DNA from BHV-2, BHV-3, BHV-4, OHV-1 or OHV-2. However, a fragment of the correct size was amplified using DNA from herpesviruses isolated from reindeer, red deer and goats. The PCR assay was able to detect virus in nasal swabs up to 14 days after experimental infection of cattle and there was a good correlation when PCR was compared with virus isolation for the detection of BHV-1 in clinical field samples. Detection of BHV-1 in fetal bovine serum and semen samples was also successful.

Cloning and expression of the 75 kDa DnaK-like protein of Chlamydia psittaci and the evaluation of the recombinant protein by immunoblotting and indirect ELISA.

The 75 kDa dnaK-like gene of Chlamydia psittaci ovine abortion strain S26/3 was isolated from an EMBL 3 chlamydial DNA library. A 7 kb DNA fragment containing the gene was subcloned into Bluescribe (M13+) plasmid and used to transform competent E. coli. These cells were found to express a cytoplasmic protein of 75 kDa. Monospecific antibodies against the protein prepared by antibody elution reacted with the native 75 kDa protein. Recombinant clones did not adhere to McCoy cell monolayers in cell adhesion studies. The 75 kDa protein purified by ion-exchange chromatography was used in immunoblotting and indirect enzyme-linked immunosorbant assay (ELISA) studies using sera previously screened for chlamydial antibodies by an indirect ELISA incorporating solubilised chlamydial elementary bodies and by microimmunofluorescence. Immunoblotting identified 6/11 sera from infected ewes that had either typical placental lesions or had been found positive on examination of stained placental smears and 1/11 sera from ewes that had no typical placental lesions. The ELISA gave positive reactions with 29 of 65 known positive sera and 15 of the 76 negative sera. It is concluded that the 75 kDa DnaK-like protein is unsuitable as an antigen for antibody detection but its potential as a component for a sub-unit vaccine against ovine enzootic abortion warrants further study.

Further evidence for a retrovirus as the aetiological agent of sheep pulmonary adenomatosis (jaagsiekte).

The infective agent of jaagsiekte was shown to be present in the fluid which accumulates in the respiratory tract of sheep during the terminal stages of the disease. The fluid also contained reverse transcriptase (RT) activity which showed a clear preference for a ribonucleic acid synthetic template over the corresponding deoxyribonucleic acid template and which utilised the RT specific template/primer poly (2'-0-methylcytidylate) oligodeoxyguanylate. This enzyme activity was associated with a particle which had typical retroviral buoyant densities in a range of gradient media.

Identification of a region of the alcelaphine herpesvirus-1 genome associated with virulence for rabbits.

The gammaherpesvirus Alcelaphine Herpesvirus 1 (AHV-1) causes the fatal lymphoproliferative disease known as malignant catarrhal fever (MCF), in susceptible hosts. The virulent C500 isolate of AHV-1 became attenuated for the laboratory model, the rabbit, as a result of serial passage in cells of bovine origin. This work describes the identification of a region of the central unique sequence of the C500 genome, located close to the terminal repeat units of the molecule, which is altered on attenuation. The virulent C500 genome contains two copies of a sequence of approximately 2 kbp, contained within a 7 kbp region of the unique DNA located adjacent to the terminal repeats at the left end of the molecule. In the genome of the attenuated virus, there are also two copies of the 2 kbp sequence but they are located at the ends of the attenuated genome unique region, adjacent to the terminally repeated sequences. One open reading frame (ORF), designated putative polypeptide 5, was altered on attenuation such that the 3' sequence was lost. The location of this ORF, coupled with the loss of its 3' sequence, suggests that this ORF may encode a gene involved in the virulent mechanisms of this virus, in a manner similar to that of the transforming proteins of Herpesvirus saimiri (HSV).

A qualitative and quantitative assessment of the humoral antibody response of the sheep to orf virus infection.

The serological response of naturally and experimentally infected lambs to orf virus infection was analysed using an enzyme-linked immunosorbent assay (ELISA) together with the Western blotting technique. The combination of these two methods permitted a qualitative and quantitative assessment of the response, which revealed considerable variation between animals. Despite this, all post-exposure sera reacted with a polypeptide (molecular weight 40 kilodaltons) which appears to be a component of the surface tubules that are characteristic of the virus.

Development and evaluation of an indirect ELISA to detect antibodies to abortion strains of Chlamydia psittaci in sheep sera.

A novel indirect enzyme-linked immunosorbent assay (ELISA) for antibodies against abortion strains of Chlamydia psittaci (C. psittaci) has been developed. The antigen used was chlamydial elementary bodies treated sequentially with N-lauroyl sarcosine and n-octyl-beta-D-glucopyranoside and finally solubilized with N-lauroyl sarcosine and dithiothreitol. Treating the antigen with sodium periodate after coating of the plates increased the specificity for antibodies to abortion strains. The test was evaluated initially with sera from experimentally infected sheep and an uninfected control group. These sheep were monitored for lambing performance and infection status. When used in conjunction with the indirect micro-immunofluorescence test (MIF), the ELISA was able to identify as negative all twenty-five sera from ewes that had no typical placental lesions and identified as positive twenty of twenty-one sera from infected ewes that had either typical placental lesions or had been found positive by isolation of chlamydia in cell culture. The combination of ELISA and MIF was also able to discriminate correctly groups of sera from six flocks with a history of infection from four known uninfected flocks.

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.

Antibody response of the ovine lymph node to experimental infection with an ovine abortion strain of Chlamydia psittaci.

After primary infection with Chlamydia psittaci in the draining area of the popliteal lymph node, viable organisms could be isolated from the efferent lymph only before the primary immune response developed. The lymph antibody response, as assayed by the complement fixation and immunofluorescence (IF) antibody tests, showed a rise in titre that peaked approximately 2 weeks after infection. Immunoblotting revealed that antibodies produced during this period were predominantly directed against the major outer membrane protein (MOMP). In secondary infection of convalescent sheep, an elevated IF antibody titre, already present in the lymph and blood, could not be boosted. Viable organisms could not be isolated from these sheep. Antibodies produced reacted to 12-14 bands in the immunoblot profile including the MOMP band. These potentially immunoprotective antigens, particularly MOMP, should be considered as useful candidates for an improved vaccine against ovine enzootic abortion, in further investigations.

Comparative studies of strains of infectious bovine rhinotracheitis virus isolated from latently infected calves.

Three strains (479 C, 778 TL, 982 LE) of infectious bovine rhinotracheitis (IBR) virus isolated from latently infected calves were compared with the prototype strain of IBR virus (LA strain) in studies which included restriction endonuclease analysis, experimental infection, and reciprocal cross protection tests in cattle. From the restriction endonuclease analysis it appeared that the 3 "latent" viruses were derived from the same isolate, and that it differed slightly from the LA strain. However, latency does not seem to have affected the pathogenicity or the immunogenicity of the virus. This is demonstrated by the identical clinical and virologic response of calves subjected to experimental infection with the various strains under study, and by the finding that when the LA strain and a "latent" strain (982 LE) were tested in cross protection tests in cattle, they proved to be mutually protective.

Pathogenesis of Chlamydia psittaci infection in sheep: detection of the organism in a serial study of the lymph node.

Sixteen sheep were inoculated subcutaneously over the left prefemoral lymph node with an abortifacient strain of Chlamydia psittaci. Groups of four animals were killed after 3, 6, 12 and 18 days. Four of eight sheep which received a control inoculum were killed on day 6 and four on day 18. The left and right prefemoral lymph nodes were removed and weighed and portions taken from each for examination by the polymerase chain reaction (PCR), by culture, and by histopathological and immunohistochemical methods. The left prefemoral lymph nodes enlarged after the injection of C. psittaci, with the group mean weight on day 6 being the greatest and that on day 18 being normal. Examination by "nested" PCR showed samples from these nodes to be positive, except for one animal killed on day 3 and one on day 12. Live organisms, however, were not cultured from any of the samples collected. C. psittaci antigen was detected immunohistochemically in three of four nodes on day 3, in each of four on day 6, and in two of four on both days 12 and 18. Nodes from the contralateral side remained normal, as did those from unchallenged control sheep, and no antigen or DNA was detected in them.

Evaluation of an immunofluorescent test for the rapid diagnosis of field infections of infectious bovine rhinotracheitis.

An indirect immunofluorescent test for the rapid detection of infectious bovine rhinotracheitis (IBR) virus in smears of nasal and ocular secretions from infected cattle, was compared with conventional virus isolation procedures using 200 swabs from 107 field outbreaks of suspected IBR. Virus was isolated from 38 per cent of the swabs and the indirect immunofluorescent test detected virus in 14.5 per cent of the positive swabs. Examination of samples from more than one animal increased the confirmation rates of infection during outbreaks to 39 per cent by virus isolation and 21.5 per cent by the immunofluorescent test. Ocular swabs were better than nasal swabs for confirming infection both by virus isolation and immunofluorescence, and agreement between the two tests increased with the number of samples collected during an outbreak.

Detection of bovine herpesvirus 1 in clinical samples by the polymerase chain reaction.

A PCR assay with primers selected from the gl gene and flanking a 468 bp DNA fragment was tested on clinical samples. Of 27 samples (nasal swabs, lung, lymph nodes, tracheal mucosa) collected from 16 different outbreaks in Scotland, 18 were positive by PCR and 13 by virus isolation. Some samples of isolated DNA had to be diluted by a factor 50-100 to obtain a positive PCR result. PCR assay could detect the BHV-1 positive samples collected from different European regions, namely from Slovakia, Italy, England, Scotland as well as the IPV sample.