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Analysis of caprine IgG1 and IgG2 subclass responses to Chlamydia psittaci infection and vaccination.

Enzyme-linked immunosorbent assays (ELISAs) specific for caprine IgG(H+L), IgG1 and IgG2 were developed and evaluated for serodiagnosis of Chlamydia psittaci infections in a Tunisian goat flock with currently occurring chlamydial abortions and a clinically inapparent goat flock of an animal research facility. Additionally, ELISAs were applied to record the IgG1 and IgG2 dynamics of four goats vaccinated with inactivated Chlamydia psittaci and Coxiella burnetii. For screening purposes, the IgG(H+L) ELISA proved to be superior to the complement fixation test because it detected a larger number of chlamydial abortions and was easier to perform and to interpret. Analysis of Chlamydia psittaci-specific IgG1 and IgG2 responses to naturally occurring infections by ELISA revealed high IgG1 levels associated with IgG2 in goats with current abortions, whereas clinically inapparent, but seropositive goats were characterized by significantly lower IgG1 levels only (P less than 0.001). Similarly, the four vaccinated goats responded initially with Chlamydia psittaci-specific IgG1, whereas second and third vaccinations induced (as in goats with chlamydial abortions) predominantly IgG1, but also IgG2. The results indicated that clinically inapparent chlamydial infection may be distinguished from overt disease by analysing specific IgG1 and IgG2 responses. Applying Coxiella burnetii- specific ELISAs on field samples, IgG1 alone could be detected in eight, IgG2 alone in one and IgG1 combined with IgG2 in nine goats. The coxiella-specific antibody response of the four vaccinated goats was--in contrast to the chlamydia-specific response--characterized by IgG2 dominance.

Abortion, Veterinary↗

Comparison of some ovine Chlamydia psittaci isolates by indirect immunofluorescence.

Four ovine abortion isolates, including the A22 vaccine isolate, and an isolate from a case of lamb arthritis, were compared by indirect immunofluorescence using antisera raised in chickens and mice. Cross titrations with homologous and heterologous antisera showed a one-way reaction between the single chlamydial isolate from a lamb with arthritis and the four isolates from cases of ovine enzootic abortion. The abortion isolates could not be distinguished.

Abortion, Veterinary↗

Comparison of DNA-spot hybridization, cell culture and direct immunofluorescence staining for the diagnosis of avian chlamydiae.

DNA-spot hybridization, cell culture and direct immunofluorescence staining were compared for the detection of avian Chlamydia psittaci strains in cell culture dilutions and in routine samples submitted for diagnosis. With dilutions of infected cell culture material, growth in BGM cells was by far the most sensitive technique, detecting 0.01 infected cells (20 elementary bodies) ml-1. DNA-spot hybridization and direct immunofluorescence staining were of approximately equal sensitivity, both detecting 16 infected cells (3.2 x 10(4) elementary bodies) per ml-1. When 27 avian liver and spleen samples were assayed, all 3 tests performed similarly (13 positive and 12 negative by all 3 tests). This suggests that in most avian samples presented for diagnosis, sufficient numbers of chlamydiae are present to allow any of the test to the be used. Thus, the direct immunofluorescence staining method is currently the test of choice for routine diagnosis since it is available in kit form, is relatively simple and quick to perform, and like DNA-spot hybridization, detects non-viable as well as viable organisms. However, if low levels of chlamydiae are to be effectively detected, such as in carrier birds or birds with recently acquired infections, then cell culture should be used.

Animals↗

Relationship between virulence of Chlamydia psittaci strains and establishment of persistent infection of McCoy cells.

The pathogenicity of chlamydial strains for their natural hosts and their ability to induce persistent infections in McCoy cells were compared. Both virulent and avirulent strains persistently infected McCoy cells, but the appearance of the cell culture varied between strains. Avirulent strains induced completely inapparent persistent infection (infection Type 1), while with invasive strains the culture alternated between periods of cell multiplication and periods of extensive cytopathic change (infection Type 2). The virulence of virulent strains was not attenuated, even after 6 months of culture, but after 2 or 3 months some avirulent strains produced infection Type 2 and became invasive for mice and abortive for ewes. This variation of virulence was accompanied by a modification of protein patterns.

Abortion, Veterinary↗

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.

Abortion, Veterinary↗

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.

Abortion, Veterinary↗

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.

Animals↗

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.

Abortion, Veterinary↗

Immunity to Chlamydia psittaci with particular reference to sheep.

Chlamydia psittaci, a zoonotic bacterium, is the causal agent of enzootic abortion of ewes, an important disease of sheep in many European countries. The major thrust of current chlamydial research is directed towards the human pathogen Chlamydia trachomatis. This review attempts to bring together relevant information concerning the host immune response to all members of the genus Chlamydiae and show how this has led to an increased understanding of the ovine humoral and cell mediated immune responses to C. psittaci while emphasising areas where there is still a lack of knowledge. Specifically the review looks at the common immuno-accessible antigens of the Chlamydiae and the antibody responses produced during infection, as well as covering the role of T cells and cytokines in the protective immune response.

Abortion, Veterinary↗

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.

Animals↗

Abortion in Japanese cows caused by Chlamydia psittaci.

An outbreak of abortion in cows occurring in Niigata Prefecture was shown to be caused by Chlamydia psittaci. Elementary bodies characteristic of Chlamydia were found in the liver of aborted fetuses and C. psittaci antigen was demonstrated by indirect immunofluorescence. Chlamydia was isolated from the liver of aborted fetuses by the yolk sac inoculation of developing chick embryos and by the intraperitoneal inoculation of guinea pigs. Abortion occurred mostly in middle or late pregnancy. Aborted fetuses showed subcutaneous edema and gelatinous infiltration, enlarged liver and spleen, and dark red pleural and ascitic fluid. Focal necrosis was shown in the liver, spleen and lymph nodes. Serological findings and isolation of Chlamydia from fecal specimens indicated a wide dissemination of C. psittaci among cows in the area.

Abortion, Veterinary↗

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.

Abortion, Veterinary↗

Antigenic and morphological differentiation of placental and intestinal isolates of Chlamydia psittaci of ovine origin.

Ewe placental and lamb intestinal isolates of Chlamydia psittaci recovered from flocks affected with ovine enzootic abortion were examined by inclusion morphology, indirect immunofluorescence (IIF) and immunoblot analysis. Chlamydiae recovered from the faeces of sheep from two flocks free of clinical disease were also examined. In cell culture ovine abortion (OA) and intestinal isolates were distinguishable by inclusion development and morphology. Similarly, in two-way IIF tests with one week mouse antisera isolates fell into two distinct groups: abortion or intestinal. Immunoblotting with convalescent sheep abortion antiserum identified 30 out of at least 40 silver staining polypeptides as antigenic both in OA and intestinal isolates. The serum produced a similar reaction pattern to the resolved proteins of each OA isolate, indicating a higher degree of antigenic conservation among these isolates. Considerable cross reactivity between the OA and intestinal isolates was identified, but the serum also showed apparent molecular weight differences between antigens of the two types in the 87-116 kDa, 38-44 kDa and 26-28 kDa regions. Furthermore, the immunoblotting analysis revealed heterogeneity among the intestinal isolates, particularly in antigens between 87-116 kDa and 38-44 kDa.

Abortion, Veterinary↗

Primary pathogenicity of an European isolate of Chlamydia psittaci from turkey poults.

Chlamydia psittaci was isolated as the sole pathogenic agent from a severe outbreak of respiratory disease in a commercial broiler turkey farm in the Netherlands. The mortality rate in the flocks was 65%. Clinical signs included conjunctivitis, swelling of the sinus infraorbitalis and sneezing. Cloacal excretion of chlamydia was demonstrated in twelve out of fifteen birds examined by a direct immunofluorescence test. In all the fifteen birds antibodies against Chlamydia psittaci were detected in the sera by a competitive ELISA. At necropsy sinusitis, rhinitis, airsacculitis, pneumonia, pericarditis and enlargement of the liver and spleen were found. Chlamydiae were demonstrated in the sinus material of all and in conjunctival smears of eight of the fifteen examined birds. Chlamydiae were isolated from all the examined birds after one to three passages on Buffalo Green Monkey (BGM) cell cultures using samples taken from lung, liver and spleen. No other pathogens were isolated. The chlamydia isolate was typed using a panel of serovar-specific monoclonal antibodies in a micro-immunofluorescence test. The isolate belonged to the avian Chlamydia psittaci serovar D. Experimental inoculation with this isolate of 7-day-old specific pathogen free (SPF) turkeys resulted in severe clinical signs, with mortality and extensive pathological lesions, similar to those seen in turkeys from the examined broiler turkey farm. From the data it was concluded that this Chlamydia psittaci isolate can cause severe disease in turkeys.

Animals↗

Comparison of serological tests for the diagnosis of Chlamydia psittaci infection of sheep.

An enzyme-linked immunosorbent assay (ELISA) was developed and compared with the complement fixation test (CFT) and the indirect immunofluorescence antibody test (IIFAT) for the detection of antibodies to C. psittaci in sheep. Field sera and experimental sera from aborting ewes, subclinically infected sheep and vaccinated sheep were used. The ELISA and IIFAT were much more sensitive than the CFT. Agreement between the CFT and ELISA varied between 50% and 98%, while agreement between the IIFAT and ELISA varied between 70.5% and 94.3%. The specificity of IIFAT and ELISA with regard to the identification of animals which may be a source of ovine abortion strains of C. psittaci was critically assessed, using antigen preparations derived from an abortion isolate and from a non-abortion isolate of C. psittaci. Indirect immunofluorescence was shown to be the more discerning test.

Abortion, Veterinary↗

Comparison of ovine abortion and non-abortion isolates of Chlamydia psittaci using inclusion morphology, polyacrylamide gel electrophoresis, restriction endonuclease analysis and reactivity with monoclonal antibodies.

Twelve reference and four Northern Ireland ovine Chlamydia psittaci isolates including ovine abortion, faecal, conjunctivitis and arthritis isolates were compared. Inclusion morphology was shown to provide a useful means of differentiating the abortion and the non-abortion isolates studied. Identical SDS-PAGE polypeptide profiles were produced by the ovine abortion isolates. The polypeptide profiles of the non-abortion isolates were similar to one another and clearly distinct from the abortion isolate profiles. The restriction endonuclease profiles of the abortion isolates were remarkably similar whereas different profiles were produced by most of the non-abortion isolates. Monoclonal antibodies were prepared and characterized. A number of these reacted with all the isolates of chlamydia tested. Three mAbs reacted exclusively with the ovine abortion isolates while four mAbs reacted exclusively with a number of the faecal isolates.

Abortion, Veterinary↗

Comparison of type I and type II Chlamydia psittaci strains infecting koalas (Phascolarctos cinereus).

The native Australian marsupial Phascolarctos cinereus, otherwise known as the koala, is prone to infection by the obligate intracellular parasite Chlamydia psittaci, which causes ocular 'pink eye' and urogenital 'dirty tail' diseases. Several chlamydial DNA probes to both chromosomal and plasmid sequences were used to type by Southern blot analysis 51 samples taken from wild and captive koalas from habitats on the eastern seaboard of Australia as far apart as Queensland and Victoria. Two types of C. psittaci were observed and called types I and II. Type II was found more frequently than type I and occurred in both ocular and urogenital samples, while type I showed a strong but not absolute preference for ocular sites. Cross-hybridization analyses indicated that type I and type II had about 10% DNA sequence identity to each other. DNA analyses showed that type II was very closely related to some ovine and bovine chlamydiae but type I could not be related to any other C. psittaci strain available. Light and electron microscopic analyses of infected BGM monolayers revealed that the two strains were similar in morphological characteristics. The type I strain was considerably more infectious than the type II strain in BGM cells and in the yolk sacs of embryonated eggs. A PCR based assay detected both type I and type II koala chlamydiae in samples that had been negative by Southern blot and tissue culture and provided the first evidence that both types can occur simultaneously at the one site of infection.

Animals↗

Experimental infections of pregnant sows with ovine Chlamydia psittaci strains.

Four sows were experimentally inoculated at 42 days pregnancy, with the BS isolate of Chlamydia psittaci from aborted ewes. Their body temperature and health were monitored until 30 days after farrowing. The sows seroconverted but their reproductive performance was not impaired. Chlamydiae were identified in the afterbirths from two sows with normal litters using stained smears, "Clearview" and "IDEIA" ELISA antigen detection tests. The presence of inclusion bodies was detected in sections of the infected fetal membranes after staining with haematoxylin and eosin, methylene blue and Streptavidin-Biotin using an anti-Chlamydia monoclonal antibody.

Animals↗