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.

Virulence of feline Chlamydia psittaci in mice is not a function of the major outer membrane protein (MOMP).

The virulent strain of feline Chlamydia psittaci, the Cello strain, produces conjunctivitis and upper respiratory disease in cats. This same strain produces a lethal disease in mice when inoculated intraperitoneally (i.p.). In this study we have shown that the Baker strain of feline C. psittaci is attenuated in the mouse model system. Intraperitoneal inoculation of mice with the Baker strain produced no disease but did stimulate an immune response that protected the mice from subsequent produced i.p. inoculation with the virulent Cello strain. To determine if the difference between these two strains was in the major outer membrane protein (MOMP), the omp1 gene which codes for this protein was sequenced for both the Baker and Cello strains. The MOMP was chosen to study because in Chlamydia trachomatis this protein has been shown to contain neutralizing epitopes and has been shown to play a role in cell attachment. These functions make it a likely structural component capable of mutating and causing altered cell tropism and virulence. The DNA sequence of the omp1 was determined by amplifying the gene with PCR, cloning the PCR product into the pCR-II cloning vector and determining the DNA sequence of the inserted gene using primers to sites in the plasmid vector. From the DNA sequence, the deduced amino acid sequence of MOMP was determined for both the attenuated Baker and the virulent Cello strains of feline C. Psittaci. The results indicated that the omp1 gene of both strains contained 1179 base pairs which coded for a protein 392 amino acids. The DNA sequences of the omp1 gene of the two strains differed by only two base pairs which resulted in two amino acid changes in the MOMP. The Baker strain had a serine instead of a tryptophan at amino acid 7 and a tyrosine instead of an aspartic acid at amino acid 125 of the uncleaved protein. Neither amino acid change was in an area of the MOMP which could logically account for the difference in biological activity. Amino acid 7 was in the leader sequence which is cleaved from the authentic MOMP and is not present in the infectious elementary body. Amino acid 125 was in a conserved hydrophobic area of one of the constant regions of the protein. A change at this location was not likely surface exposed and thus could not affect cell adhesion, tissue tropism or neutralizing epitopes. Therefore, the differences in the primary structure of the MOMP from the Baker and Cello strains of feline C. psittaci could not account for the attenuation of the Baker strain for mice. The molecular basis of their difference is yet to be determined.

Evaluation of Chlamydia psittaci subfraction and subunit preparations for their protective capacities.

A mouse toxicity potency test was used to evaluate the protective capacity of Chlamydia psittaci (Baker strain) subfraction and subunit preparations. The elementary body (EB), Chlamydial outer membrane complex (COMC), major outer membrane protein (MOMP), lipopolysaccharide (LPS), and MOMP mixed together with LPS were utilized as antigens in vaccine preparation. The EBs and COMCs were prepared by centrifugation and detergent extraction as previously described for Chlamydia trachomatis. Isolation and purification of MOMP and LPS were achieved by subjecting infected tissue culture fluids to polyacrylamide gel electrophoresis and subsequent elution from the gel. Chromatographic purification of the MOMP from EBs resulted in a preparation which also contained minor quantities of LPS. After vaccination and intraperitoneal challenge with C. psittaci (Cello strain), the MOMP presented in EBs, COMCs or in a purified form, was capable of protecting mice from death, whereas LPS did not demonstrate this capability. Further comparison of the protective capacity of these antigens indicated that the electrophoretically purified MOMP was the least protective when suboptimal levels of antigens were administered to mice. However, the level of protection afforded by electrophoretically purified MOMP could be increased to that observed with the chromatographically purified MOMP by the addition of electrophoretically purified LPS to the vaccine. These results suggest that the MOMP is the major protective antigen of C. psittaci and that the LPS component, although not protective on its own, may play a role in the potentiation of a protective immune response.