Causative Agent Of Tick-borne Fever Research Articles

Production of Tumour Necrosis Factor-alpha (TNF-α) and Reactive Nitrogen Intermediates by Ovine Peripheral Blood Leucocytes Stimulated by Ehrlichia (Cytoecetes) phagocytophila

Ehrlichia (Cytoecetes) phagocytophila, the causative agent of tick-borne fever in sheep and pasture fever in cattle, is an immunosuppressive, obligately intracellular rickettsia that invades granulocytes and monocytes of ruminants. Infected animals are known to suffer from a number of secondary infections. The mechanisms of immunosuppression are believed to be associated with physical or functional damage to leucocytes and the release of immunosuppressive substances. In the present study, the effects of E. phagocytophila on the production of tumour necrosis factor-alpha (TNF-α) and reactive nitrogen intermediates by ovine peripheral blood mononuclear cells (PBMCs) were investigatedin vivo and in vitro. The concentration of TNF-α and nitrate in ovine sera were significantly increased during infection with E. phagocytophila, peak concentrations occurring at the peak period of rickettsiaemia. The addition of E. phagocytophila to cell cultures enhanced in-vitro production of TNF-α and nitric oxide by normal ovine PBMCs.

Lymphocyte responses to mitogens and rickettsial antigens in sheep experimentally infected with Ehrlichia (Cytoecetes) phagocytophila

Infection of sheep with Ehrlichia (Cytoecetes) phagocytophila, the causative agent of tick-borne fever (TBF), was characterised by a significant reduction in lymphocyte reactivity to the mitogens phytohaemagglutinin, concanavalin A, pokeweed mitogen and Escherichia coli lipopolysaccharide during the period of rickettsiaemia. The addition of the prostaglandin inhibitor, indomethacin, or the nitric oxide inhibitor, N G-monomethyl- l-arginine, had no significant effect on the suppressive effects of E. phagocytophila on lymphocyte reactivity to the mitogens. However, peripheral blood lymphocytes obtained from primed sheep proliferated in the presence of live or heat-inactivated E. phagocytophila. Antigen-specific proliferation was detected in lymphocytes samples obtained 11 to 21 days post-inoculation with E. phagocytophila.

Inhibition of Phagosome-lysosome Fusion in Ovine Polymorphonuclear Leucocytes byEhrlichia (Cytoecetes) phagocytophila

Ehrlichia (Cytoecetes) phagocytophila, the causative agent of tick-borne fever, is an intracellular bacterium that survives and multiplies within granulocytes and monocytes. In the present study, the possible fusion of lysosomes with phagosomes containingE. phagocytophilawas investigated in poly-morphonuclear (PMN) cells of sheep infected with the agent, acid phosphatase cytochemistry and cationized ferritin being used as markers of primary and secondary lysosomal enzymes. Latex beads orCandida albicanswere incubated with infected and uninfected PMN cells and labelled with the same lysosomal markers. Lysosomal enzymes labelled with the markers were commonly found in phagosomes containing latex beads orC. albicans, but there was no evidence of phagosome-lysosome (P-L) fusion in phagosomes containingE. phagocytophila. It was significant that in cells that containedE. phagocytophila, latex beads andC. albicans, P-L fusion occurred only in phagosomes containing latex beads orC. albicans. However, evidence of P-L fusion with phagosomes containingE. phagocytophilawas obtained when PMN cells were incubated with oxytetracycline, which is known to inhibit synthesis of bacterial proteins.These findings indicate thatE. phagocytophilais capable of inhibiting P-L fusion and that oxytetracycline depresses this capability.

The effects of tick-borne fever on the susceptibility of ovine polymorphonuclear cells to P. haemolytica Cytotoxin

Tick-borne fever predisposes sheep to secondary infections by Pasteurella haemolytica. Polymorphonuclear (PMN) cells obtained from sheep 2 to 7 days after the onset of parasitaemia due to Cytoecetes phagocytophila, the causative agent of tick-borne fever, were significantly more susceptible in vitro to the cytotoxin of P. haemolytica than PMN obtained from age-matched control sheep.

Purification of Cytoecetes phagocytophila, the causative agent of tick-borne fever, from Infected ovine blood

Cytoecetes phagocytophila, the causative agent of tick-borne fever, was successfully separated by Percoll and Renografin density gradient centrifugation and by cellular affinity chromatography, from the peripheral blood leucocytes of experimentally infected sheep. After centrifugation on Renografin or Percoll density gradients, infectious particles of C. phagocytophila banded at buoyant densities which ranged between 1.063 to 1.140. Rickettsiae separated by wheat germ lectin cellular affinity chromatography retained their morphology but often lost their infectivity. Cell-free C. phagocytophila remained infective to susceptible sheep for 6 months when kept at -114 degrees C in sucrose phosphate buffer with 10 per cent dimethylsulphoxide as a cryopreservative.

Lymphocyte subpopulations in peripheral blood of sheep experimentally infected with tick-borne fever

The lymphocyte subpopulations in the peripheral blood of normal sheep and sheep experimentally infected with Cytoecetes phagocytophila, the causative agent of tick-borne fever, were analysed by flow cytometry, using a panel of monoclonal antibodies against specific lymphocyte epitopes. Experimental infection with tick-borne fever was characterised by a significant reduction in the total number of circulating lymphocytes six days after experimental infection (P<0·001). This lymphocytopenia was associated with a significant reduction in the number of B (LCAp220+) and T (CD5+) lymphocytes (P<0·001) but there was a significant increase in the number of cells which were neither T nor B (CD5-LCAp220−) cells (P<0·01). The reduction in the number of T lymphocytes was due to reduced numbers of circulating CD4+ (helper) T cells, CD8+ (cytotoxic/suppressor) T cells and those with the pan T cell marker (CD5+) but without CD4 or CD8 epitopes (CD4-CD8−). All lymphocytes returned to preinoculation levels 13 to 16 days after experimental infection.

Detection of antibodies to the agent of tick-borne fever by indirect immunofluorescence

Cytospin preparations of sheep granulocytes infected with Cytoecetes phagocytophila, the causative agent of tick-borne fever (TBF), enabled the development of an improved and reproducible indirect fluorescent antibody test to detect antibodies in the sera of sheep convalescing from TBF. The test was compared with counter-immunoelectrophoresis.

Use of counter Immunoelectrophoresis in detection of antibodies to tickborne fever

A micromethod using the counter immunoelectrophoresis technique for the detection of antibodies to Cytoecetes phagocytophila, the causative agent of tickborne fever (TBF) is described. Antibodies were first detected nine to 11 days after experimental infection of lambs with TBF and persisted for six to 10 weeks after infection. The test was also applied to approximately 440 field samples of ovine sera collected from tick infested farms in Scotland and the north of England, 16 per cent of which proved positive and showed a marked seasonal variation.

Depression of lymphocyte response to mitogens in sheep infected with tick-borne fever

Lymphocytes obtained from sheep experimentally infected with Cytoecetes phagocytophila, the causative agent of tick-borne fever, showed reduced blastogenesis induced by the mitogens phytohaemagglutinin and E. coli lipopolysaccharide. The period of reduced lymphocyte reactivity coincided with the period of parasitaemia and leucopenia.

Stages in the development of Cytoecetes phagocytophila, the causative agent of tick-borne fever

The stages of development of Cytoecetes phagocytophila were studied by light and electron microscopy. It is suggested that discrete particles represent early stages of infection while clusters are a result of subsequent division by binary fission inside cytoplasmic vacuoles.

Differentiation of strains of Cytoecetes phagocytophila, the causative agent of tick-borne fever, by complement fixation

The complement fixation test was used to assess antigenic relationships between 3 ovine strains of Cytoecetes phagocytophila. Cross-reaction suggests that the strains share common antigens and quantitative differentiation was possible.

In vitro propagation of Cytoecetes phagocytophila, the causative agent of tick-borne fever

Cytoecetes phagocytophila, a neutrophilic rickettsia which parasitizes sheep and cattle, was propagated transiently at 37°C in cultures of heparinized whole blood of sheep supplemented with Medium 199 containing HEPES buffer. Significant increases in the number of infected cells and the number of rickettsias per infected cell were observed within 24 h. Back-passage into sheep produced typical tick-borne fever.