Production and novel quantification of haemolysins produced by coagulase-negative staphylococci isolated from subclinical mastitis in sheep.

Coagulase-negative staphylococci producing cell-damaging toxins were isolated from the milk of sheep with subclinical mastitis. The haemolytic activity of Coagulase-negative staphylococcal strains was assessed on solid and liquid culture media. More than 61% and 76% of the tested strains on solid media produced evidence of alpha- and delta- haemolysins and more than 78% produced synergistic haemolysis. However almost all isolates producing haemolysin in liquid culture media produced only very few units of haemolysin compared to the positive control of five Coagulase-positive strains of staphylococci. It was concluded that solid media are better for classifying Coagulase-negative staphylococci as producers or not of haemolysins, and liquid media for measuring the size of this activity within the first few hours of intramammary infection.

Naked DNA when co-administered intranasally with heat-labile enterotoxin of Escherichia coli primes effectively for systemic B- and T-cell responses to the encoded antigen.

In this study a novel prime-boost immunisation strategy was evaluated. Priming of BALB/c mice by the intranasal route with plasmid DNA encoding beta-galactosidase (LacZ) with or without heat-labile enterotoxin (LT) of Escherichia coli as a mucosal adjuvant, resulted in the induction of weak serum antibody and proliferative T-cell responses. However, following an intraperitoneal booster injection with the beta-galactosidase protein (beta-gal), strong antibody and proliferative T-cell responses were induced in all the mice. These responses were highest in mice primed intranasally with a mixture of LacZ+LT as compared to those mice primed with DNA (LacZ) or protein (beta-gal) alone. Moreover, LacZ+LT primed mice produced high avidity antibodies and the subclasses of serum antibodies were IgG1 and IgG2a, suggesting a mixed Th1/Th2-type response. Priming of mice with either protein (beta-gal) or DNA (LacZ) alone, produced predominantly IgG1 antibodies, suggesting a Th2-type response. These findings suggest that the use of a heterologous DNA-prime, protein-boost immunisation scheme combining different routes of administration, might be an advantageous strategy for the induction of accelerated immune responses.

Mammalian granulocyte-macrophage colony-stimulating factor and some CpG motifs have an effect on the immunogenicity of DNA and subunit vaccines in fish.

A eukaryotic plasmid DNA carrying the AACGTT CpG motif in its ampR gene is a 'danger' signal for mice and caused an increase in the specific antibody titres of fish and mice after immunization with beta-galactosidase (beta-gal). A second pUC-based plasmid, which is inactive in mice and contains the GACGTC CpG motif in its cytomegalovirus (CMV) promoter, had no effect on antibody responses to beta-gal in either fish or mice. A synthetic oligonucleotide, which contains the GACGTT motif, potentiated antibody responses to co-administered beta-gal protein in mice, but not in fish. This is early evidence that lower and higher vertebrates recognize different unmethylated CpG motifs as 'danger' signals. In addition, plasmid DNA expressing mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) had a marked effect on cytotoxic T-cell-like activity in fish by reducing the average number of myofibres that expressed beta-gal, 28 days after co-injection with plasmid DNA expressing beta-gal. Although the mechanism by which the mouse GM-CSF exerted its biological effects in fish is unknown, this finding might have important implications for fish vaccination, particularly when cytotoxic T cells may play a critical role.