Study of the efficacy of a Streptococcus uberis mastitis vaccine against an experimental intramammary infection with a heterologous strain in dairy cows.

Streptococcus uberis is a worldwide pathogen that causes intramammary infections in dairy cattle. Nevertheless, commercial vaccines are currently not available and measures to control S. uberis mastitis are limited to the implementation of good management practices. The aim of the present study was to evaluate the efficacy of an S. uberis subunit vaccine against bovine mastitis (Laboratorios Hipra S.A., Amer, Spain) administered precalving against an experimental intramammary challenge with a heterologous S. uberis strain in dairy cows postcalving. With this objective, 25 gestating Holstein-Friesian heifers were randomly assigned to 1 of 2 groups: group 1 (n = 13), vaccinated by intramuscular route with the vaccine, and group 2 (n = 12), vaccinated by intramuscular route with phosphate-buffered saline as a control group. Both groups were immunized 60 and 21 d before the expected parturition date (75 and 36 d before challenge). Fourteen days after calving all cows were challenged by intramammary infusion of 100 colony-forming units of a heterologous S. uberis strain in 2 quarters per cow. Then, challenged quarters were monitored for clinical signs of mastitis, bacterial count, and somatic cell count for the following 21 d. Rectal temperature and daily milk yield per cow were also assessed. Results showed that all challenged quarters developed clinical mastitis. Nevertheless, vaccination significantly reduced the clinical signs of mastitis, bacterial count, rectal temperature, and daily milk yield losses after the intramammary infection and significantly increased the number of quarters with no bacterial isolation and somatic cell count <200,000 cells/mL at the end of the study (d 19, 20, and 21 after challenge). To confirm the efficacy of this vaccine, further studies under field conditions are needed.

Immune response after an experimental intramammary challenge with killed Staphylococcus aureus in cows and heifers vaccinated and not vaccinated with Startvac, a polyvalent mastitis vaccine.

An experimental trial was conducted to explore the effect of vaccination with a polyvalent vaccine against mastitis (Startvac) on the early immune response after experimental intramammary challenge with a heterologous killed Staphylococcus aureus strain. The effect of vaccination on milk production, clinical signs, quarter milk somatic cell count, milk polymorphonuclear neutrophilic leukocyte (PMN) concentration and viability, the concentration of antigen-specific antibodies [slime associated antigenic complex (SAAC) and J5] and their IgG1 and IgG2 subtypes in both serum and whey, and the antigen-specific IFN-γ, IL-4, and IL-17 production by blood lymphocytes after in vitro stimulation with S. aureus and Escherichia coli extracts were determined. A cohort of 8 clinically healthy end-term cows and heifers were conveniently selected, of which half was vaccinated with Startvac at 45 and 10 d before the expected calving date and half served as nonvaccinated control animals. At 15 d in milk, 2 contralateral quarters of each of the 8 animals were challenged with 2×109 cfu/mL of the formaldehyde-killed S. aureusC195strain. The 2 other quarters were infused with phosphate-buffered saline and served as control quarters. The increase in both quarter milk somatic cell count and PMN concentration and the drop in milk production after S. aureus inoculation was less pronounced in the vaccinates than in the nonvaccinates, reflecting a less severe inflammatory response. No significant differences in PMN viability between vaccinates and nonvaccinates could be demonstrated. The serum SAAC- and J5-specific antibody concentration significantly increased across the dry period in the vaccinated animals only. The whey concentration of SAAC-specific antibodies was significantly higher in vaccinates than in nonvaccinates at both 15 and 17 d in milk, independent from the challenge status of the quarters. No significant differences in the whey J5-specific antibody concentration were observed. Vaccination with Startvac seems to primarily evoke a Th2 response for S. aureus characterized by a shift toward the IgG1 antibody subtype and accompanied by a less pronounced Th1 response. The type of response against E. coli was less clear, though a weak but significant shift toward the IgG2 antibody subtype after vaccination and high IFN-γ levels after in vitro stimulation suggest a Th1 response. The increased SAAC-specific antibody concentration in whey in vaccinates compared with nonvaccinates most probably triggers the opsonization of the inoculated S. aureus bacteria, resulting in a more efficient elimination of the bacteria from the mammary gland.

Short communication: biofilm production characterization of mecA and mecC methicillin-resistant Staphylococcus aureus isolated from bovine milk in Great Britain.

Staphylococcus aureus is an important cause of contagious intramammary infection in dairy cattle, and the ability to produce biofilm is considered to be an important virulence property in the pathogenesis of mastitis. The aim of this study was to characterize the biofilm formation capacity of methicillin-resistant Staph. aureus (MRSA), encoding mecA or mecC, isolated from bulk tank milk in Great Britain. For this purpose, 20 MRSA isolates were grown on microtiter plates to determine the biofilm production. Moreover, the spa-typing and the presence of the intercellular adhesion genes icaA and icaD were analyzed by PCR. All MRSA isolates tested belonged to 9 spa-types and were PCR-positive for the ica genes; 10 of them (50%) produced biofilm in the microtiter plate assay. This is also the first demonstration of biofilm production by mecC MRSA.

Comparative efficacy of immunization with inactivated whole tachyzoites versus a tachyzoite-bradyzoite mixture against neosporosis in mice.

The worldwide economic impact of Neospora caninum infection has caused the development of effective vaccines to become one of the main goals in the field of neosporosis research. In this study, the protection conferred by antigens from inactivated whole tachyzoites (TZ) and a tachyzoite-bradyzoite mixture (TZ-BZ) of N. caninum (Nc-Spain7 isolate) incorporated into a water-in-oil emulsion (W/O) and aluminium hydroxide-ginseng extract (Al/G) was evaluated in mouse models of congenital and cerebral N. caninum infection. Immunization with TZ-BZ induced congenital and cerebral neosporosis exacerbation that was mainly characterized by reduced neonatal median survival time and increased parasite presence in adult mouse brains. The immune response of mice immunized with TZ-BZ was characterized by an increase in IFN-γ expression prior to challenge and an increase in IL-4 expression accompanied with significantly higher levels of antibodies against 2 recombinant bradyzoite-specific proteins (rNcSAG4 and rNcBSR4) after challenge. Immunization with TZ in W/O significantly reduced neonatal mortality, vertical transmission as well as parasite presence in adult mouse brains and induced a strong humoral immune response. The current study demonstrates the critical role of stage-specific antigens and adjuvants on the development of effective inactivated vaccines for the prevention of N. caninum infection.

Protection from Staphylococcus aureus mastitis associated with poly-N-acetyl beta-1,6 glucosamine specific antibody production using biofilm-embedded bacteria.

Staphylococcus aureus vaccines based on bacterins surrounded by slime, surface polysaccharides coupled to protein carriers and polysaccharides embedded in liposomes administered together with non-biofilm bacterins confer protection against mastitis. However, it remains unknown whether protective antibodies are directed to slime-associated known exopolysaccharides and could be produced in the absence of bacterin immunizations. Here, a sheep mastitis vaccination study was carried out using bacterins, crude bacterial extracts or a purified exopolysaccharide from biofilm bacteria delivered in different vehicles. This polysaccharide reacted specifically with antibodies to poly-N-acetyl-beta-1,6-glucosamine (PNAG) and not with antibodies to other capsular antigens or bacterial components. Following intra-mammary challenge with biofilm-producing bacteria, antibody production against the polysaccharide, milk bacterial counts and mastitis lesions were determined. Bacterins from strong biofilm-producing bacteria triggered the highest production of antibodies to PNAG and conferred the highest protection against infection and mastitis, compared with weak biofilm-producing bacteria and non-cellular inocula. Thus, bacterins from strong biofilm bacteria, rather than purified polysaccharide, are proposed as a cost-efficient vaccination against S. aureus ruminant mastitis.

Expression of the hemolysin operon in Escherichia coli is modulated by a nucleoid-protein complex that includes the proteins Hha and H-NS.

The Escherichia coli protein Hha is a temperature- and osmolarity-dependent modulator of the expression of the hemolysin operon. The Hha protein was purified and its DNA-binding properties analyzed. Hha binds in a non-specific manner throughout the upstream regulatory region of the hemolysin operon in the recombinant hemolytic plasmid pANN202-312. A search for interacting proteins revealed that Hha interacts with H-NS. DNA-binding studies showed that, in vitro, Hha and H-NS together form a complex with DNA that differs from those formed with either protein alone. These data, together with the effects of hha and hns mutations on the expression of the hemolysin genes, suggest that in vivo H-NS and Hha form a nucleoid-protein complex that accounts for the thermo-osmotic regulation of the hemolysin operon in E. coli.

Sequence, identification and effect on conjugation of the rmoA gene of plasmid R100-1.

The rmoA gene was recently identified from two partially overlapping sequences corresponding to a region close to the end of the tra operon of plasmid R100. Its putative amino acid sequence showed strong homology to the Hha protein of Escherichia coli and YmoA protein of Yersinia enterocolitica, which are modulators of gene expression in response to environmental stimuli. We have cloned the rmoA gene from plasmid R100-1 in pUC19 and obtained the complete nucleotide sequence, which was previously published only partially and may have contained some mistakes. The rmoA gene product has been identified in radiolabelled minicells as a protein of the predicted molecular mass. The wild-type rmoA gene of plasmid R100-1 has been mutated by gene replacement and its effect on the efficiency of conjugation has been analysed. When grown in LB medium, cells harbouring R100-1 plasmid with a disrupted copy of rmoA showed a five-fold increase in conjugation frequency compared to cells harbouring R100-1 plasmid with the wild-type rmoA gene, grown in the same conditions. When cells were grown in NaCl-free LB medium they showed a 50-fold increase in conjugation frequency.

Osmolarity modulates the expression of the Hha protein from Escherichia coli.

The effect of the osmolarity of the culture medium on the expression of the hha gene of Escherichia coli was investigated. When cells were grown in LB medium, expression reached a maximum in the exponential phase of growth and decreased in the stationary phase. Increasing the osmolarity of the LB medium had no significant effect on the expression of the hha gene, but depletion of NaCl led to a significant decrease in expression. Expression of the hha gene is thus sensitive to the osmolarity of the growth medium. High levels of expression of the hha gene when cells are grown at medium to high osmolarity are consistent with the finding that the Hha protein appears to play its main modulatory role when cells grow under these conditions.

Construction of a double hha hns mutant of Escherichia coli: effect on DNA supercoiling and alpha-haemolysin production.

A double hha hns Escherichia coli mutant was constructed. The effect of the single hns mutation and of the double hha hns mutation on the expression of the alpha-haemolysin determinant of plasmid pANN202-312 was assessed. Whereas the hns mutant moderately increased expression of the toxin, the double hha hns mutant strongly enhanced transcription of the hly operon and hence expression of the toxin. This suggests that both Hha and H-NS proteins participate in the modulation of the expression of the toxin. The enhancement of haemolysin expression in the double mutant could not be correlated to a global alteration of DNA topology: DNA preparations of a reporter plasmid isolated from this mutant gave a topoisomer distribution similar to that of the parental strain.

Complementation of the hha mutation in Escherichia coli by the ymoA gene from Yersinia enterocolitica: dependence on the gene dosage.

The Hha protein from Escherichia coli is highly similar (82%) to the YmoA protein from Yersinia enterocolitica. Both are members of a new class of proteins that modulates gene expression, probably by influencing DNA topology. In this paper, complementation of the hha mutation in E. coli by the ymoA gene from Y. enterocolitica has been studied. We show that the ymoA gene complements one of the phenotypic properties of hha mutants (high level of haemolysin production when they carry the recombinant plasmid pANN202-312) when cloned in a medium-copy-number plasmid but not when carried in a low-copy-number plasmid. Western blot analysis of the expression of YmoA in E. coli rules out inefficient expression of the protein. Surprisingly, the hha gene itself fails to complement the hha mutation when cloned in a medium-copy-number vector and causes genetic rearrangements of the E. coli chromosome as a consequence of insertion sequences mobilization.

The Hha protein as a modulator of expression of virulence factors in Escherichia coli.

We constructed hha derivatives from both a clinical uropathogenic Escherichia coli isolate (strain FVL4) and a wild E. coli strain causing bovine diarrhea (strain CCB21) and analyzed the effect of the hha allele on the expression of the different virulence factors exhibited by these strains. Expression of hemolysin and of the Vir antigen was altered in hha mutants. Whereas production of hemolysin by strain FVL4 was repressed both at a low temperature and at high osmolarity, the hha allele accounted for a significant increase of hemolysin production under these conditions. Also, the low temperature-sensitive expression of the Vir adhesin was modified in hha mutants, which were able to express this adhesin at a low temperature. Expression of other virulence factors, such as cytotoxic necrotizing factor type 1 and 2 toxins, remained unmodified in hha derivatives of strains FVL4 and CCB21.