Intramammary inoculation with lactic acid bacteria at dry-off triggers an immunomodulatory response in dairy cows.

The use of antibiotics to prevent bovine mastitis is responsible for the emergence and selection of resistant strains. Lactic acid bacteria (LAB) could be introduced into animal feed as an alternative prevention method that would bypass the risk of resistance development. In previous research, we demonstrated that two probiotic LAB strains isolated from bovine milk were capable of stimulating the production of antibodies and the host's immune cellular response in the udder. The present study aimed to elucidate whether the antibodies of animals inoculated with these strains were able to increase phagocytosis by neutrophils and inhibit the growth of different mastitis-causing pathogens. Moreover, the effect of LAB on the expression of pro-inflammatory cytokines was assessed. Ten animals were inoculated intramammarily with 106 cells of the two strains at dry-off. The blood serum was tested for its ability to opsonize bovine mastitis pathogens, the in vitro bactericidal activity of bovine blood and milk against these pathogens was determined, and cytokine mRNA expression was quantified in milk somatic cells. The inoculated animals did not show abnormal signs of sensitivity to the LAB. Their blood serum significantly enhanced the phagocytosis of Staphylococcus spp. and the LAB. Escherichia coli and Streptococcus uberis were inhibited by the milk serum but not the blood serum, whereas Staphylococcus aureus and Staphylococcus haemolyticus were inhibited by both. In regard to cytokine expression, interleukin (IL)-1ß increased markedly for up to 4 h post-inoculation, and an increase in IL-8 was observed 4, 12 and 24 h after inoculation. Tumour necrosis factor-α mRNA increased 1 and 2 h after inoculation and a significant difference was registered at 6 h for interferon-γ. This rapid immunomodulatory response shows that inoculating animals with LAB at dry-off, when they are especially susceptible, could be a useful strategy for the prevention of bovine mastitis.

Pathogenesis and inflammatory response in experimental caprine mastitis due to Staphylococcus chromogenes.

Coagulase-negative staphylococci (CNS) are the most frequently isolated bacteria in cases of subclinical mastitis in dairy cows. CNS species may differ in their pathogenicity, but very little is known about their virulence factors or their immune response in intramammary infections. To our knowledge, no experimental studies into the mastitis pathogenesis caused by CNS have been described in lactating goats. The aim of this study was to induce an experimentally Staphylococcus chromogenes mastitis in lactating goats aimed at verifying if the model can be used to evaluate the inflammatory response, the dynamics of infection and the pathological findings within the first hours of intramammary inoculation. Six Saanen goats in mid-lactation were inoculated with 1 × 107 colony forming units of S. chromogenes. Bacterial growth peaked in milk from the challenged right halves of the mammary glands (RMG) at 4 h post inoculation (PI). Shedding of viable bacteria showed a marked decrease at 12 h PI. An increase in mean somatic cell counts was observed in the milk samples from 8 h PI onwards. Mild clinical signs were evoked by intramammary inoculation. Staphylococcus chromogenes could be isolated in tissue from all RMG. Histological examination of specimens of the RMG and lymph nodes of the goats showed an increased inflammatory response throughout the experiment with respect to control halves. In conclusion, the experimental inoculation of S. chromogenes in lactating goats is capable of eliciting an inflammatory response and capable of causing pathological changes. This research represents a preliminary study for a better knowledge of the mastitis pathogenesis caused by S. chromogenes.

Bovine mastitis prevention: humoral and cellular response of dairy cows inoculated with lactic acid bacteria at the dry-off period.

The use of lactic acid bacteria (LAB) in animal feed, constitute an alternative tool for bovine mastitis prevention. Previously, two LAB strains were isolated from bovine milk and selected for their probiotics properties. So far, immune response of inoculating LAB in bovine udders at dry-off period has not been investigated. The immunoglobulin isotype levels and memory cell proliferation in blood and milk of animals inoculated with Lactobacillus lactis subsp. lactis CRL1655 and Lactobacillus perolens CRL1724 at dry-off period was studied. Ten animals were inoculated intramammarily with 106 cells of each LAB (IG) and 2 animals used as control (NIG). Milk and blood samples were taken before inoculation and 1, 2, 4, 6, 12 and 24 h and 7 and 14 days after inoculation. Somatic cell count (SCC) in milk, the presence of bovine mastitis pathogens, the levels of antibodies and lymphocyte proliferation were determined. In the IG, the SCC was <250,000 cells/ml up to 4 h after intramammary inoculation. Six and 12 h after inoculation, the SCC increased up to 600,000 and 2,000,000 cells/ml, respectively. In the NIG, the SCC reached the maximum value 7 days after inoculation. Microbiological analysis showed that all samples were negative for major bovine mastitis pathogens after 24-48 h of incubation. In general, LAB inoculation increased the amount of IgG isotypes in blood and milk, and these antibodies were able to recognise Staphylococcus aureus epitopes. Lymphocytes proliferation was significantly higher in the IG at all time points assayed, following LAB or S. aureus stimulation. The lymphocytes of animals inoculated with LAB do not react in vitro to the presence of S. aureus antigen.. The results showed that probiotic microorganisms could be a natural and effective alternative in the prevention of bovine mastitis at dry-off period and act as immunomodulatory stimulating local and systemic defence lines.

Staphylococcus aureus avirulent mutant vaccine induces humoral and cellular immune responses on pregnant heifers.

Bovine mastitis produces economic losses, attributable to the decrease in milk production, reduced milk quality, costs of treatment and replacement of animals. A successful prophylactic vaccine against Staphylococcus aureus should elicit both humoral and cellular immune responses. In a previous report we evaluated the effectiveness of a live vaccine to protect heifers against challenge with a virulent strain. In the present study the immunological response of heifers after combined immunization schedule was investigated. In a first experimental trial, heifers were vaccinated with 3 subcutaneous doses of avirulent mutant S. aureus RC122 before calving and one intramammary dose (IMD) after calving. Antibodies concentration in blood, bactericidal effect of serum from vaccinated animals and lymphocyte proliferation was determined. The levels of total IgG, IgG1 and IgG2 in colostrum and the lymphocyte proliferation index were significantly higher in vaccinated respect to non-vaccinated group throughout the experiment. The second trial, where animals were inoculated with different vaccination schedules, was carried out to determine the effect of the IMD on the level of antibodies in blood and milk, cytokines (IL-13 and IFN-γ) concentration and milk's SCC and bacteriology. The bacterial growth of the S. aureus strains was totally inhibited at 1-3×10(6) and 1-3×10(3)cfu/ml, when the strains were mixed with pooled serum diluted 1/40. The results shown that IMD has not a significant effect on the features determinate. In conclusion, a vaccination schedule involving three SC doses before calving would be enough to stimulate antibodies production in milk without an IMD. Furthermore, the results showed a bactericidal effect of serum from vaccinated animals and this provides further evidence about serum functionality. Immune responses, humoral (antigen-specific antibodies and Th2 type cytokines) and cellular (T-lymphocyte proliferation responses and Th1 type cytokines), were augmented by administration of the avirulent mutant which represent an antigenic pool.

Gliotoxinogenic Aspergillus fumigatus in the dairy herd environment.

The potential association between hygienic conditions in the environment of lactating cows and the presence of gliotoxinogenic Aspergillus fumigatus strains was studied. Milk samples (individual cow's milk [ICM], bulk tank milk [BTM]) from 44 dairy farms were sampled. In ICM samples, eight different species of Aspergillus were identified. A. flavus and A. fumigatus were predominant, with 37.8% and 26.1% relative densities, respectively. A. fumigatus strains were isolated from 61.4% of the BTM samples, and 34% of these strains were able to produce gliotoxin. Principal component analysis was used to associate the presence of A. fumigatus with some hygienic and sanitary practices. A significant and positive correlation was observed between dry cow therapy and forestripping. The presence of A. fumigatus gliotoxin producers in milk was associated with high somatic cells count (SCC) samples. Good hygienic and sanitary practices were associated with absence of A. fumigatus and relatively low SCCs of <250,000 cells/ml. In general, a high percentage of dairy farms were positive for A. fumigatus in BTM samples. This is the first work that indicates the positive effects of adequate hygienic and sanitary practices in dairy herds on the control of A. fumigatus and related species. By reducing the frequency of Aspergillus spp. in the dairy environment, the risk of farm handlers' exposure and the risk of intramammary fungal infections would also be reduced.

Effect of aeration and agitation on the protease production by Staphylococcus aureus mutant RC128 in a stirred tank bioreactor.

The modified rotating simplex method has been successfully used to determine the best combination of agitation rate and aeration rate for maximum production of extracellular proteases by Staphylococcus aureus mutant RC128, in a stirred tank bioreactor operated in a discontinuous way. This mutant has shown altered exoprotein production, specially enhanced protease production. Maximum production of proteases (15.28 UP/ml), measured using azocasein as a substrate, was obtained at exponential growth phase when the bioreactor was operated at 300 rpm and at 2 vvm with a volumetric oxygen transfer coefficient (K(L)a) of 175.75 h(-1). These conditions were found to be more suitable for protease production.

Genotyping of Staphylococcus aureus isolated from humans, bovine subclinical mastitis and food samples in Argentina.

The aim of the present study was to characterize genotypically 45 Staphylococcus aureus strains isolated from humans, bovine subclinical mastitis and food samples in Argentina by rep-PCR and PCR amplification of virulence genes. Resistances to various antibiotics could be observed for the human S. aureus, less pronounced for the bovine strains, but not for the eight S. aureus isolated from food samples. The strains could be classified genotypically by rep-PCR and by amplification of the genes encoding protein A, coagulase, clumping factor, the collagen adhesin domains A and B, capsular polysaccharide 5 and 8, the accessory gene regulator agr classes I to III, and the S. aureus gene regulator sae. rep-PCR analyses and the different gene patterns revealed that the strains could be divided into seven groups mostly matching with the origin of the isolates. The present study describes genotypic variations of S. aureus strains isolated from different origins in Argentina. The study provides a valuable insight into molecular specificities of this important pathogen.

Conventional identification of Streptococcus uberis isolated from bovine mastitis in Argentinean dairy herds.

The objective of this study was to evaluate a conventional scheme for identifying Streptococcus uberis strains isolated from bovine mastitis. Seventy-five gram-positive, catalase-negative cocci were collected from cows with mastitis from 19 dairy herds located in the east-central region of Argentina. Five American Type Culture Collection strains and bovine isolates were identified by the API 20 Strep system and by restriction fragment length polymorphism analysis of 16S rDNA. A conventional scheme based on 11 biochemical tests was selected for identification of Strep. uberis strains: the Christie-Atkins-Munch-Petersen reaction; hydrolysis of Arg, esculin, and sodium hippurate; growth in inulin, mannitol, raffinose, salicin, and sorbitol; and growth at 45 degrees C and in 6.5% NaCl. Reference strains and 25 bovine isolates were classified accurately to the species level by the conventional scheme in a blind assay. Each reference strain and each bovine isolate were identified as belonging to the same species following these 3 methods. The remaining 50 isolates identified as Strep. uberis by the API 20 Strep system and 16S rDNA RFLP were assayed by the conventional scheme. This scheme correctly identified 47 (94%) of 50 isolates as Strep. uberis by comparing their biochemical profile with that of the reference strain. Three (6%) of the 50 isolates were classified as Strep. uberis by the API 20 Strep system and by 16S rDNA RFLP and were identified as Enterococcus faecalis by the conventional scheme. Thirty percent of the Strep. uberis strains showed biochemical profiles identical to the Strep. uberis American Type Culture Collection 27958 strain. Seventy percent of the Strep. uberis strains demonstrated variability compared with the reference strain, resulting in 19 different biochemical profiles. The conventional scheme proposed in this study resulted in a relatively low number of misidentifications and could biochemically identify not only typical, but also atypical Strep. uberis strains. This conventional scheme can be considered an adequate method for identifying Strep. uberis strains isolated from bovine mastitis because of its affordable cost in developing countries, and it may contribute to determining the frequency of isolation of Strep. uberis strains in Argentinean dairy herds.

The sae locus of Staphylococcus aureus encodes a two-component regulatory system.

Sae is a regulatory locus that activates the production of several exoproteins in Staphylococcus aureus. A 3.4-kb fragment of a S. aureus genomic library, screened with a probe adjacent to the transposon insertion of a sae::Tn551 mutant, was cloned into a bifunctional vector. This fragment was shown to carry the sae locus by restoration of exoprotein production in sae mutants. The sae locus was mapped to the SmaI-D fragment of the staphylococcal chromosome by pulse-field electrophoresis. Sequence analysis of the cloned fragment revealed the presence of two genes, designated saeR and saeS, encoding a response regulator and a histidine protein kinase, respectively, with high homology to other bacterial two-component regulatory systems.

Avirulence and immunogenicity in mice of a bovine mastitis Staphylococcus aureus mutant.

An avirulent mutant, designated RC122, was derived from Staphylococcus aureus bovine mastitis strain RC108 after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. Mutant RC122, which was isolated on the basis of reduced colony size, showed diminished virulence in mice (LD50 of RC122: 3.1 x 10(10) cfu vs LD50 of RC108: 2.3 x 10(7) cfu). Mutant RC122 grew more slowly than its parental strain and showed decreased production of several exoproteins, such as alpha- and beta-hemolysin, DNAse and coagulase. The production of its capsule was induced only under in vivo growth conditions. Clearance studies performed in the mouse kidney revealed that the kinetics of disappearance of the mutant was similar to that of its parental strain. Protection experiments carried out by intraperitoneal administration in mice showed that mutant RC122 conferred a good degree of protection from challenge with homologous and heterologous strains.

Selection of a strain of Clostridium argentinense producing high titers of type G botulinal toxin.

The strain G89HT of Clostridium argentinense obtained by culture selection of the prototype G89 strain producing high titers of type G botulinal toxin was studied. Its cultural, biochemical and toxigenic characteristics and the presence of plasmids were tested. Both strains showed similar physiological features and carried a 83 MDa plasmid. A 170 MDa plasmid was also recognized in the G89HT strain. Notably, this strain was better sporulating and showed a higher toxigenicity than the prototype G89 C. argentinense strain. These two characteristics might permit a long term storage and perhaps yield high antitoxin titres.

Field trials of a vaccine against bovine mastitis. 1. Evaluation in heifers.

A vaccine was developed against bovine mastitis based on inactivated, highly encapsulated Staphylococcus aureus cells; a crude extract of Staph. aureus exopolysaccharides; and inactivated, unencapsulated Staph, aureus and Streptococcus spp. cells. This vaccine was tested on 30 heifers during a 7-mo period. The 30 heifers were randomly assigned to three groups of 10 heifers each. The prepartum group received two injections of the vaccine at 8 and 4 wk before calving, and the postpartum group received two injections at 1 and 5 wk after calving. The control group received two injections of a placebo at 8 and 4 wk before calving. The vaccine or the placebo was administered subcutaneously in the brachiocephalicus muscle of the neck. The frequencies of intramammary infections caused by Staph. aureus were reduced from 18.8% for heifers in the control group to 6.7 and 6.0% for heifers in the prepartum and postpartum groups, respectively. This protective effect was maintained for at least 6 mo. The relative risk of mastitis caused by Staph. aureus was 0.31 and 0.28 for heifers in the prepartum and postpartum groups, respectively, compared with that for heifers in the control group. The results of the trial indicated the effectiveness of the vaccine in decreasing the incidence of intrammammary infections caused by Staph. aureus. A slight but nonsignificant increase occurred in fat production in the milk of vaccinated cows. The vaccine had no observable effect on somatic cell count or streptococcal infections.

Identification of staphylococci from bovine milk in Argentina.

Two hundred and thirty three strains of staphylococci isolated from bovine milk from 17 dairies in Argentina were classified into 16 species. Most of the strains (78%) belonged to 4 species; S. aureus, S. haemolyticus, S. hominis, and S. warneri (38.5, 20.5, 9.4 and 9.0%, respectively). The remaining 12 species appeared with low frequencies. Twenty-one of the strains were isolated from mammary quarters with signs of clinical mastitis. The majority of the milk samples infected with S. aureus or S. haemolyticus showed very high somatic cell counts.