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.

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.

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.

Field trials of a vaccine against bovine mastitis. 2. Evaluation in two commercial dairy herds.

A vaccine against bovine mastitis was developed. The vaccine was based on inactivated, highly encapsulated Staphylococcus aureus cells; a crude extract of Staph. aureus exopolysaccharides; and inactivated unencapsulated Staph. aureus and Streptococcus spp. cells. In this study, the vaccine was evaluated in 164 cows from two commercial dairies (A and B) during a 4-mo period. Two doses of the vaccine were administered subcutaneously to 82 cows in the brachiocephalicus muscle of the neck within a 4-wk interval. The results of this trial revealed significantly fewer intramammary infections caused by Staph. aureus at various levels of severity (clinical, subclinical, and latent) in cows that were vaccinated. The odds ratios of all types of intrammammary infections caused by Staph. aureus for dairies A and B, which were determined by a logistic model, were 1.84 and 1.89, respectively, for quarters of vaccinated cows and quarters of control cows. The colony counts for Staph. aureus in milk from infected quarters of vaccinated cows were significantly lower than those in milk from infected quarters of control cows. Also, the somatic cell counts per milliliter in milk from vaccinated cows were significantly decreased when the initial somatic cell count was < 500,000 cells/ml at the start of the trial. The vaccine had no observable effect on fat production in milk or on streptococcal infections.

[Comparison of detection methods for the Staphylococcus aureus capsule]. / Comparación de métodos de detección de cápsula de Staphylococcus aureus.

Eleven methods for capsule detection of Staphylococcus aureus were compared. The most suitable of them were transmission electron microscopy, determination of the presence of clumping factor, determination of colonial morphology in serum-soft agar, estimation of cell volume and staining with safranine. The determination of clumping factor is a fast and effective method for presumptive diagnosis of capsulated strains, but need to be confirmed by another method. The cell volume estimation is useful for determination of capsule production in liquid cultures, while staining with safranine is suitable for genetic studies of capsule production. The other methods analyzed in this work (Indian ink staining, use of anticapsular antisera, determination of virulence for mice, lisostaphin susceptibility, resistance to phages and resistance to phagocytosis) were laborious, too slow, or need components and/or equipment not available in all laboratories. In addition, two methods of induction of capsule production were assayed, one in vitro by several passages in broth with 10% bovine serum and the other in vitro by intraperitoneal inoculation in mice. Both methods induced capsule production.

Pathogenicity in mice of Staphylococcus aureus mutants deficient in exoprotein synthesis.

Twelve mutants were isolated from a Staphylococcus aureus strain derived from bovine mastitis after mutagenesis by ultraviolet light. These mutants were found to be deficient for several characteristics such as production of most exoproteins and had altered phage type and/or colonial morphology in serum-soft agar medium. They also differed in virulence when assayed in mice by intraperitoneal administration; the ratio of the LD50 of the mutants vs. that of the parental strain ranged from 1 to 123. The different virulence of the mutants could not be associated with lack of production of exoproteins or altered colonial morphology. On the other hand, a clear correlation was evidenced between lowered virulence and slower growth rate at 37 degrees C. Three mutants were assayed in the mouse mastitis model. One of them, which was about 40 times less virulent when assayed by intraperitoneal administration, induced a histopathological lesion similar to that produced by the parent strain; the other two mutants, which were about 70 to 120 times less virulent by intraperitoneal administration, induced only a very slight lesion. Mice were vaccinated by the intraperitoneal route with two of the less virulent mutants; the LD50 in the vaccinated mice that were challenged with the parental strain increased 11 to 14 times compared with that for the unvaccinated mice.

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.

[Anti-leptospira and anti-brucella agglutinins in hares (Lepus europaeus) from Rio Cuarto Department, Province of Cordoba]. / Aglutininas anti-leptospira y anti-brucella en liebres (Lepus europaeus) del departamento de Río Cuarto, provincia de Córdoba.

Seventy-seven anti-brucella and 42 anti-leptospira agglutinin serum levels of hares in Rio Cuarto district of Córdoba Province were studied. For brucella, only one sample showed incomplete reaction at a 1:25 dilution. The isolation of brucella from liver, spleen and gastro-hepatic lymph node in this animal was negative. In the 42 serum samples examined for L. pomona, L. wolffi, L. ballum, L. hardjo, L. pyrogenes and L. grippotiphosa, 6 reacted at a dilution of 1:100 with L. ballum serovar and 1 reacted up to 1:200 dilution with L. wolffi serovar. These results indicate that the hare does not play any important role as reservoir of pathogenic brucella in this area. Meanwhile, it could act as potential reservoir for pathogenic leptospira.

Aglutininas anti-leptospira y anti-brucella en liebres (Lepus europaeus) del departamento de Río Cuarto, provincia de Córdoba. / [Anti-leptospira and anti-brucella agglutinins in hares (Lepus europaeus) from Rio Cuarto Department, Province of Cordoba]

Seventy-seven anti-brucella and 42 anti-leptospira agglutinin serum levels of hares in Rio Cuarto district of Córdoba Province were studied. For brucella, only one sample showed incomplete reaction at a 1:25 dilution. The isolation of brucella from liver, spleen and gastro-hepatic lymph node in this animal was negative. In the 42 serum samples examined for L. pomona, L. wolffi, L. ballum, L. hardjo, L. pyrogenes and L. grippotiphosa, 6 reacted at a dilution of 1:100 with L. ballum serovar and 1 reacted up to 1:200 dilution with L. wolffi serovar. These results indicate that the hare does not play any important role as reservoir of pathogenic brucella in this area. Meanwhile, it could act as potential reservoir for pathogenic leptospira.

Aglutininas anti-leptospira y anti-brucella en liebres (Lepus europaeus) del departamento de Río Cuarto, provincia de Córdoba / [Anti-leptospira and anti-brucella agglutinins in hares (Lepus europaeus) from Rio Cuarto Department, Province of Cordoba]

Seventy-seven anti-brucella and 42 anti-leptospira agglutinin serum levels of hares in Rio Cuarto district of Córdoba Province were studied. For brucella, only one sample showed incomplete reaction at a 1:25 dilution. The isolation of brucella from liver, spleen and gastro-hepatic lymph node in this animal was negative. In the 42 serum samples examined for L. pomona, L. wolffi, L. ballum, L. hardjo, L. pyrogenes and L. grippotiphosa, 6 reacted at a dilution of 1:100 with L. ballum serovar and 1 reacted up to 1:200 dilution with L. wolffi serovar. These results indicate that the hare does not play any important role as reservoir of pathogenic brucella in this area. Meanwhile, it could act as potential reservoir for pathogenic leptospira.