Mouse Mastitis Model Research Articles

Development of Mastitis in a Mouse Infection Model from Four Bovine Isolates

The goal of this study was to evaluate the ability of a commercially-available feed supplement (OmniGen-AF) to reduce development of mastitis in a mouse mastitis model. The rationale for completing this study was two-fold. First, dairy producers have reported that OmniGen-AF reduced somatic cell counts and reduced mastitis after the introduction of the product into herds. Second, recent studies have shown that the product increased markers of both innate and adaptive immunity. Our hypothesis, therefore, was that feeding the product would reduce the development and severity of mastitis in a mouse model. To assess this hypothesis, five experiments were completed with three bovine mastitis isolates. The isolates included Streptococcus uberis (S. uberis), Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus).

Identification of antimicrobial compounds active against intracellular Staphylococcus aureus

Small-colony variants (SCVs) of Staphylococcus aureus exhibit characteristics of bacteria that can penetrate mammalian cells and remain intracellular and innocuous for indefinite periods. These properties make SCVs a convenient tool that can be used to identify new antibacterial agents having activity against intracellular, quiescent bacteria. Agents active against SCVs could be useful in the treatment of chronic staphylococcal infections such as bovine mastitis. An hemB deletion mutant of S. aureus Newbould, a bovine mastitis isolate, having a stable, genetically defined SCV phenotype, was used in a screening program to identify compounds active against intracellular, gram-positive bacteria. Out of more than 260,000 compounds screened, nine compounds having the desired properties were identified. The range of MICs against gram-positive bacteria was ⩽0.12–32 μg ml −1. One of the compounds (no. 8) showed excellent activity against gram-positive (MICs ⩽0.12 μg ml −1) and gram-negative (MICs ⩽0.12–4 μg ml −1) bacteria. Each of the nine compounds demonstrated efficacy in a neutropenic mouse thigh infection model. Two compounds, including compound no. 8, reduced numbers of bacteria in a mouse mastitis model of infection. Application of a stepwise screening process has identified lead compounds that may be useful for treating persistent, intracellular infections.

Mouse mastitis model of infection for antimicrobial compound efficacy studies against intracellular and extracellular forms of Staphylococcus aureus

Despite the general in vitro susceptibility of Staphylococcus aureus isolates that cause infectious bovine mastitis, the pathogen remains difficult to eradicate with the available antibiotics. The capacity to survive intracellularly has been proposed as a factor contributing to the persistence of S. aureus in the bovine mammary gland. The costs associated with the use of cows or goats to assess the in vivo efficacy of new antibacterial compounds constitute a major drawback. Therefore, in the present study, a mouse model of intramammary infection has been characterized for in vivo testing of new experimental drugs. An inoculum of 100 CFU of S. aureus per gland caused an important level of infection with minimal tissue damage as observed at 24 h post-inoculation. By microscopy, polymorphonuclear neutrophil cell infiltration of the infected mammary glands was observed to increase over time. At 12–24 h of infection, the pathogen was primarily found alive and dividing in neutrophils and occasionally within mammary epithelial cells. Intramuscular or intraveinous injections of cephapirin at t = 0 and 10 h reduced the number of CFU/g of gland in a dose-dependent manner. In conclusion, the mouse model of infectious mastitis proposed here is suitable for primary evaluation of experimental drugs after parenteral treatment of intramammary infection with a pathogen such as S. aureus that presents both intracellular and extracellular phases of growth.

In vivo and in vitro demonstration that Staphylococcus aureus is an intracellular pathogen in the presence or absence of fibronectin-binding proteins

Staphylococcus aureus is the most significant bacterial pathogen associated with bovine mastitis. However, the relevance of intracellular infection to mastitis pathogenesis is poorly understood. We used in vitro assays and a mouse model of mastitis to demonstrate the intracellular component of the infection and to identify the importance of fibronectin-binding proteins in the processes of colonization and internalization. In vitro, a mutant strain, lacking fibronectin-binding protein (FnBPs −), had a reduced ability to bind fibronectin and to infect epithelial cells when compared to its parental wild type strain. After 2 h of infection, the internalization of the mutant bacteria into epithelial cell cultures was reduced by 60% compared with the wild type. After in vivo infection, microscopic examination using the FnBPs − strain revealed that production of a high density of live bacteria within the mammary gland epithelial cells was delayed. Both parental and mutant strains were identified within neutrophils, macrophages and epithelial cells suggesting a close similarity between the mouse mastitis model and bovine mastitis. These results demonstrate that S. aureus was able to cause an intracellular infection in the mouse model of mastitis and that the elimination of one adhesion protein delayed, but did not prevent, infection.

Lactoferrin against Staphylococcus aureus Mastitis : Lactoferrin alone or in combination with penicillin G on bovine polymorphonuclear function and mammary epithelial cells colonisation by Staphylococcus aureus

Antibiotics should combine good antibacterial activity and the capacity to work in association with the host defence system. In this study, we have investigated the effects of bovine lactoferrin alone or in combination with penicillin G on the phagocytic activity of bovine polymorphonuclear leukocytes against Staphylococcus aureus. We have shown that susceptibility of S. aureus to phagocytosis was decreased in the presence of penicillin in the medium. In a kinetic study, lactoferrin alone did not affect phagocytosis but, when used with penicillin, it reversed the negative effect of this antibiotic on phagocytosis. In addition, in an epithelial invasion assay, lactoferrin alone or in combination with penicillin reduced the invasion of mammary epithelial cells in culture by S. aureus. Lactating female CD-1 mice were infected by intra-mammary delivery of a virulent penicillin-susceptible S. aureus strain and were then randomly assigned to treatments according to a 2×2 factorial design. In this mouse mastitis model, 2 days of systemic treatments with lactoferrin and/or penicillin did not lead to a total clearance of infection by S. aureus, but bacterial number was significantly reduced by treatments with lactoferrin or penicillin. These data suggest that bovine lactoferrin, alone or in combination with penicillin G, enhances S. aureus susceptibility to immuno-defense mechanisms, which can be beneficial in the treatment of S. aureus infections.

DNA immunization against the clumping factor A (ClfA) of Staphylococcus aureus

Adhesins are considered the most important virulence factors during early phases Staphylococcus aureus infection. Antibodies induced by vaccination toward an adhesin should reduce the adherence of the pathogen and augment its phagocytosis. The present report describes the immune response of mice to a DNA vaccine directed against one of these adhesins, clumping factor A (ClfA). Injection of plasmids expressing the fibrinogen-binding region A of ClfA induced a strong and specific antibody response to ClfA in mice. In addition, splenocyte proliferation was provoked by in vitro stimulation with recombinant ClfA, thus, indicating direct implication of these cells in the immune response. Pre-incubation of S. aureus with sera of vaccinated mice reduced the pathogen’s ability to bind fibrinogen by up to 92%. These pre-incubated bacteria were phagocytosed by macrophages at an increased level in vitro and were less virulent in vivo in a mouse mastitis model. However, DNA-immunized mice were not protected against an intraperitoneal challenge. Overall, the results suggest that DNA immunization against adhesins represents a new and valuable approach to combat S. aureus infections.

Extracellular Fibrinogen-binding Protein, Efb, fromStaphylococcus aureus Blocks Platelet Aggregation Due to Its Binding to the α-Chain

Extracellular fibrinogen-binding protein (Efb) secreted by Staphylococcus aureus has previously been shown to contribute to pathogenesis in a rat wound infection model. Also antibodies against Efb exhibited a protective effect in a mouse mastitis model. The interaction between Efb and fibrinogen is divalent, with one binding site within the N-terminal repeat region in Efb and one at the C terminus. In this study we show that the distal D domain of fibrinogen contains at least one of the binding domains recognized by Efb. Efb stimulates fibrinogen binding to ADP-activated platelets. Furthermore, Efb inhibits ADP-induced, fibrinogen-dependent platelet aggregation in a concentration-dependent manner. This implies that Efb modifies platelet function by amplifying a non-functional interaction between fibrinogen and platelets. Efb recognizes the A alpha-chain of the D fragment of fibrinogen. The RGD sequence on the A alpha-chain is located close to the region recognized by Efb and contains a putative binding site for the platelet integrin GPIIb/IIIa receptor complex involved in platelet aggregation.

Mitogen-activated protein kinase-defective Candida albicans is avirulent in a novel model of localized murine candidiasis

Candida albicans strains with a deletion of the mitogen-activated protein kinase CEK1 gene are defective in the yeast to hyphal transition on solid surfaces in vitro. The virulence of a cek1Δ/cek1Δ null mutant strain was compared with its wild-type parent strain (WT) in a novel model of localized candidiasis. The mammary glands of lactating mice (at day 5 postpartum) were infected for 2, 4 and 6 days with 50 μl suspension containing 1×10 5, 1×10 6 and 1×10 7 blastospores before death. Infected and non-infected control glands were evaluated pathologically. All animals infected with cek1Δ/cek1Δ null mutant strains showed no lesions while 65% of animals infected with the WT strain had severe lesions characterized by widespread heterophilic infiltration, necrosis, and abscess formation. As an additional control, animals infected with the disrupted strain complemented with the WT CEK1, on a replicating plasmid, also showed severe pathological changes similar to the WT strain. These results clearly demonstrate that the CEK1 gene codes for a virulence determinant of C. albicans and that the mouse mastitis model is well suited for the discriminative study of the pathogenicity of different C. albicans strains.

Mitogen-activated protein kinase-defective Candida albicans is avirulent in a novel model of localized murine candidiasis.

Candida albicans strains with a deletion of the mitogen-activated protein kinase CEK1 gene are defective in the yeast to hyphal transition on solid surfaces in vitro. The virulence of a cek1 delta/cek1 delta null mutant strain was compared with its wild-type parent strain (WT) in a novel model of localized candidiasis. The mammary glands of lactating mice (at day 5 postpartum) were infected for 2, 4 and 6 days with 50 microliter suspension containing 1 x 10(5), 1 x 10(6) and 1 x 10(7) blastopores before death. Infected and non-infected control glands were evaluated pathologically. All animals infected with cek1 delta/cek1 delta null mutant strains showed no lesions while 65% of animals infected with the WT strain had severe lesions characterized by widespread heterophilic infiltration, necrosis, and abscess formation. As an additional control, animals infected with the disrupted strain complemented with the WT CEK1, on a replicating plasmid, also showed severe pathological changes similar to the WT strain. These results clearly demonstrate that the CEK1 gene codes for a virulence determinant of C. albicans and that the mouse mastitis model is well suited for the discriminative study of the pathogenicity of different C. albicans strains.

Multiple Binding Sites in the Interaction between an Extracellular Fibrinogen-binding Protein from Staphylococcus aureus and Fibrinogen

Efb (previously Fib) is a fibrinogen-binding protein secreted by Staphylococcus aureus. It has previously been shown that it plays a role in a wound infection model in the rat and that antibodies against Efb reduce the number of recovered bacteria from the mammary glands in a mouse mastitis model. Efb binds to the alpha-chain of fibrinogen and does not participate in bacterial adherence to fibrinogen. The binding of Efb to fibrinogen is divalent, with one binding site within the two repeat regions in Efb at the N terminus and one binding site at the C terminus. The divalent binding nature leads to precipitation of Efb-fibrinogen complex when the proteins are added to each other at a 1:1 molar ratio. The interaction between Efb and fibrinogen is strongly enhanced by Ca2+ or Zn2+ but not by Mg2.

Intramammary immunization with live-attenuated Staphylococcus aureus: microbiological and immunological studies in a mouse mastitis model

Mammary infection was induced in lactating mice by intramammary injection of Staphylococcus aureus. Histopathological analysis revealed infiltration and lesions of varying magnitude that were still apparent 21 days after the challenge. Concomitantly, viable S. aureus was recovered from infected mammary glands. Mice were immunized by the intramammary route with 5 × 10 6 colony forming units of a temperature-sensitive mutant of S. aureus and subsequently received a boosting injection seven days later. On day 14 mice were challenged by the intramammary route with the wild-type strain. Intramammary immunization induced a significant increase in milk IgA ( P < 0.05), serum IgG ( P < 0.05) and serum IgA ( P < 0.05) on the day of the challenge, when compared with non-immunized mice. Immunization decreased significantly ( P < 0.01) the number of S. aureus colony forming units recovered 96 h after intramammary challenge. In conclusion, the feasibility of immunizing locally with temperature-sensitive S. aureus to induce immunity in the mouse mammary gland was demonstrated. The mouse model of mastitis is proposed as a useful system for screening temperature-sensitive S. aureus strains to be utilized in the development of a vaccine.

Intramammary immunization with live-attenuated Staphylococcus aureus: microbiological and immunological studies in a mouse mastitis model.

Mammary infection was induced in lactating mice by intramammary injection of Staphylococcus aureus. Histopathological analysis revealed infiltration and lesions of varying magnitude that were still apparent 21 days after the challenge. Concomitantly, viable S. aureus was recovered from infected mammary glands. Mice were immunized by the intramammary route with 5 x 10(6) colony forming units of a temperature-sensitive mutant of S. aureus and subsequently received a boosting injection seven days later. On day 14 mice were challenged by the intramammary route with the wild-type strain. Intramammary immunization induced a significant increase in milk IgA (P < 0.05), serum IgG (P < 0.05) and serum IgA (P < 0.05) on the day of the challenge, when compared with non-immunized mice. Immunization decreased significantly (P < 0.01) the number of S. aureus colony forming units recovered 96 h after intramammary challenge. In conclusion, the feasibility of immunizing locally with temperature-sensitive S. aureus to induce immunity in the mouse mammary gland was demonstrated. The mouse model of mastitis is proposed as a useful system for screening temperature-sensitive S. aureus strains to be utilized in the development of a vaccine.

Opsonization of Staphylococcus aureus with a fibronectin-binding protein antiserum induces protection in mice

The virulence ofStaphylococcus aureusopsonized with an antiserum raised against a recombinant fibronectin-binding protein (FnBP) was compared with homologous, non-opsonized bacteria (treated with pre-immune serum) in a mouse mastitis model. Virulence was evaluated comparing the number of bacteria recovered from the infected mammary glands and according to the type of lesions produced. The average number of bacteria recovered from the mammary glands inoculated withS. aureusopsonized with FnBP-antiserum was significantly lower (up to 107cfu/ml) than the average number of bacteria recovered after inoculation with non-opsonized bacteria (up to 1010cfu/ml). Gross examination of infected mammary glands showed that 65% of glands infected with opsonized bacteria developed low grade/or had no pathological changes, and 35% developed severe mastitis whereas, 75% of glands inoculated with non-opsonized bacteria developed severe mastitis and 25% low grade mastitis or had no pathological changes. According to the histopathological examination eight out of 10 glands inoculated with opsonized bacteria produced disseminated focal necrosis or had no pathological changes and two glands produced non reactive necrotic lesions. In contrast, only three out of 10 glands inoculated with non-opsonized homologous bacteria developed disseminated focal necrosis and had no pathological changes while seven glands developed total necrosis.

Vaccination with Staphylococcus aureus fibrinogen binding proteins (FgBPs) reduces colonisation of S. aureus in a mouse mastitis model

A mouse mastitis model was used to study the effect of vaccination with fibrinogen binding proteins and collagen binding protein from Staphylococcus aureus against challenge infection with S. aureus. The mice vaccinated with fibrinogen binding proteins showed reduced rates of mastitis compared with controls. Gross examination of challenged mammary glands of mice showed that the glands of mice immunized with fibronogen binding proteins developed mild intramammary infection or had no pathological changes compared with glands from control mice. Histopathological examination of tissue sections from challenged glands showed that most glands from mice vaccinated with fibrinogen binding protein developed disseminated necrosis or had no pathological changes. A significantly reduced number of bacteria could be recovered in the glands from mice immunized with fibrinogen binding proteins as compared with controls. In a similar study, immunization of mice with collagen binding protein did not induce protection against challenge infection with S. aureus.

Vaccination with Staphylococcus aureus fibrinogen binding proteins (FgBPs) reduces colonisation of S. aureus in a mouse mastitis model.

A mouse mastitis model was used to study the effect of vaccination with fibrinogen binding proteins and collagen binding protein from Staphylococcus aureus against challenge infection with S. aureus. The mice vaccinated with fibrinogen binding proteins showed reduced rates of mastitis compared with controls. Gross examination of challenged mammary glands of mice showed that the glands of mice immunized with fibrinogen binding proteins developed mild intramammary infection or had no pathological changes compared with glands from control mice. Histopathological examination of tissue sections from challenged glands showed that most glands from mice vaccinated with fibrinogen binding protein developed disseminated necrosis or had no pathological changes. A significantly reduced number of bacteria could be recovered in the glands from mice immunized with fibrinogen binding proteins as compared with controls. In a similar study, immunization of mice with collagen binding protein did not induce protection against challenge infection with S. aureus.

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 LD 50 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°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 LD 50 in the vaccinated mice that were challenged with the parental strain increased 11 to 14 times compared with that for the unvaccinated mice.

Vaccination against Staphylococcus aureus mastitis: immunological response of mice vaccinated with fibronectin-binding protein (FnBP-A) to challenge with S. aureus

Mice were immunized with fusion proteins encompassing the fibronectin-binding domain of a staphylococcal fibronectin-binding protein (FnBP-A). A specific antibody response against the fibronectin-binding part of the fusion proteins was detected in the serum of all vaccinated animals. The protective potential of these vaccinations was evaluated in a mouse mastitis model, using Staphylococcus aureus, strain SA113, for challenge. The mice vaccinated with FnBP fusion proteins showed a decreased number of bacteria recovered from the mammary glands and significantly reduced cases of severe mastitis. Histopathological examination of tissue from challenged glands of vaccinated mice revealed either no pathological reactions or disseminated inflammatory reactions with focal necrosis whereas four of six examined tissues from challenged glands of non-vaccinated animals showed total necrosis. A combination of FnBP fusion protein with staphylococcal α-toxoid did not increase the efficacy of the vaccination and animals vaccinated with α-toxoid alone were as sensitive to challenge as those from the non-vaccinated control group. Thus vaccination of mice with recombinant FnBP resulted in significant protection against challenge with S. aureus.

Enhanced virulence of Staphylococcus aureus from bovine mastitis induced by growth in milk whey

The virulence towards mice of Staphylococcus aureus strains from bovine mastitis was enhanced upon growth in milk whey compared to homologous organisms grown in tryptic soy broth (TSB). In the mouse mastitis model, S. aureus grown in milk whey caused more severe lesions than homologous strains grown in TSB. Staphylococcus aureus strain F1440 grown in milk whey induced 75% mortality and local necrotic reaction in subcutaneously inoculated mice, whereas the homologous strain grown in TSB caused only 5% mortality and slight skin reaction. Extracellular capsule on milk whey-grown. S. aureus could not be demonstrated. However, diffuse type colony morphology could be correlated with an increased virulence of S. aureus towards mice.

Virulence of Staphylococcus aureus in a mouse mastitis model: studies of alpha hemolysin, coagulase, and protein A as possible virulence determinants with protoplast fusion and gene cloning.

Mutants of a genetically well-characterized strain of Staphylococcus aureus [SA113(83A)] were isolated after mutagenization. Alpha-hemolysin- (hla), coagulase- (coa), and protein A- (spa) negative mutants were characterized by more than 90 biochemical tests for production of extracellular proteins and biochemical profile to exclude pleiotropy. Protoplast fusion was then used to isolate double-defective (hla and coa) recombinants and recombinants with regained properties, i.e., production of alpha-hemolysin and coagulase. Studies of such mutants and recombinants in the mouse mastitis model showed that one alpha-hemolysin [SA113(83A) hla-5] and one coagulase-negative [SA113(83A) coa-147] mutant were lower in virulence compared with the wild-type strain SA113(83A). The double-negative mutant SA113(83A) hla-5 coa-147 showed a drastic decline in virulence and only induced very mild changes, as determined by microscopic examinations of infected mammary gland tissue. The recombinant with regained properties, however, was as virulent as the wild-type strain. This suggests that alpha-hemolysin and coagulase are virulence determinants of S. aureus. A high-level protein A-producing mutant (U300) showed the same virulence as the parent strain SA113(83A) in this model. One low virulence protein A-negative mutant (U320) did not markedly increase in virulence when a plasmid containing the cloned gene for protein A (pSPA15) was introduced into this mutant. By these and earlier observations, it seems likely that protein A is not an important virulence determinant in mastitis of mice. The reduced virulence of the protein A-negative mutant U320 compared with the wild-type SA113(83A) may be due to pleiotropic loss of some other unknown virulence determinant(s). Our data confirm earlier findings that pleiotropic changes are common in protein A-negative mutants.

The mouse mastitis model: observations relevant to the treatment and control of coliform mastitis.

The response to intramammary inoculation ofEscherichia coli in mice is described and the contribution of endotoxin to the clinical signs is considered. The implications of these responses for therapy are examined. The role of the neutrophil is assessed and it is considered that the rate of arrival of neutrophils in the gland is of more importance in limiting infection than the number of existing neutrophils at the time of challenge. The effect of inducing a neutrophil response by colonization of the gland by a pathogen of low virulence is described and the dangers of inducing chronic infections are emphasised. Using a modification of the mouse model in which organisms are placed on the tip of damaged or undamaged teats of suckling or non-suckling mice it is demonstrated by the pattern of response that staphylococci actively colonize the teat and subsequently invade the gland whereasE. coli infections result from the chance entrance of contaminating organisms.