Murine Mastitis Research Articles

In vitro and in vivo assessment of phage therapy against Staphylococcus aureus causing bovine mastitis

ObjectiveThe aim of this study was to assess the efficacy of lytic bacteriophages on Staphylococcus aureus causing bovine mastitis, by in vitro and in vivo assays using Galleria mellonella and murine mastitis models. MethodsBetween May and December 2016, ten S. aureus (five methicillin-resistant and five methicillin-sensitive) isolates were isolated from milk samples of cattle with mastitis in Belgium and Norway. The isolates were assessed in vitro for their susceptibility to four lytic bacteriophages (Romulus, Remus, ISP and DSM105264) and subsequently in vivo in G. mellonella larvae and in murine mastitis model. ResultsRomulus, Remus and ISP showed a lytic activity against the S. aureus isolates in vitro. A larvae survival rate below 50% was observed at 4 days post-inoculation (DPI) in the groups infected with a methicillin-sensitive S. aureus isolate and treated with these three phages in vivo. An incomplete recovery of the mouse mastitis was observed at 48h post-inoculation (HPI) in the groups infected and treated with the ISP phage in vivo. ConclusionsThe observations are much more pronounced statistically between the infected- phosphate buffered saline (PBS)-treated and infected-phage-treated groups in G. mellonella and the murine mastitis model demonstrating an effect of the phages against S. aureus associated with bovine mastitis.

Exopolysaccharides from Lactobacillus kiferi as adjuvant enhanced the immuno-protective against Staphylococcus aureus infection

Exopolysaccharides from lactic acid bacteria (LAB) have gained more attention due to their health benefits. Most research on LAB EPS focuses on antitumor and antioxidant activities. To our knowledge, the immunoadjuvant activity of LAB EPS has not been thoroughly studied. In this study, the EPS produced by Lactobacillus kiferi WXD029 were purified by ethanol precipitation and column chromatography fractionation. The molecular weight of the EPS was 3.423 × 105 Da and was mainly composed of Glu, GlcN, and GalN in a molar ratio of 3.1:1:1. In vitro, EPS could significantly enhance the proliferation and phagocytic activity as well as induce the production of NO, TNF-α, IL-1β, and IL-6 in RAW264.7 cells. In vivo, the EPS adjuvant could increase the titers of S.aureus antigen-specific antibodies and markedly enhanced T cell proliferation. Notably, EPS adjuvant also induced a strong potential Th1, Th2 and Th17-cell mixture responses. Furthermore, immunization with S.aureus antigen plus EPS adjuvant induced a protective effect when compared with S.aureus antigen alone in murine bacteremia, pneumonia and mastitis model. Collectively, these results suggest that EPS derived from probiotic Lactobacillus kiferi strain is promising as an efficient adjuvant candidate for the prevention of S. aureus infections.

Characterization of an Enterococcus faecium strain in a murine mastitis model.

The aim of the study was to characterize phenotypically and genotypically Enterococcus faecium strains collected from bovine mastitis milk and to evaluate one of them for its virulence in a murine mastitis model. A total of five E. faecium isolates were collected from cows with subclinical mastitis. EF-7A showed resistance to antibiotics tested, it presented alpha haemolysin and did not present gelatinase activity. It yielded cyA, efafm and gelE1 genes and it could be characterized as a moderate biofilm producer. It was able to internalize in MAC-T cells and 1×108 colony forming unit ml-1 was able to establish an intramammary infection in mice. The strain could be recovered from liver, kidney and blood samples. RAPD profiles showed different bands with respect to the inoculated strain. Histopathology analyses showed different grades of polymorphonuclear neutrophils infiltration in mammary glands. This is the first report that studied E. faecium strain in a lactating mouse model of mastitis and showed that the experimental inoculation was able to stimulate an inflammatory response resulting in mastitis. Results contribute to a better understanding of intramammary infections caused by E. faecium. This investigation shows that mice represent a valuable model for the study of the mastitis pathogenesis caused by E. faecium considering the high costs of using cows for mastitis research.

Nanocurcumin ameliorates Staphylococcus aureus-induced mastitis in mouse by suppressing NF‑κB signaling and inflammation

Mastitis is the inflammation of the mammary glands caused by bacteria. It causes severe economic loss to dairy industry. Curcumin, a polyphenol obtained from turmeric, has considerable anti-inflammatory effect. Since it is rapidly eliminated from the body, its oral bioavailability is low. However, nanoformulation of curcumin significantly enhances its therapeutic efficiency by improving its oral bioavailability. We evaluated whether nanocurcumin could be more effective than normal curcumin against bovine Staphylococcus aureus mastitis in mouse model. Curcumin-loaded PLGA nanoparticles (CUR-NP) were prepared by solid-in-oil-in-water emulsion method. The mouse model of mastitis was induced by inoculation of a field strain of S. aureus (bovine mastitis isolate) on the 9th day of parturition through the duct of the mammary gland. CUR-NP and curcumin were given orally for 7 days (day 2 to day 8 of parturition) prior to S. aureus inoculation. We determined the levels of inflammatory cytokines and the mRNA expression of NF‑κB. S. aureus infection increased the levels of tumor necrosis factor‑α, interleukin‑1β and myeloperoxidase in mammary tissues and C-reactive protein in serum. Both CUR-NP and curcumin significantly attenuated the levels of these cytokines. However, comparatively, the ameliorative efficiency of CUR-NP was better than normal curcumin. S. aureus infection-induced NF‑κB mRNA expression was significantly reduced to the healthy control level by CUR-NP. Our study demonstrates that the nanoformulation of curcumin can reduce pro-inflammatory mediators in S. aureus-infected mammary tissues by improving NF‑κB signaling. Besides, compared to normal curcumin, this nanoformulation appears to be a better alternative against murine mastitis.

Genomic analysis and immune response in a murine mastitis model of vB_EcoM-UFV13, a potential biocontrol agent for use in dairy cows

Bovine mastitis remains the main cause of economic losses for dairy farmers. Mammary pathogenic Escherichia coli (MPEC) is related to an acute mastitis and its treatment is still based on the use of antibiotics. In the era of antimicrobial resistance (AMR), bacterial viruses (bacteriophages) present as an efficient treatment or prophylactic option. However, this makes it essential that its genetic structure, stability and interaction with the host immune system be thoroughly characterized. The present study analyzed a novel, broad host-range anti-mastitis agent, the T4virus vB_EcoM-UFV13 in genomic terms, and its activity against a MPEC strain in an experimental E. coli-induced mastitis mouse model. 4,975 Single Nucleotide Polymorphisms (SNPs) were assigned between vB_EcoM-UFV13 and E. coli phage T4 genomes with high impact on coding sequences (CDS) (37.60%) for virion proteins. Phylogenetic trees and genome analysis supported a recent infection mix between vB_EcoM-UFV13 and Shigella phage Shfl2. After a viral stability evaluation (e.g pH and temperature), intramammary administration (MOI 10) resulted in a 10-fold reduction in bacterial load. Furthermore, pro-inflammatory cytokines, such as IL-6 and TNF-α, were observed after viral treatment. This work brings the whole characterization and immune response to vB_EcoM-UFV13, a biocontrol candidate for bovine mastitis.

Kaempferol modulates Angiopoietin-like protein 2 expression to lessen the mastitis in mice.

Mastitis is inflammation of a breast (or udder). Angiopoietin-like protein 2 (ANGPTL2) has been found as a key inflammatory mediator in mastitis. Purpose of this research was to investigate the mechanisms about repressing effect of kaempferol on mastitis. Forty mice were randomly divided into 4 groups (n=10): C57BL/6J control mice, untreated murine mastitis, 10mg/kg kaempferol treated murine mastitis (ip), and 30mg/kg kaempferol treated murine mastitis (ip). Primary cultured mouse mammary epithelial cells (MMEC) were indiscriminately divided into seven groups including control group, 10mmol/L vehicle of kaempferol group, 10μmol/L kaempferol treated group, 20μg/mL LPS treated group, 1μmol/L kaempferol plus LPS treated group, 3μmol/L kaempferol plus LPS treated group, and 10μmol/L kaempferol plus LPS treated group. In murine mastitis, kaempferol (10 or 30mg/kg) treatment prevented mastitis development, decreased myeloperoxidase (MPO) production, interleukin (IL)-6 level, tumour necrosis factor-α (TNF-α) concentration, and ANGPTL2 expression. In MMEC, kaempferol (1, 3 or 10μM) reduced MPO production, TNF-α concentration, IL-6 level, and ANGPTL2 expression. The results in present study show that kaempferol modulates the expression of ANGPTL2 to lessen the mastitis in mice.

Antibacterial, anti‐inflammatory and antioxidant effects of acetyl‐11‐α‐keto‐β‐boswellic acid mediated silver nanoparticles in experimental murine mastitis

Mastitis is an important economic disease causing production losses in dairy industry. Antibiotics are becoming ineffective in controlling mastitis due to the emergence of resistant strains requiring the development of novel therapeutic agents. In this study, the authors present the phytochemical synthesis of silver nanoparticles (AgNPs) with acetyl-11-α-keto-β-boswellic acid and evaluation of their activity in Staphylococcus aureus induced murine mastitis. Boswellic acid mediated AgNP (BANS) were oval, polydispersed (99.8 nm) with an minimum inhibitory concentration of 0.033 µg ml−1 against S. aureus, inhibitory concentration (IC50) of 30.04 µg ml−1 on mouse splenocytes and safe at an in vivo acute oral dose of 3.5 mg kg−1 in mice. Mastitis was induced in lactating mice by inoculating S. aureus (log10 5.60 cfu) and treated 6 h post-inoculation with BANS (0.12 mg kg−1, intramammary and intraperitoneal), and cefepime (1 mg kg−1, intraperitoneal). S. aureus inoculated mice showed increased bacterial load, neutrophil infiltration in mammary glands and elevated C-reactive protein (CRP) in serum. Oxidative stress was also observed with elevated malondialdehyde level, superoxide dismutase (SOD) and catalase (CAT) activities. BANS treatment significantly (P < 0.05) reduced bacterial load, CRP, SOD, CAT activities and neutrophil infiltration in affected mammary glands. BANS could be a potential therapeutic agent for managing bovine mastitis.

Angiopoietin-like protein 2 may mediate the inflammation in murine mastitis through the activation of interleukin-6 and tumour necrosis factor-α.

Mastitis is the inflammation of the mammary gland. Recent research has shown that Angiopoietin-like protein 2 (ANGPTL2) is a key inflammatory mediator. In the present study, we tested whether there is a correlation between increased ANGPTL2 expression and inflammation in response to Staphylococcus aureus in murine mastitis and the mechanisms involved. Thirty mice were divided into two groups: blank control group, challenged group. The entire infused mammary glands were removed to observe the changes of histopathology, myeloperoxidase (MPO) activity, production of tumour necrosis factor-α (TNF-α) and interleukin (IL)-6, and genes expression of ANGPTL2, TNF-α and IL-6. In challenged group, the structure of mammary glands was damaged and the large areas of cell fragments were observed. The MPO activity, IL-6 and TNF-α concentrations, ANGPTL2, IL-6, and TNF-α mRNA levels were significantly elevated in challenged group compared with blank control group. The present findings indicate ANGPTL2 may mediate the inflammation in murine mastitis through the activation of IL-6 and TNF-α.

Non-Classical ProIL-1beta Activation during Mammary Gland Infection Is Pathogen-Dependent but Caspase-1 Independent

Infection of the mammary gland with live bacteria elicits a pathogen-specific host inflammatory response. To study these host-pathogen interactions wild type mice, NF-kappaB reporter mice as well as caspase-1 and IL-1beta knockout mice were intramammarily challenged with Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The murine mastitis model allowed to compare the kinetics of the induced cytokine protein profiles and their underlying pathways. In vivo and ex vivo imaging showed that E. coli rapidly induced NF-kappaB inflammatory signaling concomitant with high mammary levels of TNF-alpha, IL-1 alpha and MCP-1 as determined by multiplex analysis. In contrast, an equal number of S. aureus bacteria induced a low NF-kappaB activity concomitant with high mammary levels of the classical IL-1beta fragment. These quantitative and qualitative differences in local inflammatory mediators resulted in an earlier neutrophil influx and in a more extensive alveolar damage post-infection with E. coli compared to S. aureus. Western blot analysis revealed that the inactive proIL-1beta precursor was processed into pathogen-specific IL-1beta fragmentation patterns as confirmed with IL-1beta knockout animals. Additionally, caspase-1 knockout animals allowed to investigate whether IL-1beta maturation depended on the conventional inflammasome pathway. The lack of caspase-1 did not prevent extensive proIL-1beta fragmentation by either of S. aureus or E. coli. These non-classical IL-1beta patterns were likely caused by different proteases and suggest a sentinel function of IL-1beta during mammary gland infection. Thus, a key signaling nodule can be defined in the differential host innate immune defense upon E. coli versus S. aureus mammary gland infection, which is independent of caspase-1.

Evaluation of therapeutic potential of nanosilver particles synthesised using aloin in experimental murine mastitis model

Nanobiotechnology is an emerging biological branch of nanotechnology. Application of nanoparticles with specific size and shape in biology has already shown unforeseen and interesting results. A study was conducted to evaluate the therapeutic potential of phytogenically derived aloin mediated nanosilver particles (AAgNPs), prepared by reduction of silver nitrate with aloin, in Staphylococcus aureus induced murine mastitis. A total of 40 female mice were divided into five groups of eight animals each. Group I served as lactating control, groups II-V were inoculated with 20 μl of 24 h broth culture of S. aureus containing 4.0 × 105 cfu/quarter under ketamine anaesthesia. After 6 h post inoculation, groups III and IV received 20 μl of aloin nanosilver (AAgNPs) through intramammary and intraperitoneal routes, respectively. Group V received antibiotic cefepime at 1 mg/kg body weight through the intra-peritoneal route. After 18 h post-treatment, serum C reactive protein, weights of mammary glands, mammary gland bacterial load, thiobarbituric acid reactive substances content, reduced glutathione content, superoxide dismutase activity and catalase activity and histopathology were determined. The compound showed a minimum inhibitory concentration of 21.8 ng/ml against S. aureus. Significant reduction (98%) in poly-morpho nuclear cell infiltration was observed with AAgNPs than antibiotic (50%).

Treatment with Gentamicin on a Murine Model of Protothecal Mastitis

The aim of this study was to establish a murine protothecal mastitis model and to evaluate the treatment efficiency of gentamicin. Challenge routes were determined with a pathogenic Prototheca zopfii genotype 2 (P. zopfii) strain. 25 BALB/c mice were inoculated in mammary glands with graded dosages (10(3), 10(4), 10(5), 10(6), 10(7) CFU of P. zopfii) and killed on the 7th day. Another 25 animals were also killed at 1, 3, 5, 7, and 9 days after inoculation of 1 × 10(6) CFU of P. zopfii, the milk somatic cell counts, pathological section of mammary glands, and P. zopfii burden were observed. The antimicrobial activity was tested using disc diffusion test and minimum inhibitory concentrations. Gentamicin was given intramuscularly to analyze the therapeutic effect. The results showed that the best infection route was intra-mammary gland, and the mastitis model was established with 1 × 10(6) CFU of P. zopfii. After infection, the somatic cell counts increased significantly. The pathological reaction mainly consisted of infiltration of inflammatory cells, destruction of acini, accumulation of lymphocyte cells and the severity of the changes was dosage and time-dependent. The P. zopfii burden revealed that P. zopfii continuously replicated. In vitro susceptibility tests indicated that the Prototheca strains were antimicrobial susceptible to gentamicin at concentrations between 0.03 and 4 μg/ml. In vivo therapeutic assay demonstrated that high concentrations of gentamicin (≥20 mg/kg) could inhibit the growth of P. zopfii. We conclude that the murine model of protothecal mastitis was established successfully and gentamicin may be an effective choice for treatment of P. zopfii.

Pam3CSK4/TLR2 signaling elicits neutrophil recruitment and restricts invasion of Escherichia coli P4 into mammary gland epithelial cells in a murine mastitis model

Mastitis-inflammation of the mammary gland is an important disease affecting dairy animals worldwide. The disease is caused by mammary pathogenic bacteria, and Escherichia coli is frequently implicated. Intramammary challenge with bacterial LPS is sufficient to elicit the disease. However, using toll-like receptor (TLR) 4-deficient mice, we previously found that mammary pathogenic E. coli is still able to elicit neutrophil recruitment, indicating the presence of bacterial virulence factors other than LPS. To date, no specific virulence factors have been identified in E. coli isolates associated with mastitis, and other microbe-associated molecular patterns (MAMPs), such as bacterial lipoproteins, are prime candidates. The synthetic analog of bacterial lipopeptides, Pam3CSK4, is recognized by TLR2 and mimics the proinflammatory properties of triacylated lipoproteins of Gram-negative bacteria.The aim of the present work was to determine the role of bacterial lipoproteins recognized by TLR2 on mammary cells as virulence factors in the mammary gland. Using the murine mastitis model, we previously showed that following intramammary LPS challenge, neutrophil recruitment is strictly dependent on alveolar macrophages. Thus, the role of alveolar macrophages in the response to intramammary bacterial lipoprotein challenge was also studied. Here, Pam3CSK4 infusion induced mastitis in wild-type mice, but not in TLR2-deficient mice. The wild-type phenotype was not restored by adoptive transfer of TLR2-expressing macrophages into the alveolar milk space of TLR2-deficient mice, indicating that cells other than alveolar macrophages are essential for Pam3CSK4/TLR2 signaling. In contrast to the Pam3CSK4 treatment, infection with E. coli P4 resulted in inflammation, even in the absence of TLR2 signaling, indicating that lipoproteins are sufficient, but not essential virulence factors in the pathogenesis of the intact bacteria. However, in the absence of TLR2, the infecting E. coli P4 invaded the alveolar epithelial cells and formed intracellular bacterial communities, indicating that intact lipoprotein/TLR2 signaling is essential to restricting bacterial invasion.

Essential role of neutrophils but not mammary alveolar macrophages in a murine model of acuteEscherichia colimastitis

Mastitis, the inflammation of the mammary gland, is an important disease affecting dairy animals worldwide. The disease is caused by mammary pathogenic bacteria and Escherichia coli are frequently implicated. Virulence factors of mammary pathogenic E. coli are only partially known and intramammary challenge with LPS elicits neutrophil recruitment in experimental bovine and murine mastitis models. We have previously shown that neutrophil recruitment in LPS-induced murine mastitis is strictly dependent on mammary alveolar macrophages. However, the relative role of alveolar macrophages and blood neutrophils in E. coli mastitis is not well defined. To this end, we selectively depleted mammary alveolar macrophages or blood neutrophils before intramammary challenge with E. coli strain P4 (ECP4). Mice depleted of alveolar macrophages prior to intramammary challenge recruited neutrophils normally and restricted bacterial growth and interstitial invasion. Importantly however, upon depletion of alveolar macrophages, ECP4 invaded the mammary alveolar epithelial cells and formed intracellular bacterial communities. In contrast, neutrophil depletion prior to intramammary infection with ECP4 was associated with unrestricted bacterial growth, tissue damage, severe sepsis and mortality. This study suggests that neutrophils but not alveolar macrophages provide essential antimicrobial defense against mammary pathogenic E. coli. Furthermore, we show here similar invasion after depletion of alveolar macrophages as in our previous studies showing that LPS/TLR4 signaling on alveolar macrophages abrogates ECP4 invasion of the mammary epithelium. Interestingly, similar ECP4 invasion and formation of intracellular communities were also observed following intramammary infection of either iNOS gene-deficient or IL-1 receptor type 1 gene-deficient mice.

Neutrophil recruitment in endotoxin-induced murine mastitis is strictly dependent on mammary alveolar macrophages

Mastitis, inflammation of the mammary tissue, is a common disease in dairy animals and mammary pathogenic Escherichia coli (MPEC) is a leading cause of the disease. Lipopolysaccharide (LPS) is an important virulence factor of MPEC and inoculation of the mammary glands with bacterial LPS is sufficient to induce an inflammatory response. We previously showed using adoptive transfer of normal macrophages into the mammary gland of TLR4-deficient C3H/HeJ mice that LPS/TLR4 signaling on mammary alveolar macrophages is sufficient to elicit neutrophil recruitment into the alveolar space. Here we show that TLR4-normal C3H/HeN mice, depleted of alveolar macrophages, were completely refractory to LPS intramammary challenge. These results indicate that alveolar macrophages are both sufficient and essential for neutrophil recruitment elicited by LPS/TLR4 signaling in the mammary gland. Using TNFα gene-knockout mice and adoptive transfer of wild-type macrophages, we show here that TNFα produced by mammary alveolar macrophages in response to LPS/TLR4 signaling is an essential mediator eliciting blood neutrophil recruitment into the milk spaces. Furthermore, using the IL8 receptor or IL1 receptor gene-knockout mice we observed abrogated recruitment of neutrophils into the mammary gland and their entrapment on the basal side of the alveolar epithelium in response to intramammary LPS challenge. Adoptive transfer of wild-type neutrophils to IL1 receptor knockout mice, just before LPS challenge, restored normal neutrophil recruitment into the milk spaces. We conclude that neutrophil recruitment to the milk spaces is: (i) mediated through TNFα, which is produced by alveolar macrophages in response to LPS/TLR4 signaling and (ii) is dependent on IL8 and IL1β signaling and regulated by iNOS-derived NO.

Toll-like receptor 4 is needed to restrict the invasion of Escherichia coli P4 into mammary gland epithelial cells in a murine model of acute mastitis

Mastitis, an inflammatory response of the mammary tissue to invading pathogenic bacteria, is a common disease in breast-feeding women and dairy animals. Escherichia coli is a leading cause of mastitis in dairy animals. During the course of the disease the host mounts a strong inflammatory response, but specific bacterial virulence factors have not yet been identified. Here we report the use of a murine mastitis model to investigate the innate inflammatory reaction of the mammary gland. We show that lipopolysaccharide (LPS) infusion induces mastitis in wild-type mice (C3H/HeN), but not in mice expressing mutated Toll-like receptor 4 (TLR4) (C3H/HeJ). The wild-type phenotype was restored by adoptive transfer of TLR4-expressing macrophages into the alveolar milk space of C3H/HeJ mice. In contrast to the LPS treatment, infection with E. coli P4 (ECP4) resulted in inflammation even in the absence of LPS/TLR4 signalling, indicating that additional factors play a role in the pathogenesis of the intact bacteria. Furthermore, in the absence of functional TLR4 the infecting ECP4 invade the epithelial cells with high efficiency, forming intracellular microcolonies. However, adoptive transfer with TLR4-expressing macrophages drastically reduced the epithelial invasion. Taken together, these results indicate that ECP4 has an invasive potential, which is restricted by alveolar macrophages in response to the LPS/TLR4 signalling.

Capsule-Negative Staphylococcus aureus Induces Chronic Experimental Mastitis in Mice

Staphylococcus aureus capsular polysaccharides (CP) have been shown to enhance staphylococcal virulence in numerous animal models of infection. Although serotype 5 CP (CP5) and CP8 predominate among S. aureus isolates from humans, most staphylococcal isolates from bovines with mastitis in Argentina are capsule negative. This study was designed to evaluate the effects of CP5 and CP8 expression on the pathogenesis of experimental murine mastitis. Lactating mice were challenged by the intramammary route with one of three isogenic S. aureus strains producing CP5, CP8, or no capsule. Significantly greater numbers of acapsular mutant cells were recovered from the infected glands 12 days after bacterial challenge compared with the encapsulated strains. Histopathological analyses revealed greater polymorphonuclear and mononuclear leukocyte infiltration and congestion in the mammary glands of mice infected with the encapsulated strains compared with the acapsular mutant, and the serotype 5 strain elicited more inflammation than the serotype 8 strain. In vitro experiments revealed that the acapsular S. aureus strain was internalized by MAC-T bovine epithelial cells in significantly greater numbers than the CP5- or CP8-producing strain. Taken together, the results suggest that S. aureus lacking a capsule was able to persist in the murine mammary gland, whereas encapsulated strains elicited more inflammation and were eliminated faster. Loss of CP5 or CP8 expression may enhance the persistence of staphylococci in the mammary glands of chronically infected hosts.

Complement activation in SCID and nude mice is related to severity of tissue inflammation in the Candida mastitis model.

A small animal model of localised candidiasis is needed for the evaluation of new antifungal compounds. Mammary glands of immunocompetent (BALB/cJ) and immunodeficient (SCID and athymic nude) mice were infected with a wild-type of Candida albicans. Some of the animals were treated with amphotericin B (AmB) while others were treated with saline and acted as controls. The histologic changes of infected mammary gland tissues and a number of other organs were evaluated. Complement (C) activation was analysed by immunoelectrophoretic quantification of molecules with C3c epitopes (C3, C3b, iC3b, and C3c) in serum. In all animals the organisms were confined to the mammary glands. Serum C3c levels were significantly higher (P<0.05) in infected untreated BALB/cJ and SCID mice, which also had severe mammary gland tissue inflammation, compared with control mice treated with AmB (4 mg kg(-1) i.p. once daily for 4 days). Both treated and control nude mice showed less tissue inflammation compared to BALB/cJ and SCID mice, and revealed insignificant activation of the complement system. It is concluded that innate immune response is important in the control of candidiasis and that the murine mastitis model is useful for immunopathological studies as well as evaluation of potential antifungal compounds.

Complement activation in SCID and nude mice is related to severity of tissue inflammation in the Candida mastitis model

A small animal model of localised candidiasis is needed for the evaluation of new antifungal compounds. Mammary glands of immunocompetent (BALB/cJ) and immunodeficient (SCID and athymic nude) mice were infected with a wild-type of Candida albicans. Some of the animals were treated with amphotericin B (AmB) while others were treated with saline and acted as controls. The histologic changes of infected mammary gland tissues and a number of other organs were evaluated. Complement (C) activation was analysed by immunoelectrophoretic quantification of molecules with C3c epitopes (C3, C3b, iC3b, and C3c) in serum. In all animals the organisms were confined to the mammary glands. Serum C3c levels were significantly higher ( P<0.05) in infected untreated BALB/cJ and SCID mice, which also had severe mammary gland tissue inflammation, compared with control mice treated with AmB (4 mg kg −1 i.p. once daily for 4 days). Both treated and control nude mice showed less tissue inflammation compared to BALB/cJ and SCID mice, and revealed insignificant activation of the complement system. It is concluded that innate immune response is important in the control of candidiasis and that the murine mastitis model is useful for immunopathological studies as well as evaluation of potential antifungal compounds.

Experimental murine mycotic mastitis: a sensitive and lenient model for studies of antifungal chemotherapy.

The murine model of mycotic mastitis was used to study the efficacy of amphotericin B (AmB). Twenty-four BALB/cJ mice at the fifth day of lactation were anesthetized and inoculated through the teat canal (two glands) with 50 microl suspension containing 5.0 x 10(7) cfu ml(-1) Candida albicans blastospores. Mice were randomly divided into two groups: untreated controls and AmB treated. Animals were euthanized 3 and 6 days after infection and treatment (4 mg kg(-1) per day intraperitoneally). The fungal burden of the mammary gland was determined by quantitative cultures. The number of C. albicans cells recovered from mammary gland homogenates were significantly lower in the AmB treated animals (both 3 and 6 days post-infection) than in the untreated controls (P<0.007 and P<0.003, respectively). The mammary glands of all untreated control animals showed marked neutrophilic infiltration, severe necrosis, and presence of blastospores, hyphae and pseudohyphae. In contrast, 10 of 12 animals treated with AmB showed only a mild neutrophilic infiltration which was restricted to alveoli and excretory ducts. All extra-mammary organs were free of infection in both groups. The results demonstrate that the murine mycotic mastitis model is suitable for investigations of new antifungal compounds. In addition, this model is more lenient than the systemic candidiasis models.

Experimental murine mycotic mastitis: a sensitive and lenient model for studies of antifungal chemotherapy

Experimental murine mycotic mastitis: a sensitive and lenient model for studies of antifungal chemotherapy