Escherichia coli inoculation of porcine mammary glands affects local mRNA expression of Toll-like receptors and regulatory cytokines

The expression of mRNAs for the Toll-like receptors (TLRs) TLR2 and TLR4, pro- and anti inflammatory cytokines and their receptors was evaluated in mammary gland biopsy material collected from sows intramammarily inoculated with Escherichia coli strain O127 at parturition. Quantitative real-time RT-PCR analysis showed increased mRNA levels for TLR2, the proinflammatory cytokines interleukin IL-1beta and tumor necrosis factor-alpha TNF-alpha, and the anti-inflammatory cytokine IL-10 in the inoculated mammary glands 24h after inoculation. Increased mRNA levels of the proinflammatory cytokine IL-6 were only observed in the inoculated mammary glands of sows that developed clinical signs of mastitis. In contrast, the expression of the anti-inflammatory cytokine, transforming growth factor-beta 1 (TGF-beta1) mRNA was unaltered, as was mRNA expression for the IL-1 receptor type I (IL-1R1). Furthermore, IL-1beta and IL-10 mRNA expression was higher in the inoculated mammary glands of sows that developed clinical signs of mastitis compared with sows that remained clinically healthy. Notably, sows that developed clinical signs of mastitis had significantly lower pre-inoculation levels of IL-1beta mRNA than sows that remained clinically healthy. These findings suggest that development of coliform mastitis is associated with the level of local expression of regulatory cytokines in response to intramammary E. coli inoculation and infection.     

Tumor necrosis factor-alpha, interleukin-6, serum amyloid A, haptoglobin, and cortisol concentrations in sows following intramammary inoculation of Escherichia coli

OBJECTIVE: To determine whether concentrations of proinflammatory cytokines, acute-phase proteins, and cortisol differ at parturition among 3 categories of sows (noninoculated, clinically affected and nonaffected following intramammary inoculation with Escherichia coll). ANIMALS: 16 sows. PROCEDURE: Sows were allocated to inoculated (n = 12) or noninoculated (4) groups. Inoculated sows received intramammary administration of E coli (serotype O127) during the 24-hour period preceding parturition. Blood samples were collected from noninoculated and inoculated sows for 3 consecutive days within 3 to 11 days before farrowing and inoculation. Samples were also collected 0, 24, 48, 72, and 96 hours after farrowing and inoculation. Inoculated sows were further categorized as affected (4 sows) or nonaffected (8 sows) based on clinical signs of disease. Serum tumor necrosis factor (TNF)-alpha, plasma interleukin (IL)-6, and serum amyloid A (SAA) concentrations were measured by use of ELISA; serum haptoglobin concentration was assayed by use of a hemoglobin-binding method; and plasma cortisol concentration was determined by use of radioimmunoassay. RESULTS: Plasma or serum concentrations of TNF-alpha, IL-6, and SAA of both categories of inoculated sows were significantly increased by 24 hours after intramammary inoculation of E coli, compared with concentrations in noninoculated sows. Concentrations of serum TNF-alpha and plasma IL-6 were significantly higher in inoculated sows that developed clinical mastitis than in nonaffected inoculated sows. CONCLUSIONS AND CLINICAL RELEVANCE: Concentrations of TNF-alpha and IL-6 are promising markers for the identification of periparturient sows with subclinical coliform mastitis. Identification of such sows should help improve the health and survival of piglets.     

Dynamics of leukocytes and cytokines during experimentally induced Streptococcus uberis mastitis

Streptococcus uberis causes a significant proportion of clinical and subclinical intramammary infections (IMI) in lactating and non-lactating dairy cows. In spite of this, its pathogenesis is incompletely understood. A study was conducted to determine leukocyte and cytokine dynamics during experimentally induced S. uberis mastitis. Five Jersey and five Holstein cows were challenged via intramammary inoculation of S. uberis into two uninfected mammary glands. Sixteen of 20 challenged mammary glands developed clinical mastitis with peak clinical signs observed at 144 h. The number of S. uberis in milk increased (P<0.05) 48 h after challenge, in spite of an increase in milk somatic cells that began at 18 h (P<0.001) and remained elevated throughout the study. Increased tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interleukin-8 (IL-8) in milk were detected 66 h after challenge (P<0.05). Peak TNF-alpha and IL-8 concentrations occurred 120 h after challenge and preceded peak clinical signs. Experimental S. uberis IMI induced local production of TNF-alpha, IL-1beta and IL-8, which may play a role in the pathogenesis of S. uberis mastitis. Other mediators may be involved in initial leukocyte recruitment to the mammary gland, since increases in milk somatic cells occurred earlier than cytokine production.     

Sows intramammarily inoculated with Escherichia coli at parturition. II. Effects on the densities of MHC class II(+), CD4(+) and CD8(+) cells in the mammary gland

The aim of this study was to determine the density of MHC class II, CD4 and CD8 positive cells in mammary glands of sows around parturition, and whether the densities were altered following intramammary inoculation with Escherichia coli prior to parturition. Also, animals developing clinical disease after inoculation were compared with animals not developing clinical disease. Fourteen cross-bred primiparous sows were subject to intramammary inoculation with E. coli bacteria 24h before estimated parturition. Mammary gland biopsies were collected and clinical observations were made. Four sows were categorised as clinically ill based on general condition, body temperature and gross mammary affection. There were no changes in density of MHC class II, CD4 and CD8 positive cells in non-inoculated glands around parturition, while significant changes in densities were shown in inoculated glands. Here, the density of MHC class II, CD4 and CD8 positive cells reached a peak at 72 h post-inoculation (p<0.01). In sows developing clinical disease, there was a tendency to an over all lower density (p=0.07) of MHC class II positive cells in inoculated glands compared with sows not developing clinical disease. When comparing the categories with respect to the density of CD4 and CD8 positive cells, the sows developing clinical disease showed a higher density (p=0.03) of CD4 and CD8 positive cells in inoculated glands than sows not developing disease. No differences were shown between categories in non-inoculated glands. It is concluded that the density of MHC class II, CD4 and CD8 positive cells seems to be unaltered around parturition. However, there is a rapid increase in density of these cells following intramammary inoculation with E. coli. Also, the data suggest that there is a difference between sows developing and sows not developing clinical disease after inoculation with respect to the increase in density of MHC class II, CD4 and CD8 positive cells in the mammary gland.     

Sows intramammarily inoculated with Escherichia coli influence of time of infection, hormone concentrations and leucocyte numbers on development of disease

The aim of this study was to identify factors that influence the development of disease in sows inoculated with Escherichia coli in the mammary gland. Ten cross-bred primiparous sows were intramammarily inoculated with living E. coli bacteria at different time points before parturition: seven sows within 48 h before parturition and three sows approximately 96 h before parturition. Before and after inoculation, blood samples and mammary gland biopsy specimens were collected and clinical observations were made. All seven sows inoculated close to parturition developed a rectal temperature of >39.5 degrees C during the first 48 h post-partum and two of them also showed other signs of clinical disease. In the sows inoculated 4 days before parturition, the rectal temperature never exceeded 39.5 degrees C during the first 48 h post-partum and none of them showed any other sign of clinical discase. There was a tendency (P < 0.1) that histological signs of mastitis were more frequent in the sows inoculated close to parturition. There were no overall differences between the two groups of sows in plasma concentrations of cortisol, oestradiol-17beta and 15-ketodihydro-PGF2alpha before inoculation. Before inoculation, the number of neutrophils in the blood was overall higher (P < 0.05) in the group of sows that were inoculated close to parturition. In comparison, the number of lymphocytes before inoculation had a tendency (P < 0.1) to be lower in that group. The data suggest that the time of infection of the mammary gland relative to parturition and the number of circulating neutrophils at the time of infection influence the development of chinical coliform mastitis in the sow.     

Experimental intramammary infection of the dairy cow with Escherichia coli during the nonlactating period

The nonlactating mammary gland was experimentally inoculated with Escherichia coli. During the first half of the nonlactating period, 32% of 34 inoculated glands were temporarily infected. All intramammary infections were eradicated by the cow without therapy and no signs of mastitis were observed. During the 30 days before parturition occurred, 88% of 42 inoculated glands in the cows became infected. Twenty-three intramammary infections were eradicated by the cow and infection in 14 glands persisted after parturition occurred. Peracute toxic mastitis occurred in those cows with infected glands.     

Sows Intramammarily Inoculated with Escherichia coli: Influence of Time of Infection,Hormone Concentrations and Leucocyte Numbers on Development of Disease

The aim of this study was to identify factors that influence the development of disease in sows inoculated with Escherichia coli in the mammary gland. Ten cross‐bred primiparous sows were intramammarily inoculated with living E. coli bacteria at different time points before parturition: seven sows within 48 h before parturition and three sows approximately 96 h before parturition. Before and after inoculation, blood samples and mammary gland biopsy specimens were collected and clinical observations were made. All seven sows inoculated close to parturition developed a rectal temperature of >39.5°C during the first 48 h post‐partum and two of them also showed other signs of clinical disease. In the sows inoculated 4 days before parturition, the rectal temperature never exceeded 39.5°C during the first 48 h post‐partum and none of them showed any other sign of clinical disease. There was a tendency (P < 0.1) that histological signs of mastitis were more frequent in the sows inoculated close to parturition. There were no overall differences between the two groups of sows in plasma concentrations of cortisol, oestradiol‐17β and 15‐ketodihydro‐PGF_2α before inoculation. Before inoculation, the number of neutrophils in the blood was overall higher (P < 0.05) in the group of sows that were inoculated close to parturition. In comparison, the number of lymphocytes before inoculation had a tendency (P < 0.1) to be lower in that group. The data suggest that the time of infection of the mammary gland relative to parturition and the number of circulating neutrophils at the time of infection influence the development of clinical coliform mastitis in the sow.     

Escherichia coli, but not Staphylococcus aureus triggers an early increased expression of factors contributing to the innate immune defense in the udder of the cow

The outcome of an udder infection is influenced by the pathogen species. We established a strictly defined infection model to better analyze the unknown molecular causes for these pathogen-specific effects, using Escherichia coli and Staphylococcus aureus strains previously asseverated from field cases of mastitis. Inoculation of quarters with 500 CFU of E. coli (n = 4) was performed 6 h, 12 h, and 24 h before culling. All animals showed signs of acute clinical mastitis 12 h after challenge: increased somatic cell count (SCC), decreased milk yield, leukopenia, fever, and udder swelling. Animals inoculated with 10 000 CFU of S. aureus for 24 h (n = 4) showed no or only modest clinical signs of mastitis. However, S. aureus caused clinical signs in animals, inoculated for 72 h-84 h. Real-time PCR proved that E. coli inoculation strongly and significantly upregulated the expression of beta-defensins, TLR2 and TLR4 in the pathogen inoculated udder quarters as well as in mammary lymph nodes. TLR3 and TLR6 were not significantly regulated by the infections. Immuno-histochemistry identified mammary epithelial cells as sites for the upregulated TLR2 and beta-defensin expression. S. aureus, in contrast, did not significantly regulate the expression of any of these genes during the first 24 h after pathogen inoculation. Only 84 h after inoculation, the expression of beta-defensins, but not of TLRs was significantly (> 20 fold) upregulated in five out of six pathogen inoculated quarters. Using the established mastitis model, the data clearly demonstrate a pathogen-dependent difference in the time kinetics of induced pathogen receptors and defense molecules.     

Protective effect of CpG-DNA against mastitis induced by Escherichia coli infection in a rat model

A mastitis model in rats, induced by Escherichia coli infection, was established and the protective effect of Cytosine-phosphate-Guanosine (CpG)-DNA was determined. An E. coli suspension containing either 2 x 10(3) colony forming units (CFU)mL(-1)(EL group), 2 x 10(5)CFU mL(-1) (EH group), or (as controls) 100 microL phosphate buffer saline (CON group), was inoculated into the mammary glands 72 h after parturition. The rats were euthanased 24 h post-infection. The histopathological changes in mammary tissue in the EL group were mild, whereas the structural changes in the EH group were severe and polymorphonuclear leukocytes (PMNs) had accumulated in the mammary alveoli. Interleukin (IL)-6 and tumour necrosis factor (TNF)-alpha and N-acetyl-beta-d-glucosaminidase (NAGase) were significantly increased in the mammary tissue from the EH group but not significantly changed in the EL group. On the basis of these findings, the potential protective effect of CpG-DNA on mammary glands was tested using a 2 x 10(5)CFU mL(-1) suspension. An intramuscular injection of either CpG-DNA (200 microg) or PBS (100 microL) was given immediately after parturition. At 72 h post-partum, 2 x 10(5)CFU mL(-1)E. coli (100 microL) were inoculated into the mammary glands of all rats. At pre-infection (0 h), and 8, 16, 24, 48 and 72 h after inoculation six rats were euthanased. CpG-DNA induced more rapid migration of PMNs from the blood to mammary tissue at the initial stage of infection, stimulated the secretion of IL-6 and TNF-alpha at different time points, reduced viable E. coli in mammary tissues and decreased the activity of NAGase. CpG-DNA also promoted the expression of its specific receptor TLR-9 mRNA in mammary tissue. The study showed that CpG-DNA protected against E. coli mastitis in this rat model.     

Sows intramammarily inoculated with Escherichia coli at parturition: I. Functional capacity of granulocytes in sows affected or non-affected by clinical mastitis

The objective of this study was to investigate if occurrence of clinical disease was related to granulocyte traits in sows. Functional capacity of granulocytes and plasma steroid hormone concentrations were assessed before inoculation with Escherichia coli in the mammary glands in sows at parturition. Blood samples were taken for 3 days approximately 1 week before parturition, and granulocyte migration, phagocytic capacity and expression of CD 18 adhesion molecules were determined. Inoculation was done within 36 h before partus. Thereafter, daily thorough clinical examinations were performed including udder health, habitus, appetite and rectal temperature, to assess the severity of disease. Based on the clinical findings four sows were classified as affected and eight as non-affected by clinical mastitis within 48 h after parturition.No difference (p>0.10) in pre-inoculation chemotaxis, phagocytosis or CD 18 expression was found between granulocytes from the sows resisting and developing clinical mastitis, respectively. However, there was an effect by the individual sow (p=0.001) on the numbers of granulocytes and white blood cells, and on plasma concentrations of estradiol-17beta and progesterone. In conclusion, these data does not suggest that impaired chemotaxis or phagocytosis by blood granulocytes contribute to the development of clinical coliform mastitis in the periparturient sow.     

The effect of experimental infectious mastitis on leukocyte subpopulations and cytokine production in non-lactating ewes.

The interactions between leukocytes and cytokines during the acute response to intramammary infections in the dry mammary gland of sheep were studied. Dry ewes were experimentally infected in one udder half with either Staphylococcus aureus or Escherichia coli, or infused with saline as control. Udder secretion samples, blood samples and udder tissue samples were collected before and 4, 8 and 24 h after infections/infusions. Total and differential leukocyte counts were calculated in both blood and mammary secretions, and flow cytometry was used to detect the presence of CD4+, CD8+, WC1+, IL-2R+, CD18+ or L-selectin + lymphocytes, CD18+ or L-selectin + neutrophils, and CD14+ leukocytes. Moreover, the concentrations of interleukin-1 beta (IL-1 beta), interleukin-8 (IL-8) and granulocyte-macrophage colony stimulating factor (GM-CSF) in udder secretions were measured using ELISA, and RT-PCR was used to detect the presence of corresponding cytokine mRNA in udder tissue biopsies. The results suggest an association between the concentrations of IL-1 beta, IL-8 and the intensity of neutrophil infiltration of the infected gland. Immunologically relevant changes in proportions of lymphocyte subpopulations might also occur in the acute phase of the inflammatory reaction of the udder. Greater cellular and cytokine responses to E. coli infection may have contributed to the milder clinical picture and more rapid resolution of infection than that seen for S. aureus. Enhancing the production of pro-inflammatory cytokines may improve defence against bacterial mastitis.     

Proinflammatory cytokines and CD14 expression in mammary tissue of cows following intramammary inoculation of Panax ginseng at drying off

The lack of efficacy of conventional strategies for the maintenance of healthy udders in domestic cattle has prompted studies on the use of immunomodulators or biological response modifiers (BRM) for this purpose. These compounds are agents that modify the host's response to pathogens leading to beneficial effects on disease outcome. The objective of this study was to evaluate the effects of a single intramammary infusion of Panax ginseng (GS) extract on the amount of pro-inflammatory cytokines and the number of monocytes/macrophages present in bovine mammary tissues at drying off. Eight mammary quarters from six nonpregnant cows in late lactation were infused with 10 mL of GS (3mg/mL), six quarters were treated with 10 mL of placebo (vehicle alone) and six quarters were maintained as uninoculated controls. The analyses of tumor necrosis factor-alpha (TNF-α) by immunohistochemistry revealed that the production of this proinflammatory cytokine significantly increased (P<0.05) in the inoculated mammary glands of cows following BRM inoculation, whereas the interleukin-1 alpha (IL-1α) and IL-6 staining area was not affected by BRM treatment. The number of monocytes/macrophages detected with CD14 antibody was significantly higher (P<0.05) in BRM-treated quarters than in placebo and uninoculated control quarters. These results indicated an immunomodulator potential of the BRM used. The beneficial effect of the extract could be used as alternative therapy in the control of mastitis at drying off, either alone or in conjunction with dry cow antibiotic therapy.     

Interleukin-8 expression by mammary gland endothelial and epithelial cells following experimental mastitis infection with E. coli

Epithelial and endothelial cells play a pivotal role in initiating and controlling the movement of leukocytes into tissues during inflammation through the production of cytokines and chemokines such as interleukin-8 (IL-8). In situ hybridization with an IL-8 riboprobe was used to determine IL-8 mRNA expression by mammary gland epithelial and endothelial cells in cows with experimental Escherichia coli mastitis. Epithelial cells of the gland, especially surrounding the alveoli, had increased IL-8 mRNA levels at all time points at which tissue samples were collected (8, 12, and 24h) after E. coli challenge. Levels of IL-8 expression in the epithelial cells decreased at 24h post-infection. IL-8 expression by mammary gland endothelial cells was low, but did increase slightly at 24h post-infection. Both epithelial and endothelial cells of the mammary gland can contribute to the production of IL-8 that is typically seen in coliform mastitis.     

脂多糖诱导哺乳期SD大鼠乳腺炎模型的建立

本试验旨在建立脂多糖(LPS)诱导哺乳期SD大鼠乳腺炎病理模型,为后续治疗乳腺炎的深入研究奠定基础。SD大鼠30只,于产后72h随机分为对照组(5只)和试验组(25只),试验组随机分为5组,各试验组大鼠经第4对乳头灌注LPS,剂量分别为10、20、30、40、50μg/侧,对照组灌注等量PBS。灌注后24h观察记录各大鼠临床症状、乳腺组织病理学变化。结果发现,在各试验组大鼠第4对乳腺均可观察到不同程度的炎症反应,且与LPS剂量呈正比例关系。结果表明,成功建立了LPS诱导哺乳期SD大鼠乳腺炎模型。     

Concentrations and specific antibodies to staphylococcal enterotoxin-C and toxic shock syndrome toxin-1 in bovine mammary gland secretions,and inflammatory response to the intramammary inoculation of these toxins

To investigate the pathological role of staphylococcal enterotoxins (SEs) and toxic shock syndrome toxin-1 (TSST-1) in bovine mastitis, the production of SEs and TSST-1 was investigated in staphylococci isolated from 120 mammary gland secretions (MGS, 51 from no clinical sign-mammary glands and 69 from staphylococcal mastitic-mammary glands) collected from dairy farms where staphylococcal mastitis frequently occurred in Miyagi and Yamagata prefectures from 1997 to 1998. Concentrations of these toxins and specific antibody titers in each MGS were also measured. Furthermore, SEC and TSST-1 were inoculated into lactating mammary glands and inflammatory responses were analyzed. A high percentage of staphylococci including Staphylococcus aureus and coagulase-negative staphylococci isolated from both no clinical sign- and mastitic-MGS produced both SEC and/or TSST-1. The concentration of SEC increased with the severity of the mastitis, and was significantly higher (P<0.05) in acute mastitic-than in no clinical signs-MGS. Titers of specific antibodies to TSST-1 in MGS were significantly higher (P<0.05) than those to SEC, regardless of whether or not the cows were lactating or mastitic. Specific antibodies purified from MGS neutralized each toxin in vitro. A significant increase (P < 0.05) in somatic cell counts was induced by the intramammary inoculation of SEC but not TSST-1. These findings indicated that SEC rather than TSST-1 plays an important role in the pathology of staphylococcal bovine mastitis. The inflammatory activity of TSST-1 was probably neutralized by specific antibodies in MGS.     

Susceptibility of sows to experimentally induced Escherichia coli mastitis

A study was conducted to compare susceptibility of sows from 2 herds to experimentally induced Escherichia coli mastitis. Four sows each from herds R and S were inoculated intramammarily at postpartum hour 8 with a strain of E coli shown previously to be capable of producing mastitis. After inoculation with E coli, sows from herd S had higher temperatures, lower WBC counts, and lower plasma protein:fibrinogen ratios than did sows from herd R. Inoculated sows from herd S lost 83% of newborn pigs due to starvation by 14 days after inoculation, whereas sows from herd R lost none. Control, noninoculated sows from both herds had normal temperatures, normal hematologic values, and minimal mortality of piglets. Levels of antibodies (complement fixing, enzyme-linked immunosorbent assay, and agglutinating) to E coli in preinoculation sera from the 2 populations of sows did not differ. Assay of lactoferrin by radial immunoassay revealed comparable concentrations in milk of sows from both herds during the first 24 hours after sows had delivered, but significantly higher values were detected in milk from sows of herd S at postpartum days 2 and 3. The basis for the marked difference in susceptibility to E coli-induced mastitis was not determined except that "susceptible" sows (herd S) were from a conventional herd and "resistant" sows (herd R) were from a specific-pathogen-free herd.     

Evaluation of the role of endotoxin and cortisol on modulation of CD18 adhesion receptors in cows with mastitis caused by Escherichia coli.

OBJECTIVE: To determine the effect of mastitis caused by Escherichia coli on expression of CD18 cell surface receptors and to evaluate the involvement and regulation of receptors by lipopolysaccharide (LPS) and cortisol. ANIMALS: 11 clinically normal lactating Holstein-Friesian cows. PROCEDURE: Binding of CD18 monoclonal antibodies to neutrophils was studied, using flow cytometry, before and after intramammary inoculation of E. coli organisms. Effect of LPS and cortisol on expression of adhesion receptors was investigated, using a whole-blood model. RESULTS: Expression of CD18 adhesion receptors on bovine neutrophils increased 35% by 12 hours after intramammary inoculation of E. coli. By 24 hours after inoculation, the number of receptors had returned to control values. High cortisol concentrations (100 nmol/L) were seen 12 to 18 hours after inoculation. Addition of LPS to blood induced a 30% increase in the number of CD18 receptors, and maximal number of receptors was expressed at an LPS concentration of 0.1 ng/ml. A decrease in the number of CD18 receptors was induced by incubation with cortisol or dexamethasone before challenge-exposure with LPS. CONCLUSIONS: An increase in the number of CD18 receptors on neutrophils is mediated by local production of LPS. Subsequent endogenous release of cortisol may prevent additional increases in the number of receptors. CLINICAL RELEVANCE: During acute mastitis caused by E. coli, there is an increase in the number of CD18 receptors on circulating neutrophils. Cortisol induces a decrease in the number of CD18 receptors, probably modulating the acute inflammatory response in mammary glands of lactating cows.     

Experimental staphylococcal mastitis in bitches: clinical, bacteriological, cytological, haematological and pathological features

The objectives of the work were to study the features of experimentally induced canine mastitis and to present hypotheses regarding the pathogenesis of the disease. The right caudal abdominal mammary gland of six bitches was inoculated on day 8 after whelping with Staphylococcus intermedius to induce mastitis; adjacent mammary glands were used as controls. Clinical examination, bacteriological and cytological (whiteside test, Giemsa) examination of mammary secretion, as well as haematological tests were performed from 5 days before until 34 days after challenge. Mastectomy was sequentially performed 1, 2, 4, 18, 26 and 34 days after challenge in each of the bitches, in order to carry out a pathological examination of mammary glands. All animals developed clinical mastitis: challenged glands became painful, hot, enlarged and oedematous; secretion was brownish, purulent, with flakes or clots, subsequently becoming yellowish and thick. Staphylococci were isolated from all inoculated glands (up to 22 days). WST was positive in 41/46 samples from inoculated glands and 66/138 samples from control glands; neutrophils predominated during the acute stage. Blood leukocyte counts increased, whilst platelet counts decreased. Gross pathological findings initially included congestion, purulent discharge and subcutaneous oedema; then abscesses, brownish areas and size decrease were seen. Salient histopathological features were initially neutrophilic infiltration, haemorrhages, destruction of mammary epithelial cells and alveoli, and then infiltration by lymphocytes, shrunken alveoli, loss of glandular architecture and fibrous tissue proliferation. We conclude that in bitches, intrammamary inoculation of Staphylococcus intermedius can induce clinical mastitis, followed by subclinical disease. The disorder is characterized by bacterial isolation and leukocyte influx in challenged glands, by leukocyte presence in adjacent mammary glands, by increased blood leukocyte counts and by destruction of mammary parenchyma.