Cofilin-1, peroxiredoxin-1, and galectin-3: Major proteins released by macrophages infected with Corynebacterium pseudotuberculosis.

Corynebacterium pseudotuberculosis, a broad host-spectrum zoonotic pathogen, causes caseous lymphadenitis (CLA) in small ruminants and is responsible for considerable economic losses in the livestock industry worldwide. Macrophages play a pivotal role in the immunopathogenesis of CLA. However, the immunoregulatory mechanisms of macrophages against C. pseudotuberculosis remains poorly understood. In the present study, for the first time, the partial exoproteome of murine peritoneal macrophages infected with C. pseudotuberculosis was profiled and the differential expression of the identified proteins was analyzed. In macrophages, infection with C. pseudotuberculosis, rather than with heat-killed bacteria, induced release of diverse proteins. Three unconventional proteins: cofilin-1, peroxiredoxin-1, and galectin-3 were significantly expressed and released by infected macrophages into the culture supernatant. These proteins are involved in the host inflammatory response and may be responsible for the excessive inflammation of CLA. In C. pseudotuberculosis-infected macrophages, the release of cofilin-1 and peroxiredoxin-1 was predominant at later stages of infection, while the release of galectin-3 was independent of time. Taken together, the present work contributes to our understanding of the functional role of macrophage response to C. pseudotuberculosis infection.

Clinico-pathological responses and PCR detection of Corynebacterium pseudotuberculosis and its immunogenic mycolic acid extract in the vital organs of goats.

Caseous lymphadenitis is an infectious disease of almost all animals, particularly small ruminants that are caused by Corynebacterium pseudotuberculosis. The organism causes the formation of suppurative abscesses in superficial and visceral lymph nodes and in visceral organs. This current study was designed to elucidate the clinicopathological responses and PCR detection of the aetiological agent in the vital organs of goats challenged with C. pseudotuberculosis and its immunogenic mycolic acid extract. A total of twelve clinically healthy crossbred Boer female goats were divided into three groups: A, B, and C (four goats per group). Group A was inoculated intradermally with 2 ml of sterile phosphate buffered saline (PBS) pH 7 as a control group. Group B was inoculated intradermally with 2 ml of mycolic acid extract (1 g/ml), while group C was inoculated intradermally with 2 ml of 109 colony-forming unit (cfu) of live C. pseudotuberculosis. The experimental animals were observed for clinical responses for 90 days post-inoculation and the clinical signs were scored according to the severity. The clinical signs observed in this study were temperature, heart rate, respiratory rate, rumen motility, enlargement of lymph nodes, and body condition score. The experimental animals were euthanised and tissue samples from different anatomical regions of the vital organs were collected in 10% buffered formalin, processed, sectioned, and stained with H&E. Results of both C. pseudotuberculosis and mycolic acid treated groups indicated a significant difference (p < 0.05) in body temperature. Group C showed a significant increase in temperature (p < 0.05) at week 1 (39.59 ±â€¯0.29 °C), week 2 (39.67 ±â€¯0.27 °C) and week 3 (40.22 ±â€¯0.15 °C). Whereas group B showed a significant increase in temperature (p < 0.05) only at week 1 (39.36 ±â€¯0.14 °C). Heart rate in group C showed a significant increase between week 1 (93.35 ±â€¯0.42 bpm) and week 11 (86.52 ±â€¯1.32 bpm), and the mean heart rate of group B showed a significant increase (p < 0.05) between week 1 (89.90 ±â€¯0.60 bpm) and week 9 (86.90 ±â€¯0.99 bpm). Group C showed a significant increase of respiratory rate (p < 0.05) at week 1 (36.85 ±â€¯0.14 bpm), week 2 (36.90 ±â€¯0.62), week 3 (30.80 ±â€¯1.97 bpm), and week 4 (34.85 ±â€¯1.19 bpm). The mean of the respiratory rate of group B only increased at week 1 (32.98 ±â€¯1.30 bpm) and week 2 (31.87 ±â€¯0.48 bpm). Both groups C & B showed significant decreases in rumen motility and body condition score as compared to the control. The histopathological changes were significant in group C, this was shown by mild to severe haemorrhage, congestion, degeneration and necrosis, oedema, infiltration with inflammatory cells mainly lymphocytes and macrophages, while group B was less affected and showed mild to moderate haemorrhage, congestion, degeneration and necrosis, infiltration of inflammatory cells and oedema as compared to the control group. This study concluded that C. pseudotuberculosis caused typical CLA disease with a short incubation period in the experiment. While the mycolic acid extracted from C. pseudotuberculosis caused mild clinical signs, no abscess formation, and negative PCR result. Moreover, evidence of mild to moderate histopathological changes in vital organs was also observed.

Responses of testosterone hormone concentration, semen quality, and its related pro-inflammatory cytokines in bucks following Corynebacterium pseudotuberculosis and its mycolic acid infection.

Corynebacterium pseudotuberculosis is the causative agent of caseous lymphadenitis, a debilitating chronic disease of sheep and goats. Little is known about the buck's reproductive pathophysiology with respect to inoculation with Corynebacterium pseudotuberculois and its immunogen mycolic acid extract. Therefore, this present study was designed to determine the concentration of testosterone hormone, pro-inflammatory cytokines, and semen quality of the experimental animals. A total of 12 bucks, divided into groups 1, 2, and 3 (Negative control group, Positive control group and Mycolic acid group respectively), were enrolled in this study. Following inoculation, all goats were observed for clinical responses and monitored for 60 days post-challenge and were then sacrificed. Blood samples were collected via the jugular once before inoculation and on a weekly basis post-challenge. Semen samples were collected 2 weeks post-challenge and prior to the sacrifice of the experimental animals. During the post inoculation period of 60 days, the concentration of testosterone hormone for group 2 was increased significantly (p < 0.05) in weeks 5, 6, and 9 but decreased in weeks 2 and 7 post inoculation. In group 3, the mean concentration of testosterone was increased significantly (p < 0.05) in weeks 5, 6, 7, and 9 post inoculation but decreased in week 2. The concentration of interleukin 6 (IL 6) in treated group 2 did not show any significant difference (p > 0.05) but increased significantly (p < 0.05) in week 2 post inoculation in group 3. For concentration of interleukin 1ß (IL1ß) in both treated groups 2 and 3 showed significant difference (p < 0.05) in weeks 2 and 3 post inoculation. The tumor necrosis factor-alpha (TNF-α) concentration in both treated groups 2 and 3 did not show any significant difference (p > 0.05) as compared to group 1. The concentration of interferon-γ (IFNγ) significantly increased (p < 0.05) for group 2 for weeks 2, 3, 4, and 5 where else for group 3 was not in significant difference (p > 0.05) compared to group 1. Both group 2 and group 3 showed a reduction in semen qualities as compared to group 1, but the severity was more intense in group 2 if compared to group 3. In conclusion, therefore, the present study concluded that the mycolic acid group revealed significant responses of testosterone hormone concentration, semen quality, and its related pro-inflammatory cytokines in bucks following infection but the severity lesser compared to Corynebacterium pseudotuberculosis group.

Factors impairing cell proliferation in the granulation tissue of pressure ulcers: Impact of bacterial burden.

The authors aimed to assess the factors that impair cell proliferation in the granulation tissue of pressure ulcers using immunohistochemistry for the cell proliferation marker Ki-67. This was a single center, cross-sectional study. The study included 86 patients with stage III or IV pressure ulcers. Two granulation tissue biopsy specimens were obtained from 86 patients. The specimens were used for histological examination, Ki-67 immunohistochemistry, and bacterial count assessment. The % of Ki-67-stained cells was considered as the Ki-67 index. Pearson's product-moment correlation coefficient (r) was used to assess the relationship between the Ki-67 index and other quantitative variables, including age, body mass index, bacterial count (Log10 CFU/g), serum albumin level, hemoglobin level, white blood cell count, and C-reactive protein level. The Mann-Whitney U test was used to compare the mean Ki-67 index according to gender, diabetes, smoking status, and wound culture. Univariate and multivariate linear regression analyses were used to assess the association between the Ki-67 index and other parameters. The Mann-Whitney U test revealed that the bacteria-positive group had a lower Ki-67 index (p = 0.045). Bacterial count demonstrated a significant negative correlation with the Ki-67 index (r = -0.325, p = 0.002). Multivariate linear regression analysis showed that bacterial count was a significant predictor of the Ki-67 index. The adjusted ß-coefficient was -1.34 (95% confidence interval, -2.01 to -0.66, p < 0.001). Among the isolated bacteria, Corynebacterium spp. and Staphylococcus aureus were significantly associated with a low Ki-67 index, but Pseudomonas aeruginosa was not. These results suggest a negative relationship between bacterial count and cell proliferation in pressure ulcer granulation tissue, as indicated by the Ki-67 index. Granulation tissue formation in pressure ulcers may be accelerated if high bacterial load is treated appropriately.

Encrusted cystitis causing postrenal failure.

Encrusted cystitis is characterized by chronic inflammation of the bladder with encrustation of the mucosa, induced by urea-splitting bacterial infection. However, encrusted cystitis in itself is not well known. We report a case of encrusted cystitis causing postrenal failure. An 81-year-old man with pneumonia complained of pollakisuria, micturition pain, and gross hematuria. Bladder calculi were found, and transurethral lithotripsy was performed. However, because his symptoms did not improve, he was referred to our hospital. His urine pH was 8.5, and urine culture grew Corynebacterium and Proteus. Computerized tomography and cystoscopy revealed bladder "encrustation," caused by bladder wall calcification, and bilateral hydronephrosis. Hence, he was diagnosed with postrenal failure resulting from encrusted cystitis. Immediate bilateral nephrostomy was constructed, with continuous bladder perfusion with an acid solution for acidification of his urine, followed by intravenous administration of ceftriaxone. After 2 weeks of treatment, the calcification disappeared and his bladder mucosa was normalized. The postrenal failure also improved and thus the nephrostomy tubes were removed. Encrusted cystitis is curable by prompt treatment with acidification of urine. Therefore, precise diagnosis and therapy are critical.

Differential arabinan capping of lipoarabinomannan modulates innate immune responses and impacts T helper cell differentiation.

Toll-like receptors (TLRs) recognize pathogens by interacting with pathogen-associated molecular patterns, such as the phosphatidylinositol-based lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM). Such structures are present in several pathogens, including Mycobacterium tuberculosis, being important for the initiation of immune responses. It is well established that the interaction of LM and LAM with TLR2 is a process dependent on the structure of the ligands. However, the implications of structural variations on TLR2 ligands for the development of T helper (Th) cell responses or in the context of in vivo responses are less studied. Herein, we used Corynebacterium glutamicum as a source of lipoglycan intermediates for host interaction studies. In this study, we have deleted a putative glycosyltransferase, NCgl2096, from C. glutamicum and found that it encodes for a novel α(1→2)arabinofuranosyltransferase, AftE. Biochemical analysis of the lipoglycans obtained in the presence (wild type) or absence of NCgl2096 showed that AftE is involved in the biosynthesis of singular arabinans of LAM. In its absence, the resulting molecule is a hypermannosylated (hLM) form of LAM. Both LAM and hLM were recognized by dendritic cells, mainly via TLR2, and triggered the production of several cytokines. hLM was a stronger stimulus for in vitro cytokine production and, as a result, a more potent inducer of Th17 responses. In vivo data confirmed hLM as a stronger inducer of cytokine responses and suggested the involvement of pattern recognition receptors other than TLR2 as sensors for lipoglycans.

Acute peritonitis due to Corynebacterium ulcerans in a patient receiving continuous ambulatory peritoneal dialysis: a case report and literature review.

A 55-year-old Japanese woman receiving continuous ambulatory peritoneal dialysis (CAPD) was admitted to our service with abdominal pain and cloudy peritoneal fluid. Laboratory data revealed a white blood cell count of 7.20 × 10(9 )cells/L, hemoglobin 9.8 g/dl, hematocrit 29.0%, platelet count 284 × 10(9 )cells/L, and C-reactive protein (CRP) 0.109 g/L. Peritoneal fluid white blood cell count of 2,000 cells/µl suggested acute peritonitis. An empiric trial of cefazolin and ceftazidime, subsequently switched to meropenem, vancomycin, minocycline, and amikacin, did not improve the patient's symptoms. The peritoneal fluid collected before initiation of antibiotic therapy grew Corynebacterium ulcerans. Ampicillin/sulbactam was started based on the culture and sensitivity data. On hospital day 8, the CAPD catheter was removed due to no clinical improvement and persistently increased levels of CRP to 0.0174 g/L. A 14-day course of ampicillin/sulbactam improved her clinical condition and laboratory data. Microbiological analysis revealed that C. ulcerans isolated from this patient did not produce diphtheria toxin. C. ulcerans was not isolated from her dog's oral and nasal cavities during a search for the route of her infection. We recommend that in patients with peritoneal dialysis, special attention should be paid to Corynebacterium peritonitis, especially due to C. ulcerans, which may produce diphtheria toxin, be resistant to multiple antibiotics, and frequently become recurrent.

The isolation of Corynebacterium coyleae from clinical samples: clinical and microbiological data.

Fourteen Corynebacterium coyleae isolates were recovered from 12 in-patients during a 5-years period. In six patients, the isolates were considered as clinically significant, three definite (sepsis), two probable (sepsis and soft tissue infection), and one possible (post-transfusional bacteremia). In the remaining 6 patients (all neonatal bacteremias), there was not enough data for considering the isolates as clinical significant. API Coryne identified all isolates as C. jeikeium, while Biolog GP2 correctly identified 7 out of the 14 isolates. Definitive identification was achieved in all isolates by the sequencing of a fragment of 724 to 1423 pb of 16S rDNA. Successive isolations from two patients presented identical random amplified polymorphic DNA (RAPD) profiles. All of the isolates were in-vitro-sensitive to beta-lactams, gentamicin, rifampin, tetracycline, vancomycin, linezolid, and resistant to clindamycin. Resistance to erythromycin occurred in 83.3% of isolates, all of them presenting phenotype cMLS and harboring the gene ermX.

Hydrophobicity and biofilm formation of lipophilic skin corynebacteria.

Lipophilic corynebacteria isolated as natural flora of human skin were examined. Among 119 assayed strains 94% presented a hydrophobic cell surface and 75.6% were able to form biofilms. These attributes, as well as aggregation in liquid media, were statistically connected with each other and promote the developing of biofilms on solid surfaces. This was characteristic of all the lipophilic Corynebacterium species found on human skin that were examined in this study. C. jeikeium and CDC group G2 strains dominated in this population, and they could be responsible for investigated features in the whole lipophilic skin bacterial population. These two groups are the most common coryneform bacteria isolated from nosocomial infections and these attributes most likely promote them to cause opportunistic infections.

Experimentally induced orchitis associated with Arcanobacterium pyogenes: clinical, ultrasonographic, seminological and pathological features.

The objectives of this study were to describe the features of experimentally induced orchitis associated with Arcanobacterium pyogenes and confirm the pathogenicity of the organism for the ovine testicle. One testicle of each of nine rams was inoculated with 1.3 +/- 10(4) colony-forming-units of an A. pyogenes isolate and regular clinical, ultrasonographic, bacteriological and seminological examinations were carried out up to 204 days after challenge. The rams were sequentially euthanatized 3, 6, 9, 18, 30, 50, 71, 113 and 204 days after challenge and a gross- and histopathological examination of their testicles was performed. All rams developed clinical orchitis and general signs. The initial ultrasonographic findings were changes of size and echogenicity of the genitalia, whilst in the long-standing phase they were wider appearance of the mediastinum testis, presence of hyperechogenic foci, changes of echogenicity of the genitalia and increased echogenicity of the scrotum and tunics. The following changes in semen evaluation parametres were recorded: the pH, the percentage of dead sperms, the percentage of abnormal sperms and the number of nonsperm round cells increased, whilst the mass motility, the individual motility and the sperm concentration decreased; the following sperm defects were observed: misshapen or piriform heads, sperms with coiled tails, sperms without tail and sperms with proximal cytoplasmic droplet; at the early stages neutrophils were the prevailing nonsperm round cell type, later the proportion of immature germ cells increased and in the long-standing phase there were enlogated spermatids and leucocytes; it is noteworthy that semen evaluation parametres were restored to normal at the late stages of the disease. A. pyogenes was consistently isolated from the semen samples after challenge, as well as from the dissected genitalia. The salient post-mortem findings were: initially, subcutaneous oedema, fluid into the vaginal cavity, congested and distended vessels, increased size of the genitalia and a hard dark area inside the testicles; subsequently, there were changes of size of the genitalia, thickening of scrotum and tunics and presence of fibrin on the testicular surface; in the long-standing phase of the disorder, there were induration of scrotum and tunics with adhesion between the tunics and discolouration of the surface of the genitalia. The prominent histopathological changes were observed in the inoculated testicles; milder changes were seen in the respective epididymides; interstitial oedema, diffuse neutrophilic infiltration and extravasation were observed in the early stages after challenge; lymphocytic infiltration with concurrent fibrosis, mineralization and inspissation of the tubular elements of the seminiferous tubules and presence of vacuolated Sertoli cells were seen later; finally, regeneration of the epithelium and presence of Sertoli cells and spermatogonia with various degrees of spermatogenic activity were evident. These findings, allied to the isolation of A. pyogenes from field cases of ovine orchitis, provide clear evidence that A. pyogenes is pathogenic for the ovine genitalia; however, the mechanisms of transition of the organism from commensal to pathogenic state are not clear. It is also noteworthy that some degree of fertility was restored in the late stages of the disorder. Ultrasonography appeared to be useful for the diagnosis of intra-scrotal abnormalities, especially during investigation of the long-standing stage of the disease, after clinical findings have subsided.

Lactoferrin concentrations in milk from normal and subclinical mastitic cows.

The concentrations of lactoferrin (Lf) in quarter milk from normal lactating cows and subclinical mastitic cows were measured to determine whether the Lf concentration in milk is influenced by the age of the cow, the stage of lactation, number of milk somatic cells and the presence of pathogens. Lf concentrations in 111 quarter milk samples from 28 normal lactating cows and 270 quarter milk samples from 198 subclinical mastitic cows were measured by means of a single radial immunodiffusion test. Lf concentrations (means +/- standard deviations; logarithmic form) in normal cows and subclinical mastitic cows were 2.23 +/- 0.39 and 2.70 +/- 0.39, respectively. The mean milk Lf concentration (log) in subclinical mastitic cows was significantly (p<0.01) higher than that in normal cows. The mean milk Lf concentration (log) in normal lactating cows aged 5 years was lower than those in normal lactating cows aged 2 years (p<0.01) and 3 years (p<0.05). The results showed that the milk Lf concentration (log) is associated with age of the dairy cow (one-way analysis of variance test, p<0.01). The mean milk Lf concentration (log) in the latter lactational period tended to be higher than those in the peak and middle periods. Milk Lf concentrations (log) tended to be proportional to the level of the somatic cell count (SCC) score. Mean milk Lf concentrations (log) in subclinical mastitic cows infected with Staphylococcus aureus and with other streptococci species were significantly (p<0.01) higher than those in cows infected with coagulase-negative staphylococci and with Corynebacterium bovis.

Infection of the skin caused by Corynebacterium ulcerans and mimicking classical cutaneous diphtheria.

Extrapharyngeal infections caused by Corynebacterium ulcerans have rarely been reported previously, and diphtheria toxin production has usually not been addressed. This case demonstrates that strains of C. ulcerans that produce diphtheria toxin can cause infections of the skin that completely mimic typical cutaneous diphtheria, thereby potentially providing a source of bacteria capable of causing life-threatening diseases in the patient's environment. Therefore, it is recommended to screen wound swabs for coryneform bacteria, identify all isolates, carefully assess possible toxin production, and send questionable strains to a specialist or a reference laboratory.

Inflammatory liver disease shortens atracurium-induced neuromuscular blockade in rats.

BACKGROUND: and objective Inflammatory liver dysfunction in rats leads to a prolonged vecuronium-induced neuromuscular blockade due to insufficient metabolism. A coexisting resistance against the drug partly counteracts this prolongation. The present study investigates the pharmacodynamics of atracurium whose metabolism does not depend on liver function. METHODS: Male Sprague-Dawley rats (n=14; 290 +/- 30 g) were randomly allocated to either a group in which liver inflammation was induced by intravenous injection of 60 mg kg(-1) heat-killed Corynebacterium parvum or to a control group. On day 5 after injection, liver function was assessed using the aminopyrine breath test. Under propofol anaesthesia, duration of action of atracurium (4.8 mg kg(-1)) was measured by evoked mechanomyography (stimulation of the sciatic nerve; contraction of the gastrocnemius muscle). Nitric oxide concentrations, as variables for the severity of the inflammation, were assessed by measurement of nitrite/nitrate plasma concentrations. RESULTS: In C. parvum-injected rats, nitrite/nitrate plasma concentrations were increased (972 +/- 597 vs. 25 +/- 7 micromol L(-1)), the aminopyrine turnover was depressed (1.7 +/- 0.4% vs. 3.5 +/- 0.5%), and the atracurium-induced neuromuscular blockade was shortened (372 +/- 128 s vs. 1081 +/- 234 s). CONCLUSIONS: A systemic inflammatory response syndrome with liver dysfunction results in decreased sensitivity to atracurium. Further investigations are needed regarding a possible up-regulation of acetylcholine receptors or an increased protein binding of atracurium during sepsis to clarify reasons behind this phenomenon.

Corynebacterium jeikeium native valve endocarditis following femoral access for coronary angiography.

We present a unique case of rapidly fatal native aortic-valve endocarditis due to Corynebacterium jeikeium, with inoculation as a complication of repeated femoral vascular access for coronary angiography.

[Diphtheria and infections caused by Corynebacterium diphtheriae in 1997]. / La diphtérie et les infections liées à Corynebacterium diphtheriae en 1997.

INTRODUCTION: Diphtheria is a reemerging disease. Two epidemics recently occurred in Algeria and Independent States Community, not so far from Europe. Imported cases were diagnosed in contiguous European countries. This review focuses on the data obtained from these epidemics, with particular emphasis on new clinical forms of Corynebacterium diphtheriae infections. CURRENTS KNOWLEDGE AND KEY POINTS: Sore throat with membranes is no longer the only clinical feature of diphtheria. However, patients' management is identical, with combination of antibiotics, injection of specific antisera, and immunization of patients' close contacts and relatives. French and American sero-epidemiological studies showed that antibody levels does not provide protection, particularly in the elderly. Adult populations would therefore be at risk every 10 years. Recent advances in molecular biology led to the development of gene amplification with polymerase chain reaction, that may be used for the detection of the toxin gene. They also promoted epidemiological surveys of circulating strains via ribotyping. Although this technic evidenced predominant strains in the various countries, genotypes encountered during an epidemics may differ. Besides diphtheria which has apparently been eradicated in France, systemic infections with non-toxigenic strains of C diphtheriae, such as endocarditis, septicemia and arthritis, are evenly diagnosed. FUTURE PROSPECTS AND PROJECTS: A French national reference center for C diphtheriae has been recently created. This center collects most of the strains isolated in France, clinical data and assesses the toxigenicity of bacteria, allowing strict epidemiological survey.

Sex differences in susceptibility of ICR mice to oral infection with Corynebacterium kutscheri.

Sex difference in susceptibility to oral infection with Corynebacterium (C.) kutscheri was experimentally studied in ICR mice. Immature (4-week-old) and adult (14-week-old) mice were inoculated with two infecting doses of C. kutscheri, and necropsied for bacteriological and serological survey 4 weeks after the bacterial infection. No macroscopic lesions at necropsy were demonstrated, except for one adult male given 10(9) bacteria. In immature mice, C. Kutscheri isolated from the oral cavity and cecum with FNC agar, were recovered in only 40.0% of female mice but in 90.0% of male mice given 10(6) bacteria (p < 0.05), and in only 55.6% of female mice but in 80.0% male mice given 10(8) bacteria. In adult mice given 10(9) bacteria, the organism were recovered in only 45.5% of female mice but in 90.9% of male mice (p < 0.05), furthermore, the mean number of organisms in the cecum of male mice harboring the organism was significantly higher than that in females (p < 0.01). Castration caused an increase in host resistance in adult male mice. These results indicated that ICR male mice were more susceptible than females, in terms of bacterial colonization in the cecum and the oral cavity, to oral infection with C. kutscheri.

Characteristics of dairy cows during episodes of bacteriologically negative clinical mastitis or mastitis caused by Corynebacterium spp.

OBJECTIVES: To use clinical and lactational characteristics to determine whether bacteriologically negative (BN) clinical mastitis episodes are more apt to be caused by gram-negative or gram-positive bacteria, and to investigate severity of clinical mastitis caused by Corynebacterium spp (COR). DESIGN: Case series. SAMPLE POPULATION: 300 clinical mastitis episodes affecting 123 dairy cows vaccinated against lipopolysaccharide core antigens. PROCEDURE: Cows were examined at onset of clinical mastitis, and 23 characteristics, including rectal temperature, heart rate, rumen contraction rate, degree of dehydration, udder and milk characteristics, lactation number, stage of lactation, and season of year, were recorded. Milk production and milk constituent concentrations before onset of mastitis were obtained from herd records. Values for cows with BN milk were compared with values for cows from which milk yielded gram-negative bacteria (GNB) or gram-positive cocci (GPC); logistic regression was used to predict which pathogen type was causing BN mastitis. Characteristics for cows from which milk yielded COR were compared with those of cows from which milk was BN or yielded GPC. RESULTS: BN clinical mastitis episodes differed significantly from episodes caused by GPC, and were similar to, but milder than, episodes caused by GNB. COR were isolated in a substantial proportion of mastitis episodes, but clinical signs were milder than when GPC were isolated. CLINICAL IMPLICATIONS: Most BN mastitis episodes in cows receiving lipopolysaccharide core antigen vaccines appear to be caused by low-grade infection with GNB, and treatment and management decisions should be made accordingly. The COR may be economically important clinical mastitis pathogens in some herds.