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.     

Pathophysiology of enteropathogenic Escherichia coli during a host infection

Enteropathogenic Escherichia coli (EPEC) is a major cause of infantile diarrhea in developing countries. However, sporadic outbreaks caused by this microorganism in developed countries are frequently reported recently. As an important zoonotic pathogen, EPEC is being monitored annually in several countries. Hallmark of EPEC infection is formation of attaching and effacing (A/E) lesions on the small intestine. To establish A/E lesions during a gastrointestinal tract (GIT) infeciton, EPEC must thrive in diverse GIT environments. A variety of stress responses by EPEC have been reported. These responses play significant roles in helping E. coli pass through GIT environments and establishing E. coli infection. Stringent response is one of those responses. It is mediated by guanosine tetraphosphate. Interestingly, previous studies have demonstrated that stringent response is a universal virulence regulatory mechanism present in many bacterial pathogens including EPEC. However, biological signficance of a bacterial stringent response in both EPEC and its interaction with the host during a GIT infection is unclear. It needs to be elucidated to broaden our insight to EPEC pathogenesis. In this review, diverse responses, including stringent response, of EPEC during a GIT infection are discussed to provide a new insight into EPEC pathophysiology in the GIT.     

Acute phase response in dairy cows with experimentally induced Escherichia coli mastitis.

Six Finnish Ayrshire cows were challenged intramammarily with 1500 CFU of Escherichia coli (E. coli) into single udder quarters, and the challenge was repeated into contralateral quarters 3 weeks later. All cows received flunixine meglumine once, and 3 of them were also treated with enrofloxacin. At the 2nd challenge, treatments were changed vice versa. The development of mastitis was followed by monitoring of systemic and local clinical signs, and with serial milk and serum samples. Intramammary challenge with E. coli produced clinical mastitis in all cows, the severity of the disease varying greatly between the animals. No significant changes between the 2 treatment regimens or sequent challenges were found for any of the clinical parameters. The response of each cow followed the same pattern after both challenges; three of the cows became mildly and the other 3 either moderately or severely affected. Two severely affected cows had to be euthanized because of severe mastitis. Serum haptoglobin and amyloid-A concentrations peaked 2-3 days after bacterial challenge. Serum haptoglobin did not correlate with the severity of the disease. Serum amyloid-A rose gradually in the severely affected cows, and significant differences were found between severely versus moderately or mildly affected cows at day 4. Serum tumor necrosis factor alpha concentrations increased only in the severely affected cows. Serum cortisol response was prolonged in the severely diseased animals, and was significantly lower after the second challenge. Serum nitrite/nitrate concentration increased in the severely affected cows. This indicated excess nitric oxide production during acute E. coli mastitis. Strongly decreased milk production, and high bacterial growth in the infected quarters were best predictors for the outcome from acute E. coli mastitis.     

Controlling acute Escherichia coli mastitis during the periparturient period with recombinant bovine interferon gamma

The efficacy of recombinant bovine interferon (rBoIFN)-gamma against experimentally induced Escherichia coli mastitis during the periparturient period was investigated. Dairy cows intramammarily treated with rBoIFN-gamma 24 h before the E. coli challenge had fewer infected quarters, lower clinical scores, and infections of shorter duration when compared to placebo-treated animals. All rBoIFN-gamma treated cows survived the experimental E. coli challenge. However, placebo treated cows had a 42% mortality rate attributed to coliform mastitis within 3 days of the challenge. Results from this study suggest that intramammary infusion of rBoIFN-gamma can prevent the rapid, unrestricted growth of E. coli within the mammary gland and inhibit the subsequent development of an unlimited inflammatory response under experimental conditions. It is likely that controlling severe local inflammatory reactions may also decrease the pathological alterations to mammary parenchymal tissue that often accompanies acute coliform mastitis during the periparturient period. The potential for prophylactic treatment of perinatal dairy cows with rBoIFN-gamma to regulate the rate, severity, and duration of naturally occurring coliform mastitis during periods of heightened susceptibility is discussed.     

Apoptosis of bovine neutrophils during mastitis experimentally induced with Escherichia coli or endotoxin

OBJECTIVE: To determine whether apoptosis of neutrophils was accelerated during mastits experimentally induced by use of Escherichia coli or E coli endotoxin and whether differences were apparent in the response to E coli or endotoxin. ANIMALS: 11 healthy lactating Holstein cows. PROCEDURE: Blood samples were collected from cows at various intervals after intramammary inoculation with E coli or endotoxin. Percentage of apoptotic neutrophils detected after in vitro incubation for 3 hours was determined. Fluorescein isothiocyanate-labeled annexin-V in combination with propidium iodide was used to distinguish apoptosis and necrosis of neutrophils. Total and differential circulating leukocyte counts and rectal temperature were determined at the time of collection of blood samples. Milk yield and milk somatic cell counts were determined at the time of milking. RESULTS: Inoculation of endotoxin did not accelerate in vitro induction of neutrophil apoptosis. However, inoculation of E coli increased the percentage of apoptotic neutrophils. At 18 hours after inoculation, 20% of the neutrophils were apoptotic, compared with 5% before inoculation. Milk somatic cell count and rectal temperature increased, milk production and total leukocyte count decreased, and percentage of immature neutrophils increased after inoculation with E coli or endotoxin. However, kinetics of the responses were more rapid, more severe, and of shorter duration during endotoxin-induced mastitis. CONCLUSIONS AND CLINICAL RELEVANCE: In vitro induction of apoptosis of neutrophils was accelerated only during E coli-induced mastitis and not during endotoxin-induced mastitis. Endotoxin inoculation as a model for studying coliform mastitis in dairy cows should be viewed with caution.     

Identification of a bovine mastitis Escherichia coli subset

Eleven Escherichia coli isolates from clinical bovine mastitis cases (mastitic strains) and 11 from the cowshed environment (environmental strains) were compared, to determine if the former were a subset of the latter. The mastitic and environmental strains could not be distinguished according to O antigen and antibiotic sensitivity. All mastitic isolates showed significantly (P<0.0001) faster growth in milk and faster lactose fermentation than most (approximately 64%) environmental strains, but growth rates in nutrient broth did not differ. The rates of lactose fermentation and growth in milk were positively correlated. Adhesion and phagocytosis of mastitic strains by bovine PMN were significantly (P<0.0001) lower than those of environmental strains, and correlated negatively with growth in milk and lactose fermentation. The average percentages of killing by bovine leukocytes in the two sources were not statistically different. All mastitic strains were serum sensitive, whereas most ( approximately 72%) environmental ones were resistant. Finally, pulse-field gel electrophoresis revealed two main pulse type clusters, sharing a similarity coefficient of 79%. Cluster 1 comprised only environmental strains, whereas cluster 2 comprised mostly mastitic strains and only three environmental ones. Four mastitic strains shared a similarity coefficient of less than 74% with the other strains and were not included in the clusters. Our results suggest that clinical bovine mastitis E. coli isolates may form a subset of the general environmental E. coli population; they seem better able to multiply in the udder medium and to evade the host cellular innate immune response, and are genetically distinct from most environmental strains.     

Characterization and purification of the F17 adhesin on the surface of bovine enteropathogenic and septicemic Escherichia coli

The F17 antigen from bovine enterotoxigenic Escherichia coli strain (E coli 25KHO9), which adhered to calf intestinal villi, was isolated. An enterotoxin-negative derivative (25KHO9st) was used for further studies. Using an immunogold-labeling technique, the F17 antigen was characterized as a fimbrial protein. Pure fimbriae with a subunit molecular weight of 20,000 were obtained by homogenization and use of a sucrose gradient. The adhesion of E coli 25KHO9st was mediated by the F17 fimbriae, as both F17 antibodies and F17 protein blocked the adhesion of the strain 25KHO9st. The F17 fimbriae were serologically distinct from K88, K99, F41, and 987P fimbriae and did not agglutinate bovine, ovine, guinea pig, human, or chicken erythrocytes. Peptide fingerprint analysis revealed F17 and F(Y) adhesins to be homologous, if not identical.     

CpG-ODN enhances mammary gland defense during mastitis induced by Escherichia coli infection in goats

Seven healthy native goats in early lactation, weighing 30–40 kg, were used in this study. The right mammary gland of the seven does were infused with CpG-ODN at a dosage of 100 μg kg⁻¹ body weight on the day 5 postpartum (PP). The left glands were used as controls and infused with sterile phosphate-buffered saline (PBS). On day 8 PP, the same dosage of CpG-ODN or PBS was again infused. On day 9 PP, the mammary glands (both right and left) of the seven does were infused with 6 × 10⁶ colony-forming units (CFU) Escherichia coli and, at 0, 8, 16, 24, 48 and 72 h postinfection (PI), milk samples were collected from all glands. Goats were euthanized at 72 h PI and the mammary tissue harvested. Infusion with 6 × 10⁶ CFU ml⁻¹ E. coli induced acute mastitis. Histopathological evaluations showed that polymorphonuclear neutrophils (PMNs) were still present in alveoli at 72 h PI, but PMNs in the CpG-ODN-treated glands has disappeared. Bacteria counts in milk peaked at 16 h PI and CpG-ODN induced a significant decrease in viable bacteria from 16 h PI until the end of the experiment. This study showed that CpG-ODN promoted the expression of its specific receptor (TLR-9 mRNA) in mammary tissue, stimulated IL-6 production, reduced bacteria counts in milk, attenuated the impact of inflammation mediators on cells and significantly shortened the inflammation course. These results suggest that the CpG-ODN improved mammary gland defense and, thereby, had a beneficial effects against mastitis caused by E. coli infection in goats.     

Epigenetic mechanisms contribute to enhanced expression of immune response genes in the liver of cows after experimentally induced Escherichia coli mastitis

Endotoxins, such as lipopolysaccharide (LPS), are released during infection with Gram-negative bacteria, which can result in excessive activation of toll-like receptor (TLR) signalling. The aim of the present study was to investigate whether epigenetic mechanisms are involved in controlling the onset and progression of the systemic inflammatory response. Using chromatin accessibility by real-time (CHART) PCR to assess livers from cows with experimentally induced Escherichia coli mastitis, this study demonstrated that the chromatin at the site of the promoters of the genes encoding TLR2, TLR4, lipopolysaccharide binding protein (LBP) and haptoglobin (HP) was opened up 24 h after infection, accompanied by enhanced mRNA expression by these genes. Such modulation did not occur in the same samples for the αS1-casein promoter, which served as a negative control. Demethylation of the TLR4 promoter accompanied opening up of chromatin. These data suggest that modulation of epigenetic factors might offer a novel approach to treating adverse systemic reactions elicited in cows with E. coli mastitis.     

Hyperacute Escherichia coli mastitis of cattle in the immediate post-partum period

The pathology of hyperacute coliform mastitis was studied in 5 post-parturient cows. In all infected quarters infiltration of neutrophils was negligible. In all except one case there was severe damage to the ductular and secretory system, involving most areas of the gland. Bacteria were dense in infected alveoli, and there was evidence of substantial phagocytosis of bacteria by the secretory epithelium. The exception showed a large lesion in the middle of the gland from which the spread was ductular; other infections were consistent with spread via the teat canal. The organisms were largely confined to the ductular/secretary lumen and there was little invasion of the parenchyma. The severity of the disease was considered due to the absence of the inflammatory response seen in mid lactation.     

AFA and F17 adhesins produced by pathogenic Escherichia coli strains in domestic animals.

AFA and F17 are afimbrial and fimbrial adhesins, respectively, produced by pathogenic Escherichia coli strains in domestic animals. F17-related fimbriae are mainly detected on bovine and ovine E. coli associated with diarrhoea or septicaemia. The F17-G adhesin subunits recognize N-acetyl-D-glucosamine (GlcNAc) receptors present on bovine intestinal cells. Some F17 subtypes also bind to GlcNAc receptors present on human uroepithelial and intestinal Caco-2 cells or to the laminin contained in the basement of mammalian membranes. F17 is often associated with other virulence factors (aerobactin, serum resistance, CNF2 toxin, K99, CS31A or AFA adhesins) on pathogenic E. coli. A cluster of only four genes is required to synthesize functional F17-related fimbrial structures. The hypothesis of multifunctional F17 fimbrial subunits is supported by the fact that: i) the N-terminal part of the adhesin subunit participates in receptor recognition, whereas the C-terminal part is required for biogenesis of the fimbrial filament; and ii) the interaction between structural and adhesin subunits seems to be crucial for the initiation of monomer polymerization. Recently, determinants related to the afa gene clusters from human pathogenic E. coli associated with intestinal and extra-intestinal infections were identified in strains isolated from calves and piglets with diarrhoea and septicaemia. Two afa-related gene clusters, designated afa-7 and afa-8, that encode afimbrial adhesins were cloned and characterized from bovine pathogenic E. coli. These animal afa gene clusters were plasmid and chromosome borne and were expressed by strains that produced other virulence factors such as CNF toxins, F17, PAP and CS31A adhesins. A high frequency of afa-8 and a low prevalence of afa-7 among bovine E. coli isolates were suggested by preliminary epidemiological studies. As with the human afa gene clusters, the animal ones encode an adhesive structure composed of two proteins: AfaE which mediates adhesion to epithelial cells and AfaD which is an invasin.     

A study of relationships among F17 a producing enterotoxigenic and non-enterotoxigenic Escherichia coli strains isolated from diarrheic calves

We investigated the clonal relationships among 41 enterotoxigenic (ETEC) or non-enterotoxigenic (NETEC) Escherichia coli strains producing the F17 a fimbriae isolated from diarrheic calves in France or Belgium in the early 1980s. Twenty-three of the 26 ETEC strains were highly clonally related, most of them with a O101:K32:H9-serotype. The NETEC strains were also divided in clonal subgroups, most of them with O101:H-serotype. The F17 a positive ETEC strains are no longer isolated from diarrheic calves in these countries. It is postulated that the use of a vaccine including O101, K32 and H9 antigens in addition to K99 (F5) explains the strongly reduced isolation of the O101:K32:H9, K99 (F5) E. coli clone.     

Cow milk yield and composition before development of Escherichia coli mastitis

High milk yield, low milk fat and low milk protein were considered as possible predisposing factors to bovine Escherichia coli mastitis. Morning and afternoon milk yields were recorded in 46 Friesian cows later developing E coli mastitis and compared with 92 uninfected controls. Animals developing E coli mastitis gave a significantly higher milk yield than controls. The overall morning: afternoon ratio was (mean +/- se) 1.66 +/- 0.41, with no difference in ratio for the two groups. Further studies on 85 animals later developing E coli mastitis, and 192 controls, in four Friesian herds did not reveal differences in milk fat content (except as related to yield), milk protein or in the interrelationship of days of lactation, milk protein or in the interrelationship of days of lactation, milk fat and milk protein in the two groups. Again there was a correlation between high milk yield and a tendency to develop E coli mastitis but this may have been an age effect in both investigations. No correlation between milk yield and mastitis severity was detected. High yielders which succumbed to E coli mastitis in three herds were producing less milk than mastitis-free controls in the fourth herd which suggests that the correlation is not with yield per se.