Host-pathogen gene expression profiles during infection of primary bovine mammary epithelial cells with Escherichia coli strains associated with acute or persistent bovine mastitis

Escherichia coli intramammary infection (IMI) is often acute with local and systemic clinical manifestations that clear within 7 days. However, if not diagnosed early and treated, E. coli IMI could result in generalized systemic reaction and death. Persistent E. coli IMI is characterized by mild clinical manifestations followed by acute episodes of clinical mastitis during lactation. Factors responsible for pathogenesis of E. coli IMI and variation in clinical manifestations are not known. There are studies indicating that the outcome of E. coli IMI is mainly determined by cow factors. However, recent research demonstrated that virulence attributes of E. coli strains have significant impact on the outcome of E. coli IMI. The aims of this study were; (a) to compare gene expression profiles of PBMEC cocultured with strains of E. coli associated with acute or persistent IMI and; (b) to identify genes of E. coli induced during bacterial interaction with PBMEC. Utilizing cDNA we analyzed gene expression patterns of PBMEC cocultured with strains of E. coli using non-treated PBMEC as negative control. We evaluated also expression patterns of virulence associated genes of E. coli after co-culture with PBMEC using qRT-PCR. Our results showed that infection by both strains induced increased expression of pro-inflammatory cytokines, chemokines and innate immune response and apoptosis related genes. Our qRT-PCR results showed significant up-regulation of ler, eae, flic and iutA genes mainly in the strains of E. coli associated with persistent IMI. The pathogenesis and clinical severity of E. coli IMI may be determined by combined effects of host-pathogen factors.     

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.     

奶牛乳房炎金黄色葡萄球菌小菌落突变株的表型鉴定及其遗传基础分析

金黄色葡萄球菌(Staphylococcus aureus)是导致奶牛乳房炎的主要病原菌,其在抗菌药物的作用下可形成小菌落突变株(small colony variants,SCVs)。为了探讨牛源性金黄色葡萄球菌在表型转变过程中的遗传基础,对临床分离的奶牛乳房炎金黄色葡萄球菌野生型及其SCV、人工诱导的SCV、临床SCV的表型回复株进行了相关特性及基因组序列的分析。结果发现:金黄色葡萄球菌的SCV和野生型差异显著,野生型菌株在庆大霉素及磺胺甲噁唑/甲氧苄啶(SXT)的诱导下绝大多数产生甲萘醌缺陷型SCV,仅有1株经SXT诱导产生的SCV为胸腺嘧啶核苷(胸苷)缺陷型。菌株基因与表型之间存在明显关联,与野生型基因组相比,临床SCV均显示喹啉/醌代谢相关基因menA的碱基缺失,可能导致蛋白表达提前终止。庆大霉素诱导的SCV在基因menB处出现碱基位点突变;SXT诱导的SCV在代谢相关基因MW1485和aroA处分别出现基因片段的缺失和插入。回复株可能通过在代谢相关基因menA二次突变来恢复功能。本研究首次利用全基因组测序技术分析牛源性金黄色葡萄球菌SCV表型改变的遗传基础,有助于对奶牛乳房炎金黄色葡萄球菌SCV产生更深入的理解。     

Curli expression by Escherichia coli strains isolated from bovine mastitis

The aim of the study was to determine the expression of mannose-sensitive and mannose-resistant adhesins by agglutination of cattle, sheep, goat, rabbit, horse, and chicken red blood cell assay, and curli fimbriae by Congo red binding assay among 341 E. coli strains isolated from 51 milk samples of clinically recognized bovine mastitis. Curli fimbriae expression within biofilms created on an inert surface was also investigated. To determine whether curli fimbriae are expressed both in conditions optimal for their production and in conditions resembling the host organism, the study was conducted in anaerobic atmosphere at 37 degrees C, and at room temperature in aerobic atmosphere. The results demonstrated that although the E. coli isolates examined were deprived of mannose-sensitive and mannose-resistant adhesins they were able to produce curli fimbriae in both aerobic and anaerobic conditions at room and higher temperature, indicating that these adhesins may be involved in the pathogenesis of bovine mastitis.     

Studies about the mechanism of internalization by mammary epithelial cells of Escherichia coli isolated from persistent bovine mastitis

The purpose of this study was to investigate the interaction between Escherichia coli and primary mammary epithelial cell cultures derived from cows with persistent intramammary infection (IMI). Two strains of E. coli, isolated from the milk of two different cows suffering from persistent E. coli IMI were tested for adhesion to and invasion of three primary mammary epithelial cell cultures derived from mammary biopsies of the two infected cows. Intracellular E. coli were detected during five days post infection in vitro. Both strains of E. coli adhered to and invaded monolayers of all three primary mammary epithelial cell cultures. One strain adhered less but invaded more than the other. Comparison with other mammary pathogens indicated that E. coli invaded the cells less efficiently than Staphylococcus aureus, about as efficiently as Streptococcus dysgalactiae and more efficiently than Streptococcus uberis. The mechanism of E. coli invasion was studied using the cytoskeleton disrupting agents colchicine and cytochalasin D. These compounds inhibited the invasion of E. coli. Invasion of E. coli could also be inhibited by the phosphokinase inhibitors genistein and staurosporin in a dose-dependent fashion. Phorbol-myristyl-acetate (PMA) had no effect on the invasion of E. coli. Histology of mammary tissue revealed chronic inflammatory changes in quarters that were persistently infected by E. coli. Intracellular bacteria were not detected in mammary tissue sections. Polymerase chain reaction (PCR) analysis suggested that the two strains of E. coli lacked genes encoding for bundle-forming pili (bfpA), intimin (eae) and translocated intimin receptor (tir), which are characteristic for enteropathogenic E. coli (EPEC).     

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.     

Virulence factors of Escherichia coli isolated from bovine clinical mastitis.

Escherichia coli isolates from bovine mastitis were examined for a selection of virulence factors. The strains originated from Finland and Israel, which have differences in the proportion of mastitis caused by E. coli, clinical pictures of coliform mastitis, environmental conditions and herd management. The genes of nine virulence factors were detected by polymerase chain reaction. Presence of K1 and K5 capsules was assessed by use of specific bacteriophages. Serum resistance was tested by a turbidimetric assay. Out of 160 Finnish isolates, 37% had traT, 14% cnf2, 8% cnf1, 11% aer, 9% f17, 8% sfa, 7% pap, 1% afa8D and 1% afa8E. Out of 113 Israeli isolates, 41% had traT, 4% aer, 3% cnf2, 1% cnf1, 1% sfa and 1% f17. Some of the genes were distributed among two major pathotype groups, with either f17 family or sfa, pap and cnf1 as major determinants. Genes for F17a, CS31A, Afa7D and Afa7E were not detected. Altogether 49% of Finnish and 42% of Israeli isolates had at least one virulence gene, but genes other than traT were present in only 24% of Finnish and 5% of Israeli isolates. Serum resistance was more common among Finnish (94/160) than Israeli isolates (19/113). K1 and K5 capsules were not detected.     

Phylogroup and lpfA influence epithelial invasion by mastitis associated Escherichia coli

Escherichia coli infection is one of the most common causes of bovine mastitis in well managed dairies. Although E. coli infections are usually transient, E. coli can also cause persistent intramammary infections. We sought to determine whether E. coli isolates recovered from either transient or persistent intramammary infections differed both genetically and in their ability to invade mammary epithelial cells. E. coli isolates from transient (EC(trans), n=16) and persistent (EC(pers), n=12) mastitis cases were compared for differences in overall genotype, virulence genes, serotype, phylogroup (A, B1, B2, D), and invasion of bovine mammary epithelial cells, MAC-T by microarray analysis, suppressive subtractive hybridization, PCR and gentamicin protection assays. EC(trans) and EC(pers) were diverse in overall genotype and serotype, and were predominantly of phylogroups A and B1. Both EC(trans) and EC(pers) contained genes encoding type II, IV and VI secretion systems, long polar fimbriae (lpfA) and iron acquisition, and lacked genes associated with virulence in diarrheagenic E. coli. EC(trans) had fewer virulence genes than EC(pers) (p<0.05), but no individual virulence genes were unique to either group. In phylogroup A, EC(pers) were more invasive than EC(trans) (p<0.05), but no difference was observed between them in phylogroup B1. Enhanced epithelial cell invasion was associated with lpfA (p<0.05). Our findings indicate that a genetically diverse group of E. coli is associated with transient and persistent mastitis. We did not identify a set of bacterial genes to account for phenotypic differences. However, we found that mastitis phenotype, phylogroup and presence of lpfA were associated with the ability to invade cultured bovine mammary epithelial cells.     

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.     

Pathogenesis of experimental bovine mastitis following a small inoculum of Escherichia coli

Mastitis was produced in four quarters of two lactating cows by the inoculation of 50 or 200 viable Escherichia coli. Changes were investigated by light microscopy and scanning electron microscopy. After 10 hours the changes were confined to the superficial layer of the epithelium of the teat and lactiferous sinuses; single cells or small groups of cells were damaged and were being extruded from the tissue. By 14 hours there was extensive necrosis and sloughing of the epithelial cells which did not extend beyond the basement membrane, and an intense inflammatory response associated with the epithelial damage. The somatic cells in the milk, mainly polymorphonuclear leucocytes, increased 40 to 250 times and strongly inhibited the survival of E coli. Epithelial damage appeared first in the ventral portions of the gland and then extended through the lactiferous sinus to the large ducts and secreting tissue. This pattern of damage together with the failure to observe bacteria attached to the epithelia was consistent with the production of diffusible toxins by the organisms.     

Role of prostaglandins in pathogenesis of bovine mastitis induced by Escherichia coli endotoxin

Four doses (5 to 100 micrograms, 1 dose/quarter) of Escherichia coli endotoxin were introduced into lactating mammary glands of 2 cows. There was no effect on milk prostaglandin (PG) E2 concentration, except that the concentration was increased from 200 pg/ml of milk to 1,060 pg/ml at post-treatment hour (PTH) 8 in cow 1 and from 75 to 420 pg/ml at PTH 4 in cow 2 after the highest dose 100 micrograms. Endotoxin caused a dose-dependent increase in milk PGF2 alpha concentrations in both cows. After the highest dose, PGF2 alpha was maximally increased from 200 to 3,500 pg/ml at PTH 4 in cow 1 and from 250 to 2,000 pg/ml in cow 2 at PTH 8. The instillation of 50 micrograms of endotoxin in all 8 quarters of 2 more lactating cows caused no significant (P greater than 0.05) changes in milk PGE2 and thromboxane B2 concentrations, whereas milk PGF2 alpha was significantly increased from the base-line value of 642 to 2,683, 1,189, and 2,281 pg/ml at PTH 4, 8, and 12, respectively. The 6-keto-PGF1 alpha was also significantly increased from the base-line value of 305 to 871, 631, and 600 pg/ml at the corresponding times, respectively. A marked increase in vascular permeability, as judged by high concentrations of serum albumin in the whey, was observed as early as PTH 4 and peaked at PTH 12 followed by a gradual decline, although it remained significantly increased over the control for 48 hours after treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Depression of polymorphonuclear leukocyte function associated with experimentally induced Escherichia coli mastitis in sows

In a study of susceptibilities of sows from 2 herds to experimentally induced Escherichia coli mastitis, a marked difference was seen. The "susceptible" sows were from a conventional herd and "resistant" sows were from a specific-pathogen-free herd. The purpose of the study was to determine whether deficient neutrophil function was associated with increased susceptibility to E coli-induced mastitis. Four in vitro procedures were used to evaluate polymorphonuclear leukocyte (PMN) function: (i) random migration under agarose, (ii) ingestion of 125I-iododeoxyuridine-labeled Staphylococcus aureus, (iii) quantitative nitroblue tetrazolium reduction, and (iv) iodination. After parturition and intramammary inoculation with E coli, sows from the susceptible herd were neutropenic and the neutrophils which were present in the peripheral blood had reduced function. Specifically, there were depressed random migration under agarose, S aureus ingestion, and iodination when compared with PMN function in resistant sows. These data indicate that susceptibility to E coli mastitis was associated with deficiencies in PMN numbers and function. Potential causes of the neutrophil dysfunction are discussed and include possible systemic hormonal aberrations or the presence of an inapparent viral or bacterial infection.     

Characterization of integrons-mediated antimicrobial resistance among Escherichia coli strains isolated from bovine mastitis

To assess the prevalence of antimicrobial resistance and class I integrons in Escherichia coli strains (n=58) isolated from bovine mastitis in Inner Mongolia, antimicrobial susceptibility and the presence of various types of integrons were characterized. Most isolates were susceptible to amikacin, colistin, ceftazidime, gentamicin and kanamycin, while those also exhibited high resistant incidence rates to ampicillin, amoxicillin, sulfadiazine and sulfamethoxydiazine. The integrase gene of integrons was amplified by PCR using degenerate primers. The integrons were confirmed by restriction fragment length polymorphism (RFLP) analysis of positive PCR products. Neither class II nor class III integron was detected, while 56.90% (n=33) of the isolates were positive for the presence of intI1 gene. Sequencing analysis of gene cassettes revealed that seven gene cassettes were found, which encoded resistance to trimethoprim (dfrA1 and dfrA17), aminoglycosides (aacA4, aadA1 and aadA5) and chloramphenicol (catB3), respectively. Of them, the gene cassette array dfrA17-aadA5 was found most prevalent (62.96%). The percentage of positive-integron among the isolates whose resistant profile was relatively broad (n> or =7) is 100.00%, while the one in narrow-profile isolates (n=2-6) is 30.56%. The correlation analysis revealed the incidence of integrons among the isolates were highly related to the resistant profile, indicating integrons play an important role in the dissemination and spread of the antimicrobial resistant strains.     

The efficacy of bovine lactoferrin in the treatment of cows with experimentally induced Escherichia coli mastitis

The effect of bovine lactoferrin (Lf) was studied in experimental Escherichia coli mastitis, using enrofloxacin as a comparator. Mastitis was induced in six clinically healthy primiparous dairy cows by infusing 1500 colony-forming units of E. coli into a single udder quarter. The challenge was repeated into a contralateral quarter of the same cows 3 weeks later. At the first challenge, three cows were treated with 1.5 g of bovine lactoferrin intramammarily three times (12, 20 and 36 h postchallenge, PC), and the other three cows received 5 mg/kg of enrofloxacin (Baytril) parenterally (12, 36 and 60 h PC). Flunixin meglumine (2.2 mg/kg) was administered to all cows twice at 24-h intervals. During the second challenge, the treatments for the two groups were reversed. Intramammary challenge with E. coli produced clinical mastitis in all cows, but the severity of the disease varied markedly. No statistically significant differences between treatment groups were observed in clinical signs such as rectal temperature, rumen motility and general attitude. Milk somatic cell count, daily milk yield and bacterial counts in cows treated with Lf and those receiving enrofloxacin also did not differ significantly. However, a trend for a more rapid elimination of bacteria was seen in the cows treated with enrofloxacin. Milk NAGase activity also decreased significantly faster in the group treated with enrofloxacin. The concentration of lipopolysaccharide in milk compared with the number of bacteria was significantly lower in Lf than in enrofloxacin-treated cows (20 h PC).     

Prevalence of verotoxin-producing Escherichia coli (VTEC) in bovine coli mastitis and their antibiotic resistance patterns.

Between December 1996 and October 1997, milk samples from a total of 145 cows with coli mastitis were screened for the presence of verotoxin-producing E. coli (VTEC). VTEC were found in four (2.8%) out of the 145 samples. The four isolated strains proved to be verotoxin (VT) 1-, VT2- or VT1- and VT2-positive. However, no strain contained all three virulence factors tested. Further strain characterization was carried out by serotyping as well as by resistance pattern analysis.