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1.
Bovine mastitis caused by Escherichia coli has traditionally been viewed as a transient infection. However, E. coli can also cause clonal persistent intramammary infection (IMI) in dairy cows. In this study, we explored the possibility that E. coli strains associated with persistent IMI are better able to adhere to, invade, survive and replicate in cultured mammary epithelial cells (MAC-T) than transient strains, and examined their serotype, overall genotype, phylogenetic group, and the presence of known virulence genes. Both transient and persistent E. coli strains adhered to MAC-T cells, but persistent strains invaded MAC-T cells 2.6-63.5 times more than transient strains. Blocking the adhesin/invasin FimH with mannose diminished but did not eliminate adhesion and invasion of any strain. Cytoskeletal and protein kinase inhibitors cytochalasin D, colchicine, genistein and wortmannin dramatically reduced invasion of MAC-T cells by both strains. All of the persistent strains, but only one transient strain, were able to survive and replicate intracellularly in MAC-T cells over 48 h. Transient and persistent strains displayed heterogeneous serotypes and overall genotypes, but similar phylogeny (group A), and lacked virulence genes of invasive E. coli. We have found that E. coli strains associated with persistent IMI are better able to invade and replicate within cultured mammary epithelial cells than transient strains. The invasion process involves the host cytoskeleton and signaling cascades and is not FimH dependent. Our findings suggest that the invasion of mammary epithelial cells and intracellular survival play an important role in the pathogenesis of persistent E. coli mastitis.  相似文献   

2.
We previously showed that Staphylococcus aureus cells adhered mainly to an elongated cell type, present in cultures of bovine mammary gland cells. Moreover, we showed that this adhesion was mediated by binding to fibronectin. The same in vitro model was used here, to study adhesion of other important mastitis pathogens. Like the S. aureus strains, the Streptococcus dysgalactiae strains adhered mainly to elongated cells, which seemed to be mediated by fibronectin binding. In contrast, Streptococcus uberis strains adhered mainly to cubic cells. Since the cubic cells did not express fibronectin and S. uberis cells bound fibronectin less efficiently, the adhesion of S. uberis cells was independent of fibronectin binding. Streptococcus agalactiae strains adhered poorly to both cell types. The specificity and efficiency of adhesion of Escherichia coli strains was strongly strain dependent. None of the S. agalactiae and E. coli strains tested was able to bind fibronectin efficiently. The results suggest that the different mastitis pathogens have different target cell specificities and use different mechanisms to adhere to cells of the bovine mammary gland.  相似文献   

3.
Seven strains of Escherichia coli, originating from clinical cases of bovine mastitis, and one Salmonella typhimurium control strain were tested for their ability to adhere to, and invade, bovine mammary epithelial cells (MAC-T cells) in vitro. Four of the seven strains were isolated from cows with chronic intramammary infections with recurrent episodes of clinical mastitis and three strains were isolated from single cases of clinical mastitis. Both adhesion and invasion of all strains were dose and time dependent. The four E. coli strains isolated from recurrent cases of clinical mastitis invaded twice as frequently as and three times faster than the strains isolated from single cases of clinical mastitis. By contrast, there was no difference in the amount or speed of adhesion between the two types of strains of E. coli. Adhesion and invasion curves of E. coli resembled a two-step chain reaction, where invasion was the rate-limiting step. Although adhesion and invasion of E. coli has not been demonstrated in vivo yet, the results of the present study may contribute to an understanding of the pathogenesis of chronic intramammary infections caused by E. coli.  相似文献   

4.
Escherichia coli is a major pathogen in the aetiology of bovine mastitis. Although classically considered to be an environmental pathogen causing mainly transient infection, the incidence of persistent E. coli mastitis infections may be increasing, suggesting an adaptation of this pathogen to the bovine udder environment. Mastitis E. coli strains have been demonstrated to enter bovine mammary cells in vitro but little is known about the invasion mechanism or the intracellular fate of the bacteria. In order to further understand the pathogenesis of persistent E. coli bovine mastitis we investigated the intracellular trafficking of mastitis E. coli isolates in primary bovine mammary cells using confocal microscopy and fluorescent markers of endocytic compartments. Consistent with other studies, mastitis E. coli were found to invade primary bovine mammary cells in vitro. This process did not involve in the rearrangement of the actin cytoskeleton. Intracellular bacteria were observed within membrane-bound compartments that labelled with the early endosomal marker phosphatidylinositol 3-phosphate (PtdIns(3)P) and also within late endosome-like compartments labelled with the small GTPase Rab7, indicating an endocytic mechanism of bacterial internalization. Bacteria were not observed within acidified lysosomal compartments or autophagic vacuoles, suggesting that the internalized bacteria are not targeted for lysosomal degradation via either the classical endocytic pathway or the autophagic response. Our findings are consistent with an endosomal survival niche for the internalized bacteria, allowing them to evade host immune responses and establish an infection reservoir that could later re-emerge as a recurrent clinical mastitis episode.  相似文献   

5.
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.  相似文献   

6.
Isolation of Shiga-toxin (Stx) positive Escherichia coli O157:H7 from commercially grown pigs has been reported. Furthermore, experimental infection studies have demonstrated that Stx-positive E. coli O157:H7 can persist in 12-week-old experimentally orally inoculated conventional pigs for up to 2 months and that persistence was not dependent upon intimin. We have shown that the flagellum of Stx-negative E. coli O157:H7 does not have a role to play in pathogenesis in ruminant models whereas, in poultry, the flagellum of E. coli O157:H7 was important for long-term persistent infection. The contribution of the flagellum of Stx-negative E. coli O157 in the colonisation of pigs was investigated by adherence assays on a porcine (IPI-21) cell line, porcine in vitro organ culture (IVOC) and experimental oral inoculation of conventional 14-week-old pigs. E. coli O157:H7 NCTC12900nal(r) and isogenic aflagellate and intimin deficient mutants adhered equally well to IPI-21 cells. In porcine IVOC association assays, E. coli O157:H7 NCTC12900nal(r) was associated in significantly higher numbers to tissues from the caecum and the terminal rectum than other sites. The aflagellate and intimin deficient mutants significantly adhered in greater numbers to more IVOC gastrointestinal tissues than the parent. Groups of 14-week-old pigs were dosed orally with 10(10)CFU/10ml of either E. coli O157:H7 NCTC12900nal(r) or isogenic aflagellate and intimin deficient mutants and recovery of each test strain was similar. Histological analysis of pig tissues at post mortem examination revealed that E. coli O157 specifically stained bacteria were associated with the mucosa of the ascending and spiral colon. These data suggest that colonisation and persistence of Stx-negative E. coli O157:H7 in pigs, involves mechanisms that do not require the flagellum or intimin.  相似文献   

7.
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.  相似文献   

8.
Two experiments were done to determine the rate of intramammary infection (IMI) in dairy cows during the nonlactating period. In experiment 1, all glands were equally exposed to Klebsiella pneumoniae and Streptococcus agalactiae for the first 3 weeks after the start of the nonlactating period. Nearly all new IMI was caused by S agalactiae. In experiment 2, the susceptibility of nonlactating mammary glands to new IMI by S agalactiae after direct inoculation into the lactiferous sinus was determined. During the 1st month of the nonlactating period, 30% of inoculated glands became infected. During the last month of the nonlactating period, 90% of inoculated glands became infected.  相似文献   

9.
The caprine arthritis-encephalitis virus (CAEV) and Visna Maedi virus cause persistent infections with long latent periods and induce degenerative and chronic inflammatory lesions of the central nervous system, joints, lungs and udder. Monocyte/macrophage lineage is the main target cell for CAEV and Visna Maedi virus but we speculate that mammary epithelial cells may also be infected. Primary cultures of milk cells, mammary tissues of experimentally and naturally infected goats and ewes were used. Primary cultures of mammary tissue from ewes and goats were infected with the CAEV Cork strain. The lentiviral infection of the primary culture was demonstrated by a typical cytopathic effect in mammary epithelial cells and the presence of an infectious virus in coculture with permissive fibroblasts. To identify the epithelial cells in explants and demonstrate the antigenic expression of CAEV, primary cultures were immunostained with polyclonal anti-keratin and monoclonal anti-CAEV p30. Colocalisation studies under a UV fluorescence microscope and by epifluorescence microscopy showed the expression of specific viral antigens in mammary epithelial cells from the eight animals used. Infected mammary epithelial cells may act as a reservoir for the virus which may play an important role in the virus dissemination and in the pathogenesis of the mammary lentiviral disease.  相似文献   

10.
Using a simple in vitro test it was demonstrated that staphylococci, Streptococcus agalactiae, and micrococci, the species of bacteria which are commonly isolated from udder infections, adhered to mammary gland epithelial cells readily and in large numbers. Some strains of organisms which are associated with sporadic outbreaks or occur less commonly, like Str. dysgalactiae and Str. uberis, adhered moderately. Escherichia coli, Klebsiella spp., Corynebacterium pyogenes, C. bovis, Str. bovis, and Str. faecalis, species which are isolated occasionally, adhered poorly. From these studies, it appears that selective adherence of bacteria to the epithelial cells is a factor contributing to the ability of organisms to infect the mammary gland and may, therefore, be considered an important stage in the pathogenesis of bovine mastitis.  相似文献   

11.
One hundred and five strains of Escherichia coli that were isolated from calves with diarrhea in the state of São Paulo, Brazil, and were negative for enterotoxins and cytotoxins, were examined for the eae gene. Four (3.8%) strains were positive by polymerase chain reaction (PCR) and were shown to produce intimin by using Western blot with specific antiserum against the conserved N-terminal region of intimin. Subtyping of the intimins was done by PCR with specific primers and by Western blot with specific antisera against the C-terminal variable region of the protein. Three of these isolates (O?:H11, O26:H-, O123:H1) produced the beta subtype of intimin, and the 4th (0103:H2) produced intimin that was not typable. The 0103:H2 and the O26:H-isolates adhered to HEp-2 cells with diffuse adherence and localized-like adherence patterns, respectively. The other strains did not adhere to HEp-2 cells. To our knowledge, this is the first report of the occurrence of a subtype of intimin described for human enteropathogenic E. coli among bovine diarrheogenic E. coli. It is also the first report from Brazil demonstrating the presence of bovine E. coli harboring the eae gene.  相似文献   

12.
An assay for the adhesion of tritiated thymidine-labelled Staphylococcus aureus and Escherichia coli to bovine mammary ductular epithelial cell lines was developed. The relative adhesion of 15 strains of S. aureus to these cell lines was examined. Four strains did not adhere and the remaining 11 adhered at variable levels. Adhesion to different cell lines was generally similar. Adhesion to freshly collected bovine mammary epithelial cells was significantly greater than that to cells maintained in tissue culture. The system described was demonstrated to be a suitable model for studying adhesion of mastitis-causing organisms to bovine mammary epithelial cells.  相似文献   

13.
The role of fimbria in adherence of an avian pathogenic Escherichia coli (APEC) O78 strain 789 to chicken intestine was studied. Bacterial adhesion to tissue sections representing the regions within the chicken intestinal tract was determined by using immunohistochemical methods. E. coli 789 grown to express the type 1 fimbria adhered efficiently to the crop epithelium, to the lamina propria of intestinal villi, and to the apical surfaces of both the mature as well as the crypt-located enterocytes in intestinal villi, whereas no adhesion to mucus-producing goblet cells was detected. The adhesion was inhibited by mannoside and the role of type 1 fimbriae in the observed adhesion was confirmed with a recombinant strain expressing type 1 fimbriae genes cloned from E. coli and Salmonella enterica. E. coli 789 strain grown to favor AC/I fimbriae expression as well as the recombinant E. coli strain expressing the fac genes adhered to goblet cells but only poorly to the other epithelial sites. E. coli strain 789 as well as S. enterica serovar Typhimurium IR715 and S. enterica serovar Enteriditis TN2 strains were able to multiply in ileal mucus medium. The type 1 fimbria expressing bacteria adhered to the ileal mucus, whereas the AC/I fimbriated strains showed poor adherence to the mucus. The adhesion of E. coli 789 onto the crop epithelium and the follicle associated epithelium of the chicken ileum was efficiently inhibited by an adhesive strain ST1 of Lactobacillus crispatus isolated from chicken, whereas poor inhibition of E. coli adherence was observed with the weakly adhesive L. crispatus strain 134mi. The type 1 fimbriae may be important in colonization of the chicken intestine by APEC and Salmonella.  相似文献   

14.
Quarter samples (n = 6,328) of mammary secretions were collected from 160 cows during physiologic transitions of the udder to determine the frequency of isolation of mastitis-causing pathogens and the incidence of new intramammary infections (IMI) during the nonlactating period. None of the cows in the herd was infected with Streptococcus agalactiae, and the prevalence of Staphylococcus aureus was low. Cows were not treated with antibiotics at cessation of milking. A threefold increase in the percentage of quarters infected with major mastitis-causing pathogens developed from late lactation to early involution. Coliforms and streptococci other than Str agalactiae accounted for 94% of major pathogen infections. The number of quarters infected with coagulase-negative staphylococci increased slightly from late lactation to early involution, whereas the number of quarters infected with Corynebacterium bovis decreased markedly. Major pathogens caused 101 of 153 IMI at parturition and greater than 90% were caused by streptococci and coliforms. At parturition, 51 of 52 minor pathogen IMI were caused by coagulase-negative staphylococci. During early lactation, there was a marked decrease in quarters infected with major pathogens; however, the number of quarters with major pathogen IMI during early lactation was 2.3 times higher than the number of quarters infected before cessation of milking. The number of quarters with minor pathogen IMI during early lactation was the same as at parturition, but a marked decrease in quarters infected with coagulase-negative staphylococci and a marked increase in C bovis IMI developed from parturition to early lactation.  相似文献   

15.
From 1987 to 1991, almost 36,000 quarter samples of mammary secretion representing 1790 lactations of 510 dairy cows from a research herd were collected for bacteriological examination. The percentage of cows infected with Streptococcus uberis ranged from 12 to 16% of cows/year. S. uberis was isolated from 14.2% of lactations over the 5-year period. The prevalence of S. uberis intramammary infection (IMI) was significantly higher in cows with > or = 4 lactations than in cows with 3 or fewer lactations. Regardless of lactation number, the prevalence of S. uberis was highest before parturition, during early lactation and near drying off. The prevalence of S. uberis infected quarters ranged from 1.3 to 2.3% of quarters/year; the prevalence rate for the 5-year period was 2% of quarters. The quarter prevalence of S. uberis was lowest in cows with < or = 3 lactations, increased significantly with lactation number and was highest in cows with > or = 6 lactations. The percentage of quarters infected with S. uberis varied significantly by year. The majority (95%) of S. uberis IMI were subclinical. The ratio of subclinical IMI to clinical IMI was lowest during early lactation, and increased with days in milk, and with lactation age except for cows in their 5th and 6th lactations. Results of this epidemiological investigation suggest that opportunities exist where suitable control measures could be applied to reduce the impact of S. uberis infections in the dairy herd.  相似文献   

16.
OBJECTIVE: To determine whether lactoferrin (LF) or milk influenced adherence of Streptococcus uberis to bovine mammary epithelial cells. SAMPLE POPULATION: Three strains of S uberis from cows with mastitis, pooled milk samples from 3 clinically healthy Jersey cows early in the lactation period, and bovine mammary epithelial cells from a clonal cell line. PROCEDURES: Adherence of S uberis to bovine mammary epithelial cells in the presence of various concentrations of LF or milk and after pretreatment of bacteria with LF or milk was tested. Bacteria were cultured with mammary epithelial cell monolayers for 1 hour. The culture supernatant was removed, and the epithelial cells were lysed. Adherence index was calculated as number of colony-forming units (CFU) in the cell lysate divided by number of CFU in the supernatant times 10,000. RESULTS: All 3 strains of S uberis were found to bind to purified LF and LF in milk. Addition of LF to the culture medium enhanced adherence of all 3 strains to mammary epithelial cells, whereas addition of milk enhanced adherence of 2 strains and decreased adherence of the third. Pretreatment of bacteria with LF or milk increased adherence of 1 of the strains but decreased adherence of the other 2. Increased adherence was antagonized by rabbit antibovine LF antibody. CONCLUSIONS: Results suggest that LF may function as a bridging molecule between S uberis and bovine mammary epithelial cells, facilitating adherence of the bacteria to the cells.  相似文献   

17.
It is difficult to use tissue culture assays to investigate adherence and other properties of Edwardsiella tarda because the organism is invasive and produces a potent hemolysin. We therefore relied on polymerase chain reaction (PCR) to determine the occurrence of genes for enterotoxins (LT-I, EAST-1), Shiga toxin (Stx-1, Stx-2), cytotoxic necrotizing factors (CNF-1, CNF-2), aerobactin, invasion plasmid of enteroinvasive Escherichia coli, EPEC adherence factor (EAF), intimin (Eae), enterohemolysin (EntHly) and hemolysin (Hly) in 53 isolates of E. tarda from humans and fish from several countries. All isolates were negative for all genes investigated by PCR. Adhesion to and invasion of HeLa cells were determined by using the unusually short incubation time of 1h or 30 min. All isolates adhered and invaded in these tests. Finally, a random amplified polymorphic DNA (RAPD) test distinguished, with a few exceptions, isolates of human and fish origin.  相似文献   

18.
Adherence of Moraxella bovis to cell cultures of bovine origin   总被引：5，自引：0，他引：5  
The adherence of five strains of Moraxella bovis to cell cultures was investigated. M bovis adhered to cultures of bovine corneal epithelial and Madin-Darby bovine kidney cells but not to cell types of non-bovine origin. Both piliated and unpiliated strains adhered but piliated strains adhered to a greater extent than unpiliated strains. Antiserum against pili of one strain inhibited adherence of piliated strains but caused only slight inhibition of adherence to the unpiliated strains. Treatment of bacteria with magnesium chloride caused detachment of pili from the bacterial cell and markedly inhibited adherence of piliated strains but caused only slight inhibition of adherence by the unpiliated strains. The results suggested that adhesion of piliated strains to cell cultures was mediated via pili but that adhesins other than pili may be involved in the attachment of unpiliated strains of M bovis to cells.  相似文献   

19.
From 1987 to 1991, almost 36 000 quarter samples of mammary secretion representing 1790 lactations of 510 dairy cows from a research herd were collected for bacteriological examination. The percentage of cows infected with Streptococcus uberis ranged from 12 to 16 % of cows/year. S. uberis was isolated from 14.2 % of lactations over the 5-year period. The prevalence of S. uberis intramammary infection (IMI) was significantly higher in cows with ≥4 lactations than in cows with 3 or fewer lactations. Regardless of lactation number, the prevalence of S. uberis was highest before parturition, during early lactation and near drying off. The prevalence of S. uberis infected quarters ranged from 1.3 to 2.3 % of quarters/year; the prevalence rate for the 5-year period was 2 % of quarters. The quarter prevalence of S. uberis was lowest in cows with ≤3 lactations, increased significantly with lactation number and was highest in cows with ≥6 lactations. The percentage of quarters infected with S. uberis varied significantly by year. The majority (95 %) of S. uberis IMI were subclinical. The ratio of subclinical IMI to clinical IMI was lowest during early lactation, and increased with days in milk, and with lactation age except for cows in their 5th and 6th lactations. Results of this epidemiological investigation suggest that opportunities exist where suitable control measures could be applied to reduce the impact of S. uberis infections in the dairy herd.  相似文献   

20.
In this paper, we analyzed a very large field data set on intramammary infections (IMI) and the associated somatic cell count (SCC) in dairy cows. The objective of the study was to analyze the impact of coagulase-negative staphylococci (CNS) IMI on cow SCC, both mean and variability, and on the potential of these infections to have a major impact on the bulk milk SCC (BMSCC). Data and milk samples for bacterial culture were collected by Quality Milk Production Services (QMPS) between 1992 and March of 2007. The QMPS program services dairy farms in New York State and other states in the Northeastern USA and operates in conjunction with Cornell University. Only records from cows where SCC and milk production data were available, and where only one organism was isolated from bacterial cultures of milk samples (or where culture was negative) were used for this analysis. A total of 352,614 records from 4200 whole herd mastitis screening sampling qualified for this study. Within herds an average of 15% (S.D. 12%) of cows sampled were infected with CNS, ranging between 0 and 100%. Average within herd prevalence of cows with a CNS IMI and an SCC over 200,000 cells/ml was 2% (S.D. 4%) with a minimum of 0% and a maximum of 50%. Results of linear mixed models showed three distinct populations of IMI statuses: negative cultures with the lowest SCC; CNS and Corynebacterium bovis with a moderate increase in SCC, and Streptococcus agalactiae, Streptococcus spp. and Staphylococcus aureus showing an important increase in SCC. Surprisingly, milk production was slightly but significantly higher in CNS infected cows compared to culture-negative cows, whereas it was strongly reduced in cows with a major pathogen IMI. The percentage contribution of CNS infections to the BMSCC was 17.9% in herds with a BMSCC less than 200,000 cells/ml. This value decreased to 11.9 and 7.9% in herds with bulk milk SCC between 200,000 and 400,000 and over 400,000 cells/ml, respectively. We concluded that very few herds with milk quality problems would have an important increase in BMSCC that could be mostly attributed to CNS infections. On the other hand, in herds with low BMSCC, CNS infections may be an important contributor to the total number of somatic cells in the bulk milk.  相似文献   

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