Comparative proteomic analysis of proteins expression changes in the mammary tissue of cows infected with Escherichia coli mastitis

Cows infected with Escherichia (E.) coli usually experience severe clinical symptoms, including damage to mammary tissues, reduced milk yield, and altered milk composition. In order to investigate the host response to E. coli infection and discover novel markers for mastitis treatment, mammary tissue samples were collected from healthy cows and bovines with naturally occurring severe E. coli mastitis. Changes of mammary tissue proteins were examined using two-dimensional gel electrophoresis and label-free proteomic approaches. A total of 95 differentially expressed proteins were identified. Of these, 56 proteins were categorized according to molecular function, cellular component, and biological processes. The most frequent biological processes influenced by the proteins were response to stress, transport, and establishment of localization. Furthermore, a network analysis of the proteins with altered expression in mammary tissues demonstrated that these factors are predominantly involved with binding and structural molecule activities. Vimentin and α-enolase were central "functional hubs" in the network. Based on results from the present study, disease-induced alterations of protein expression in mammary glands and potential markers for the effective treatment of E. coli mastitis were identified. These data have also helped elucidate defense mechanisms that protect the mammary glands and promote the pathogenesis of E. coli mastitis.     

Therapeutic effect of anti-TNF-α antibody and levofloxacin (LVFX) in a mouse model of enterohemorrhagic Escherichia coli O157 infection

The ability of an anti-TNF-α antibody to confer protection against enterohaemorrhagic Escherichia coli (EHEC) O157 was investigated in germfree IQI mice. The use of an antibiotic levofloxacin (LVFX) alone or with the antibody was also studied. Protection included an increase in survival rate. Treatment with the anti-TNF-α antibody inhibited the histological signs associated with EHEC infection but did not prevent the colonization of EHEC or production of Shiga toxin (Stx). No clinical signs were observed and EHEC was completely eliminated in the mouse model receiving both anti-TNF-α antibody and LVFX. Anti-TNF-α antibody suppressed inflammatory cytokine response in the mouse kidney and brain by EHEC infection.     

The influence of anti-inflammatory therapy on bacterial clearance following intramammary Escherichia coli challenge in goats

Coliform mastitis in dairy cattle frequently results in systemic disease with occasional deaths in association with endotoxic shock. Systemic anti-inflammatory therapy has been used to alter the course of endotoxic shock in severe cases. Use of anti-inflammatory therapy has been questioned on the basis that such treatment may compromise immune function and decrease clearance of bacteria from infected mammary glands. The purpose of this investigation was to determine whether anti-inflammatory therapy influenced bacterial clearance following intramammary challenge of lactating goats with Escherichia coli.Standardized quantities of a pathogenic coliform culture were infused through the teat canal into one half of the mammary gland in 18 goat does. The does were then randomly assigned to receive one of three intravenous treatments: saline (controls), one dose of steroid (dexamethasone), or two doses of a non-steroidal anti-inflammatory agent (flunixin meglumine). The clinical signs, milk production, complete blood counts, serum clinical chemistry values, milk bacterial cultures and milk somatic cell concentrations were monitored sequentially.Goats treated with anti-inflammatory agents exhibited some improvement in clinical response to challenge with E. coli (e.g. rectal temperature, degree of appetite suppression) as compared to saline controls. There were no significant differences between treatments in the degree of inflammation present in the mammary glands or supramammary lymph nodes examined at necropsy. The most important finding was that anti-inflammatory therapy did not adversely influence the clearance of E. coli from challenged glands.     

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

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

Essential role of neutrophils but not mammary alveolar macrophages in a murine model of acute Escherichia coli mastitis

Mastitis, the inflammation of the mammary gland, is an important disease affecting dairy animals worldwide. The disease is caused by mammary pathogenic bacteria and Escherichia coli are frequently implicated. Virulence factors of mammary pathogenic E. coli are only partially known and intramammary challenge with LPS elicits neutrophil recruitment in experimental bovine and murine mastitis models. We have previously shown that neutrophil recruitment in LPS-induced murine mastitis is strictly dependent on mammary alveolar macrophages. However, the relative role of alveolar macrophages and blood neutrophils in E. coli mastitis is not well defined. To this end, we selectively depleted mammary alveolar macrophages or blood neutrophils before intramammary challenge with E. coli strain P4 (ECP4). Mice depleted of alveolar macrophages prior to intramammary challenge recruited neutrophils normally and restricted bacterial growth and interstitial invasion. Importantly however, upon depletion of alveolar macrophages, ECP4 invaded the mammary alveolar epithelial cells and formed intracellular bacterial communities. In contrast, neutrophil depletion prior to intramammary infection with ECP4 was associated with unrestricted bacterial growth, tissue damage, severe sepsis and mortality. This study suggests that neutrophils but not alveolar macrophages provide essential antimicrobial defense against mammary pathogenic E. coli. Furthermore, we show here similar invasion after depletion of alveolar macrophages as in our previous studies showing that LPS/TLR4 signaling on alveolar macrophages abrogates ECP4 invasion of the mammary epithelium. Interestingly, similar ECP4 invasion and formation of intracellular communities were also observed following intramammary infection of either iNOS gene-deficient or IL-1 receptor type 1 gene-deficient mice.     

Cellular and humoral immune responses during intrathoracic paracoccidioidomycosis in BALB/c mice

Paracoccidioidomycosis is a chronic infection that primarily affects the lungs. Here we investigated cellular and humoral immune responses after intrathoracic Paracoccidioides brasiliensis infection in BALB/c mice. P. brasiliensis-colony-forming units (CFUs), fungal DNA and granulomas in lungs increased progressively, peaking at day 90 postinfection (p.i.). IFN-γ production was highest on day 15 p.i., declining thereafter. The kinetics of the NO production was similar to that described for IFN-γ. In contrast, IL-10 increased from day 45 p.i. reaching a peak at day 90. Levels of serum IgG1 were higher than IgG2a between days 30 and 90 p.i. 30% of mice died by day 90 p.i. These data indicate that infection with P. brasiliensis by the intrathoracic route shows high IFN-γ and NO production at day 15 p.i., unable to control multiplication of fungi, which appears to be associated with a progressive increase in IL-10 and in the number and complexity of granulomas.     

Characterization of shiga toxin-producing Escherichia coli strains isolated from calves in Poland

Fecal samples from 67 3–5-months-old calves with diarrhea were screened for the presence of shiga toxin-producing Escherichia coli (STEC). Several accessory virulence factors genes were also tested. Among 192 E.coli isolates tested, 15 (7.6%) were found to harbour the shiga toxin 1 or 2 (stx1 or stx2) genes. The stx2-carrying samples were further subtyped by PCR for the stx2c, stx2d, and stx2e toxin variants. It was shown that stx2-positive bacteria mainly possessed the stx2c shiga toxin type gene. The enterohemolysin (hlyA) and intimin (eae) genes were found in seven (46.7%) STEC strains whereas the cytotoxic necrotizin factor 1 and 2 or the P fimbrial genes were detected in two isolates only. This study confirmed that calves are a reservoir of STEC strains (with all pathogenicity genes) that may be virulent for humans.     

Field trial evaluating changes in prevalence and patterns of antimicrobial resistance among Escherichia coli and Enterococcus spp. isolated from growing broilers medicated with enrofloxacin, apramycin and amoxicillin

The present study investigates, under field conditions, the influence of antimicrobial administration on prevalence and patterns of antimicrobial resistance among Escherichia coli and Enterococcus spp. isolated from growing broilers. For this purpose, a group of 16,000 commercial broiler chickens was treated with enrofloxacin from day 1 to day 3, gentamicin from day 19 to day 21, and ampicillin from day 26 to day 28. A control group of 16,000 broilers was placed in the same controlled environment poultry house. Fecal (from both groups) and feed samples were collected at regular intervals. Few E. coli isolates were obtained from either farm environment or poultry feed samples, while enterococci were found to be ubiquitous among these samples. The frequency of resistance against most antimicrobials tested was significantly higher (P < 0.05) in E. coli isolated from broilers receiving intermittent antimicrobial pressure than that from non-medicated broilers, whereas in enterococci these differences were only observed among structurally related antimicrobial drugs and over a short period of time. By the time the broilers reached market age (33 days), several multi-resistant E. coli and enterococci were detected in the feces of the medicated group. Results suggest that antimicrobial resistance in E. coli was mainly medication-dependent, whereas among enterococci, changes observed over time were apparently influenced by factors apart from antimicrobial exposure, namely the resistance organisms previously present in farm environment and those present in feedstuffs.     

Detection of fluoroquinolone resistance level in clinical canine and feline Escherichia coli pathogens using rapid real-time PCR assay

Fluoroquinolones are used to treat infections caused by Escherichia coli in canine and feline veterinary patients, particularly those infecting the urinary tract. The gyrA gene is a primary target causing fluoroquinolone resistance in Gram negative coliforms, with mutations in codons 83 and 87 generally associated with high-level of resistance E. coli clinical isolates. We have developed a fluorescence resonance energy transfer (FRET) quantitative PCR to identify enrofloxacin-resistance in clinical E. coli isolates that carry mutations in codons 83 and 87 of gyrA. This real-time quantitative PCR assay is rapid, economical, and sensitive compared with cultured antimicrobial susceptibility testing. The assay identified as few as four genome copies per reaction from culture and 19 genome copies in urine. For the 70 isolates tested, the sensitivity was 87.5% (95% CI = 75–95.3%) (n = 42/48), specificity was 100% (95% CI = 87.3–100%) (n = 22/22), whereas accuracy was 91.4% (95% CI = 82.3–97%) (n = 64/70). Furthermore, we were able to accurately differentiate between the wild type and mutants E. coli directly from infected canine urine samples (n = 5) within 2 h. These results were confirmed by sequence alignments of the PCR products and comparison with the susceptibility testing. The FRET-PCR assay appears to have promising clinical application as an early diagnostic tool for rapid and sensitive detection and differentiation of the level of fluoroquinolone resistance among clinical E. coli isolates that may facilitate design of the dosing regimen.     

Staphylococcus aureus and Escherichia coli elicit different innate immune responses from bovine mammary epithelial cells

Escherichia coli and Staphylococcus aureus are the most important pathogenic bacteria causing bovine clinical mastitis and subclinical mastitis, respectively. However, little is known about the molecular mechanisms underlying the different host response patterns caused by these bacteria. The aim of this study was to characterize the different innate immune responses of bovine mammary epithelium cells (MECs) to heat-inactivated E. coli and S. aureus. Gene expression of Toll-like receptor 2 (TLR2) and TLR4 was compared. The activation of nuclear factor kappa B (NF-κB) and the kinetics and levels of cytokine production were analyzed. The results show that the mRNA for TLR2 and TLR4 was up-regulated when the bovine MECs were stimulated with heat-inactivated E. coli, while only TLR2 mRNA was up-regulated when the bovine MECs were stimulated with heat-inactivated S. aureus. The expression of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6 and IL-8 increased more rapidly and higher when the bovine MECs were stimulated with heat-inactivated E. coli than when they were stimulated with heat-inactivated S. aureus. E. coli strongly activated NF-κB in the bovine MECs, while S. aureus failed to activate NF-κB. Heat-inactivated S. aureus could induce NF-κB activation when bovine MECs cultured in medium without fetal calf serum. These results were confirmed using TLR2- and TLR4/MD2-transfected HEK293 cells and suggested that differential TLR recognition and the lack of NF-κB activation account for the impaired immune response elicited by heat-inactivated S. aureus.     

Resistance patterns and detection of aac(3)-IV gene in apramycin-resistant Escherichia coli isolated from farm animals and farm workers in northeastern of China

The aminoglycoside apramycin has been used widely in animal production in China since 1999. This study was aimed to investigate the resistance pattern of apramycin-resistant Escherichia coli isolated from farm animals and farm workers in northeastern of China during 2004–2007 and to determine whether resistance to apramycin was mediated by plasmid containing the aac(3)-IV gene and the mode for the transfer of genetic information between bacteria of farm animals and farm workers. Thirty six E. coli isolates of swine, chicken, and human origins, chosen randomly from 318 apramycin-resistant E. coli isolates of six farms in northeastern of China during 2004–2007, were multi-resistant and carried the aac(3)-IV gene encoding resistance to apramycin. Conjugation experiments demonstrated that in all 36 cases, the gene encoding resistance to apramycin was borne on a mobilisable plasmid. Homology analysis of the cloned aac(3)-IV gene with the sequence (accession no. X01385) in GenBank showed 99.3% identity at a nucleotide level, but only with a deletion of guanosine in position 813 of the gene in all 36 cases. The results indicted that resistance to apramycin in these isolates was closely related to aac(3)-IV gene. Therefore, the multi-resistance of E. coli could complicate therapeutic practices for enteric infections in both farm animals and human.     

Some properties of beta toxin produced by Clostridium haemolyticum strain IRP-135

Six culture media were evaluated for the optimization of β-toxin production by Clostridium haemolyticum (strain IRP-135) using both batch and dialysis culture techniques. The lethal component of β-toxin remained active for 13 days when maintained at 37°C but was inactivated by heating at 60° for 20 min. A 1 : 10,000 dilution of trypsin inactivated the toxin in 15 min. Preliminary data from electrophoresis in SDS acrylamide gel indicate the molecular weight of the β-toxin to be approximately 32,000.     

Changes in various metabolic parameters in blood and milk during experimental Escherichia coli mastitis for primiparous Holstein dairy cows during early lactation

Background

The objective of this study was to characterize the changes in various metabolic parameters in blood and milk during IMI challenge with Escherichia coli (E. coli) for dairy cows during early lactation. Thirty, healthy primiparous Holstein cows were infused (h = 0) with ~20-40 cfu of live E. coli into one front mammary quarter at ~4-6 wk in lactation. Daily feed intake and milk yield were recorded. At –12, 0, 3, 6, 12, 18, 24, 36, 48, 60, 72, 96, 108, 120, 132, 144, 156, 168, 180 and 192 h relative to challenge rectal temperatures were recorded and quarter foremilk was collected for analysis of shedding of E. coli. Composite milk samples were collected at -180, -132, -84, -36, -12, 12, 24, 36, 48, 60, 72, 84, 96, 132 and 180 h relative to challenge (h = 0) and analyzed for lactate dehydrogenase (LDH), somatic cell count, fat, protein, lactose, citrate, beta-hydroxybutyrate (BHBA), free glucose (fglu), and glucose-6-phosphate (G6P). Blood was collected at -12, 0, 3, 6, 12, 18, 24, 36, 60, 72, 84, 132 and 180 h relative to challenge and analyzed for plasma non-esterified fatty acids (NEFA), BHBA and glucose concentration. A generalized linear mixed model was used to determine the effect of IMI challenge on metabolic responses of cows during early lactation.

Results

By 12 h, E. coli was recovered from challenged quarters and shedding continued through 72 h. Rectal temperature peaked by 12 h post-challenge and returned to pre-challenge values by 36 h post-IMI challenge. Daily feed intake and milk yield decreased (P <0.05) by 1 and 2 d, respectively, after mastitis challenge. Plasma BHBA decreased (12 h; P <0.05) from 0.96 ± 1.1 at 0 h to 0.57 ± 0.64 mmol/L by 18 h whereas concentration of plasma NEFA (18 h) and glucose (24 h) were significantly greater, 11 and 27%, respectively, after challenge. In milk, fglu, lactose, citrate, fat and protein yield were lower whereas yield of BHBA and G6P were higher after challenge when compared to pre-challenge values.

Conclusions

Changes in metabolites in blood and milk were most likely associated with drops in feed intake and milk yield. However, the early rise in plasma NEFA may also signify enhanced adipose tissue lipolysis. Lower concentrations of plasma BHBA may be attributed to an increase transfer into milk after IMI. Decreases in both milk lactose yield and % after challenge may be partly attributed to reduced conversion of fglu to lactose. Rises in G6P yield and concentration in milk after challenge (24 h) may signify increased conversion of fglu to G6P. Results identify changes in various metabolic parameters in blood and milk after IMI challenge with E. coli in dairy cows that may partly explain the partitioning of nutrients and changes in milk components after IMI for cows during early lactation.     

Dietary high protein-induced diarrhea and intestinal inflammation by activation of NF-κB signaling in piglets

The present study aimed to investigate whether inflammation-associated responses in piglets are induced by high protein (HP) through activating nuclear factor kappa B (NF-κB) signaling. Sixteen piglets (35 d of age, Duroc × [Landrace × Yorkshire], weaned at d 21, initial BW = 9.70 ± 0.11 kg) were allocated to 18% and 26% CP (HP group) at random, comprising 8 replicate pens per treatment. The piglets were slaughtered to collect intestinal tissues when apparent, persistent, and stable diarrhea syndromes happened (on d 12). No significant differences were observed in their growth performance (P > 0.05), but reduction by 19.11%, 25.31%, 23.64% of ADFI, ADG, and G:F, respectively was detected in the HP group. The HP group had greater (P = 0.002) diarrhea rates. Furthermore, dietary HP had lower ileal villus height (VH; P = 0.048), ratio of villus height to crypt depth (VH/CD ratio; P = 0.016), and colonic CD (P = 0.034), as well as had the trend (P = 0.075) to reduce the ileal villus absorptive area. Moreover, HP diets significantly elevated the goblet cell numbers in the ileal villi (P = 0.016) and colonic crypts (P < 0.001) and up-regulated (P = 0.012) the mRNA expression of mucin2 (Muc2) in the ileum. In addition, HP diets increased the myeloperoxidase concentration in the ileum (P = 0.002) and colon (P = 0.007) of piglets. Dietary HP significantly down-regulated the mRNA expression of tumor necrosis factor-α (TNF-α; P < 0.001) in the ileum, induced nitric oxide synthase (iNOS; P = 0.040) and interleukin-22 (IL-22; P = 0.008) in the colon, and inclined to down-regulate interleukin-1β (IL-1β; P = 0.076) expression in the colon. The relative protein abundance of Galectin-3 (P = 0.046) in the colon and the ratio of phosphorylation NF-κB to NF-κB (p–NF–κB/NF-κB ratio) in the ileum of HP piglets were also greater (P = 0.038). These results suggest that dietary HP may cause diarrhea in piglets by activating NF-κB signaling induced intestinal inflammation.     

Role of nitric oxide production in dairy cows naturally infected with Mycobacterium avium subsp. paratuberculosis

Nitric oxide (NO) is a crucial mediator in host defense and is one of the major killing mechanisms within macrophages. Its induction is highly affected by the types of cytokines and the infectious agents present. In the current study, NO production was evaluated after in vitro infection of unfractionated peripheral blood mononuclear cells (PBMCs) with Mycobacterium avium subsp. paratuberculosis (MAP) after 8 h, 3 and 6 days of culture for cows in different stages of disease. In addition, the effects of in vitro exposure to inhibitory cytokines such as interleukin-10 (IL-10) and transforming growth factor β (TGF-β) as well as the pro-inflammatory cytokine IFN-γ were correlated with the level of NO production. Nitric oxide production was consistently higher in cell cultures from subclinically infected animals at all time points. An upregulation of NO production was demonstrated in unfractionated cell cultures from healthy control cows after exposure to MAP infection as compared to noninfected cell cultures. A similar increase in NO due to the addition of MAP to cell cultures was also noted for clinically infected cows. NO level among subclinically infected cattle was greater at all time points tested and was further boosted with the combination of both in vitro MAP infection and IFN-γ stimulation. Alternatively, nonspecific stimulation with LPS from Escherichia coli O111:B4-W resulted in an upregulation of NO production in all infected groups at 3 and 6 days after in vitro infection. Finally, the in vitro exposure to inhibitory cytokines such as IL-10 and TGF-β prior to MAP infection or LPS stimulation resulted in the downregulation of this inflammatory mediator (NO) in all experimental groups at all time points. In summary, a higher level of NO production was associated with cows in the subclinical stage of MAP infection. As well, the results demonstrated an increase in NO production upon infection with MAP and in the presence of exogenous IFN-γ. Finally, the results suggest an important role of IL-10 and TGF-β on the profile of NO production which may explain the low NO production in MAP clinically infected cows.     

Differential responses of cells from human skin keratinocyte and bovine mammary epithelium to attack by pore-forming Staphylococcus aureus alpha-toxin

Human skin keratinocytes HaCat attacked by Staphylococcus aureus α-toxin showed a transient drop of cellular ATP levels whereas in toxin-perforated bovine mammary epithelial cells (BMEC), the ATP levels dropped more slowly. Morphologically, during the ATP level depletion, HaCat cell developed a spacious intracellular vacuole together with the transient influx of trypan blue. WST-1 signal, which tested the function of mitochondrial enzyme in viable cells, also decreased concomitantly. On the other hand, BMEC excluded trypan blue and vacuolation was not observed throughout the experiment. We conclude that mammary epithelial cells resist the toxin better than keratinocytes. This is the first report showing that α-toxin enhances transient membrane permeability to large molecules, temporary vacuole formation and the transient defect of mitochondrial enzyme in viable cells without cell lysis.     

Characterization of integrons in multiple antimicrobial resistant Escherichia coli isolates from bovine endometritis

To assess the prevalence of antimicrobial resistance and three classes of integrons in Escherichia coli (E. coli) strains (n = 57) isolated from bovine endometritis in Inner Mongolia of China, antimicrobial susceptibility and the presence of three types of integrons were characterized. Most isolates were susceptible to ceftiofur, furazolidone, ciprofloxacin and enrofloxacin, while 57 isolates were all resistant to sulfamethoxydiazine and trimethoprim. High resistant incidence rates were exhibited to sulfadiazine, tetracycline, oxytetracycline, cefazolin, chloramphenicol. Forty-six of 57 E. coli strains were resistant to above 10 antibiotics (80.70%). The integrase gene and gene cassettes of integrons were amplified by PCR. DNA sequencing and analysis were used to identify the genetic content of the integron-variable regions. Neither class II nor class III integron was detected, while 36.8% (n = 21) of the isolates were positive for the presence of intI1 gene. Analysis of gene cassettes revealed that six gene cassettes were found, which encoded resistance to trimethoprim (dhfr, dhfrI, dfrA17) and aminoglycosides (aadA1, aadA2, aadA5). Among them, the gene cassette array dfrA17–aadA5 was found most prevalent (66.7%). The resistance profile of positive-integron isolates was relatively broad and they were resistant to more than eight antimicrobials (n ? 8). The correlation analysis revealed the incidence of integrons among the isolates were related to the multiple antibiotic resistance profile, indicating integrons play an important role in the dissemination and spread of the antimicrobial resistant strains.     

Induction of inflammatory host immune responses by organisms belonging to the genera Chlamydia/Chlamydophila

Chlamydia/Chlamydophila are a family of intracellular gram-negative bacteria that infect their hosts primarily via mucosal epithelia. Chronic disease associated with bacterial persistence, inflammation and tissue damage are common sequelae of infection with these organisms. Human epithelial cell lines respond to infection by releasing pro-inflammatory cytokines and chemokines such as interleukin (IL)-6 and IL-8, and upregulating the expression of mRNA encoding Iκ-Bα, the endogenous inhibitor of NF-κB. However, Iκ-Bα is not upregulated in response to bacterial lipopolysaccharide (LPS). The failure of epithelial cells to respond to LPS is associated with the absence of surface expression of CD14. Identification of the components of Chlamydia/Chlamydophila that can induce pro-inflammatory mediators coupled with the mechanisms by which epithelial cells detect infection and respond accordingly will advance the development of preventative strategies.     

The Effect of Experimental Infectious Mastitis on Leukocyte Subpopulations and Cytokine Production in Non-lactating Ewes

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