Effect of intravenous lipid infusion on biomarkers of insulin resistance and immune functions of dry and nonpregnant dairy cows.

During the transition period, dairy cows often experience negative energy balance, which can induce metabolic and immunological disturbances. Previous work has shown that there is a relationship between the dysfunction of immune cells and the increase in blood nonesterified fatty acid (NEFA) concentration. Nevertheless, it is difficult to determine the exact effect of NEFA on the immune system, as other metabolic and hormonal perturbations occur simultaneously during the transition period. In the present study, we have determined the effect of NEFA on immune functions using an experimental model designed to assess the effects independently of energy balance, as well as hormonal and metabolic changes due to parturition. Six dry and nonpregnant cows were infused with either sterile water (control treatment) or a lipid emulsion (Intralipid 20%, Frenesius Kabi, lipid treatment) at a rate of 1 mL/kg per hour for 6 h according to a crossover design. Blood concentrations of NEFA, ß-hydroxybutyrate (BHB), and glucose were measured every hour throughout the infusion period, and 1 and 18 h after the end of infusion. Proliferation and interferon-γ secretion of lymphocytes, phagocytosis, and oxidative burst of neutrophils and blood insulin concentration were evaluated before, during, and at the end of the infusion. For NEFA, BHB, and glucose, treatment × time interactions were present. When compared with the control condition, NEFA and BHB levels were greater in the plasma of cows infused with lipids from 1 h after the start of infusion until 1 h after the end of infusion. Glucose level also increased in response to lipid infusion from 2 h of infusion until 1 h after the end of treatment. For sterile water and lipid infusions, respectively, maximal concentrations were 0.06 ± 0.10 mM and 1.39 ± 0.10 mM for NEFA, 0.70 ± 0.05 mM and 1.06 ± 0.05 mM for BHB, and 4.56 ± 0.27 mM and 6.90 ± 0.27 mM for glucose. For all blood metabolites, there were no differences between treatments 18 h postinfusion. Lipid infusion significantly increased blood insulin concentration at 3 and 6 h of infusion. However, it returned to its basal concentration 18 h after the end of the infusion. Lymphoproliferation declined as early as 3 h after the start of the lipid infusion. At 3 and 6 h of infusion, lipid treatment significantly reduced INF-γ concentration in the culture cell supernatant. The lipid infusion did not affect neutrophil phagocytosis. Nevertheless, the efficacy of the response was affected by a reduction of neutrophils' oxidative burst. These results confirm that NEFA inhibits immune functions independently of energy balance and other changes that occur during the transition period. They also indicate that high blood lipid concentration causes insulin resistance.

Effect of different fatty acids on the proliferation and cytokine production of dairy cow peripheral blood mononuclear cells.

During the transition period, dairy cows often experience negative energy balance, which induces metabolic and immunological disturbances. Our previous work has shown a relationship between the inhibition of immune functions and increased blood nonesterified fatty acid (NEFA) levels. In this study, we evaluated the effect of 11 fatty acids (palmitoleic, myristic, palmitic, stearic, oleic, linoleic, docosahexaenoic, conjugated linoleic, lauric, eicosapentaenoic, and linolenic acids) as well as a mix that represented the NEFA profile observed during the transition period at different concentrations (0, 50, 100, and 250 µM) on proliferation and cytokines secretion of lymphocytes. To assess lymphoproliferation, peripheral blood mononuclear cell (PBMC) from 5 healthy cows (166-189 d in milk) were isolated, stimulated with the mitogenic lectin concanavalin A (ConA), incubated for 72 h with or without fatty acids, and subjected to flow cytometry analysis. Our results showed that all fatty acids, except lauric acid, significantly reduced proliferation of PBMC in a dose-dependent manner. The most detrimental effect was observed with conjugated linoleic and stearic acids, where proliferation of PBMC was already inhibited at the lowest dose (50 µM). For cytokine secretion, we found that levels of IL-4 in culture supernatant of ConA-stimulated PBMC were reduced after a 24-h exposure to the lowest dose (50 µM) of oleic and palmitoleic acids. A dose of 100 µM of eicosapentaenoic acid, NEFA mixture, and myristic acid was necessary to observe a reduction in IL-4 levels. The PBMC also showed a decrease in the secretion of IFN-γ in response to lauric, linolenic, palmitoleic, and stearic acids at 50 µM and myristic acid at 100 µM. Overall, polyunsaturated fatty acids were more potent inhibitors of cytokine secretions than saturated fatty acids. In addition, we detected an inverse relationship between the melting points of fatty acids and their ability to inhibit IL-4 and IFN-γ secretions, as evidenced by greater inhibition with low-melting point fatty acids. Overall, our study confirmed that NEFA have a negative effect on some lymphocyte functions, and that their inhibitory effect on cytokine secretions increases with the degree of unsaturation.

Susceptibility to Nisin, Bactofencin, Pediocin and Reuterin of Multidrug Resistant Staphylococcus aureus, Streptococcus dysgalactiae and Streptococcus uberis Causing Bovine Mastitis.

Antibiotics are the most effective strategy to prevent and treat intramammary infections. However, their misuse has led to the dissemination of multidrug resistant bacteria (MDR) for both animals and humans. Efforts to develop new alternative strategies to control bacterial infections related to MDR are continuously on the rise. The objective of this study was to evaluate the antimicrobial activity of different bacteriocins and reuterin against MDR Staphylococcus and Streptococcus clinical isolates involved in bovine mastitis. A bacterial collection including S. aureus (n = 19), S. dysgalactiae (n = 17) and S. uberis (n = 19) was assembled for this study. Antibiotic resistance profiles were determined by the disk diffusion method. In addition, sensitivity to bacteriocins and reuterin was evaluated by determining minimum inhibitory concentrations (MIC). A total of 21 strains (37.5%) were MDR. MICs ranged from ≤1.0 µg/mL to ≥100 µg/mL for nisin and 2.0 to ≥250 µg/mL for bactofencin. Reuterin was active against all tested bacteria, and MICs vary between 70 and 560 µg/mL. Interestingly, 20 MDR strains were inhibited by bactofencin at a concentration of ≤250 µg/mL, while 14 were inhibited by nisin at an MIC of ≤100 µg/mL. Pediocin did not show an inhibitory effect.

Immune and experimental infection responses of dairy cows vaccinated with the combination of six Staphylococcus aureus proteins that are expressed during bovine intramammary infection and a triple adjuvant.

Staphylococcus aureus is a leading cause of bovine intramammary infections (IMI). Standard antibiotic treatments are not very effective and currently available vaccines lack tangible efficacy. Developing a vaccine formulation for S. aureus mastitis is challenging and selection of target antigens is critical. The gene products of six S. aureus genes that are highly expressed during IMI were selected as antigens for this study. The vaccine contained six recombinant proteins formulated with Emulsigen®-D, a CpG oligodeoxynucleotide and indolicidin. Nine cows in mid-lactation received the vaccine while ten received saline (placebo). Two immunizations were performed 10 weeks apart. All the antigens induced an immune response. A balanced immune response (IgG2/IgG1 ratio of 1) was observed for antigen SACOL0442 while a predominant Th2 response was observed for the other antigens (IgG2/IgG1 ratio <1). Immunizations induced CD4+ cell proliferation in response to SACOL0442, SACOL0029, SACOL0720 and SACOL1912 while a CD8+ cell proliferation was induced by SACOL0720. Four weeks after the second immunization, three quarters per animal were experimentally infused with ∼60 CFU of S. aureus. Although no difference in S. aureus counts was observed between the two groups after this robust infectious challenge, a sustained reduction in milk somatic cells counts (SCC) was observed in vaccinated cows. A correlation between SCC and S. aureus counts in milk was also observed. Altogether, this indicates that the collective immune responses induced by the antigens certainly contribute to the observed benefits of the whole vaccine. More work is needed to understand how different antigens stimulate a different response using the same adjuvant.

A targeted genotyping approach to enhance the identification of variants for lactation persistency in dairy cows.

Lactation persistency (LP), defined as the ability of a cow to maintain milk production at a high level after milk peak, is an important phenotype for the dairy industry. In this study, we used a targeted genotyping approach to scan for potentially functional single nucleotide polymorphisms (SNPs) within 57 potential candidate genes derived from our previous genome wide association study on LP and from the literature. A total of 175,490 SNPs were annotated within 10-kb flanking regions of the selected candidate genes. After applying several filtering steps, a total of 105 SNPs were retained for genotyping using target genotyping arrays. SNP association analyses were performed in 1,231 Holstein cows with 69 polymorphic SNPs using the univariate liner mixed model with polygenic effects using DMU package. Six SNPs including rs43770847, rs208794152, and rs208332214 in ADRM1; rs209443540 in C5orf34; rs378943586 in DDX11; and rs385640152 in GHR were suggestively significantly associated with LP based on additive effects and associations with 4 of them (rs43770847, rs208794152, rs208332214, and rs209443540) were based on dominance effects at P < 0.05. However, none of the associations remained significant at false discovery rate adjusted P (FDR) < 0.05. The additive variances explained by each suggestively significantly associated SNP ranged from 0.15% (rs43770847 in ADRM1) to 5.69% (rs209443540 in C5orf34), suggesting that these SNPs might be used in genetic selection for enhanced LP. The percentage of phenotypic variance explained by dominance effect ranged from 0.24% to 1.35% which suggests that genetic selection for enhanced LP might be more efficient by inclusion of dominance effects. Overall, this study identified several potentially functional variants that might be useful for selection programs for higher LP. Finally, a combination of identification of potentially functional variants followed by targeted genotyping and association analysis is a cost-effective approach for increasing the power of genetic association studies.

Non-antimicrobial approaches at drying-off for treating and preventing intramammary infections in dairy cows. Part 1. Meta-analyses of efficacy of using an internal teat sealant without a concomitant antimicrobial treatment.

Use of antimicrobial approaches at drying-off for preventing new intramammary infections (IMI) during the dry period in dairy cows could be replaced by non-antimicrobial approaches. Such approaches would be of interest not only for organic but also for conventional dairy producers. The objective of the current review was to quantify the effect of non-antimicrobial internal teat sealant (ITS)-based approaches at drying-off for treating and preventing IMI, when compared with no treatment or with an antimicrobial-based approach. The protocol for this review was published before initiating the review. A total of 18 trials from 16 articles could be used to investigate the effect of an ITS-based approach. With the available results, we conclude with a high level of confidence that non-antimicrobial ITS-based dry-off approaches are efficient for preventing new IMI during the dry period when compared with no treatment, and would reduce risk of new IMI by 52%. Moreover, we are relatively confident that a bismuth subnitrate-based ITS performed better than an antimicrobial for preventing new IMI during the dry period (a risk reduction of 23%). Similarly, we are relatively confident that an ITS-based approach would only slightly or not at all reduce the prevalence of IMI at calving compared with untreated quarters.

Effect of stage of lactation and gestation on milking-induced hormone release in lactating dairy cows.

Three experiments were conducted to better understand why milking-induced prolactin (PRL) release decreases as lactation advances. Experiment 1 compared the effects of milking, 2-min manual stimulation of the mammary gland (without milking), or injection of 1 IU of oxytocin (without milking) on hormonal release in early lactation cows, late-lactation and nongestating cows, and late-lactation and gestating cows (n = 6 per physiological status). Blood samples were collected from 20 min before the start of the treatments to 60 min after. During milking, PRL release (area under the curve above the baseline) was greater in the early lactation cows than in the late-lactation cows but was unaffected by gestation. Lactation stage and gestation did not affect PRL release by manual stimulation. Oxytocin did not induce a significant release of PRL or cortisol. Cortisol release was unaffected by physiological status and was similar for milking and mammary stimulation. Milking-induced ß-endorphin release was not affected by physiological status. Experiment 2 compared the effects of milking, 2-min manual stimulation, or 10-min manual stimulation in cows in early (n = 6) and late (n = 6) lactation. Prolactin release was greater in the early lactation cows than in the late-lactation cows for all 3 treatments. A 10-min manual stimulation induced greater PRL release than a 2-min stimulation did. Cortisol release was greater in the early lactation cows but was similar among the 3 treatments. Experiment 3 compared the effects of a 5-min manual stimulation and the injection of domperidone (a dopamine antagonist) in cows in early (n = 6) and late (n = 6) lactation. Manually induced PRL release was greater in the early lactation cows than in the late-lactation cows. Prolactin release was greater with domperidone injection than with manual stimulation and was not affected by lactation stage. Thus, the reduction of milking-induced PRL release in late lactation is not a consequence of the lower sensitivity of the mammary gland to stimulation, a shorter milking time, the gestation stage, or the reduced capacity of the pituitary gland to secrete PRL.

Antibiofilm and antibacterial effects of specific chitosan molecules on Staphylococcus aureus isolates associated with bovine mastitis.

Staphylococcus aureus is one of the major pathogens causing bovine intramammary infections (IMIs) and mastitis. Mastitis is the primary cause for the use of antibiotics in dairy farms but therapeutic failure is often observed. One of the reasons for the lack of effectiveness of antibiotic therapy despite the observed susceptibility of bacterial isolates in vitro are bacterial biofilms. In this study, we used chitosan of well-defined molecular weight (0.4-0.6, 1.3, 2.6 and 4.0 kDa) and investigated their antibiofilm and antibacterial activities in in vitro and in vivo models related to S. aureus IMIs. A chitosan of at least 6 units of glucosamine was necessary for maximum antibacterial activity. The 2.6 and 4.0 kDa forms were able to prevent biofilm production by the biofilm hyperproducer strain S. aureus 2117 and a bovine MRSA (methicillin-resistant S. aureus). The intramammary administration of the 2.6 kDa chitosan showed no adverse effects in mice or in cows, as opposed to the slight inflammatory effect observed in mammary glands with the 4.0 kDa derivative. The 2.6 kDa chitosan killed bacteria embedded in pre-established biofilms in a dose-dependent manner with a >3 log10 reduction in CFU at 4 mg/ml. Also, the 2.6 kDa chitosan could prevent the persistence of the internalized MRSA into the mammary epithelial cell line MAC-T. An in vitro checkerboard assay showed that the 2.6 kDa chitosan produced a synergy with the macrolide class of antibiotics (e.g., tilmicosin) and reduced the MIC of both molecules by 2-8 times. Finally, the intramammary administration of the 2.6 kDa chitosan alone (P<0.01) or in combination with tilmicosin (P<0.0001) reduced the colonization of mammary glands in a murine IMI model. Our results suggest that the use of chitosan alone or in combination with a low dose of a macrolide could help reduce antibiotic use in dairy farms.

Non-antibiotic approaches at drying-off for treating and preventing intramammary infections: a protocol for a systematic review and meta-analysis.

Intramammary infection (IMI) treatment and prevention at drying-off is one of the leading causes for using antimicrobials on dairy farms. The objective of the current paper is to describe the protocol used for conducting a systematic review of the literature on non-antibiotic strategies that can be used on dairy cows at dry off to treat and prevent IMI. Relevant literature will be identified using a combination of database search strategies and iterative screening of references. To be included in the review, articles will have to: (1) be published after 1969; (2) be written in English, French, or Spanish; (3) use a study design such as a controlled trial, an observational study, or an experimental study conducted in vivo; (4) be conducted on commercial dairy cows; (5) investigate a non-antibiotic intervention used at dry off; and finally, (6) report on a relevant mastitis outcome. Titles and abstracts, then full articles will be reviewed for inclusion. Specific data will be extracted and risk of bias will be assessed for all included articles. The planned systematic review will be the first to colligate, in a coherent whole, studies investigating non-antibiotic strategies for treating and preventing IMI at drying-off.

mRNA Expression of lipogenic enzymes in mammary tissue and fatty acid profile in milk of dairy cows fed flax hulls and infused with flax oil in the abomasum.

In the present study, the effect of flax hulls with or without flax oil bypassing the rumen on the expression of lipogenic genes in the mammary tissue of dairy cows was investigated. A total of eight dairy cows were used in a replicated 4 × 4 Latin square design. There were four periods of 21 d each and four treatments: control diet with no flax hulls (CONT); diet with 9·88 % flax hulls in the DM (HULL); control diet with 500 g flax oil/d infused in the abomasum (COFO); diet with 9·88 % flax hulls in the DM and 500 g flax oil/d infused in the abomasum (HUFO). A higher mRNA abundance of sterol regulatory element binding transcription factor, fatty acid (FA) synthase, lipoprotein lipase (LPL), PPARγ1, stearoyl-CoA desaturase (SCD) and acetyl-coenzyme A carboxylase-α was observed in cows fed HULL than in those fed CONT, and HUFO had the opposite effect. Compared with CONT, COFO and HUFO lowered the mRNA abundance of SCD, which may explain the lower proportions of MUFA in milk fat with flax oil infusion. The mRNA abundance of LPL in mammary tissue and proportions of long-chain FA in milk fat were higher in cows fed COFO than in those fed CONT. The highest proportions of trans FA were observed when cows were fed HULL. The present study demonstrates that flax hulls with or without flax oil infusion in the abomasum can affect the expression of lipogenic genes in the mammary tissue of dairy cows, which may contribute to the improvement of milk FA profile.

Mammary gene expression and activity of antioxidant enzymes and oxidative indicators in the blood, milk, mammary tissue and ruminal fluid of dairy cows fed flax meal.

The effects of flax meal (FM) on the activity of antioxidant enzymes (superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT)) in the blood, mammary tissue and ruminal fluid, and oxidative stress indicators (thiobarbituric acid-reactive substances(TBARS) and 1,1-diphenyl-2-picrylhydrazyl-scavenging activity) in the milk, plasma and ruminal fluid of dairy cows were determined.The mRNA abundance of the antioxidant enzymes and oxidative stress-related genes was assessed in mammary tissue. A total of eight Holstein cows were used in a double 4 x 4 Latin square design. There were four treatments in the diet: control with no FM(CON) or 5% FM (5FM), 10% FM (10FM) and 15% FM (15FM). There was an interaction between treatment and time for plasma GPx and CAT activities. Cows supplemented with FM had a linear reduction in TBARS at 2 h after feeding, and there was no treatment effect at 0, 4 and 6 h after feeding. TBARS production decreased in the milk of cows fed the 5FM and 10FM diets. There was a linear increase in nuclear factor (erythroid-derived 2)-like 2 (NFE2L2) mRNA abundance in mammary tissue with FM supplementation.A linear trend for increased mRNA abundance of the CAT gene was observed with higher concentrations of FM. The mRNA abundance of CAT, GPx1, GPx3, SOD1, SOD2, SOD3 and nuclear factor of k light polypeptide gene enhancer in B-cells (NFKB) genes was not affected by the treatment. These findings suggest that FM supplementation can improve the oxidative status of Holstein cows as suggested by decreased TBARS production in ruminal fluid 2 h post-feeding and increased NFE2L2/nuclear factor-E2-related factor 2 (Nrf2) mRNA abundance in mammary tissue.

The expression of a putative exotoxin and an ABC transporter during bovine intramammary infection contributes to the virulence of Staphylococcus aureus.

Staphylococcus aureus is a leading cause of intramammary infections (IMI) and bovine mastitis is an important disease for the dairy industry. As this bacterium probably expresses specific genes for establishment of IMI, we studied the transcriptional profile of four S. aureus strains recovered from experimentally infected cows. Microbial RNA was extracted from bacteria isolated from milk, reverse-transcribed and labeled for hybridization to sub-genomic microarrays to detect candidate genes for further investigations. Several S. aureus genes were expressed during IMI; some were detected in samples from more than one strain, more than one cow and at more than one time point during infection. A selection of four genes showing strong expression and with putative functions in pathogenesis was further studied by qPCR. By comparing the expression in different media in vitro, we found that gene SACOL2171 was induced by iron restriction whereas the expression of the transcriptional regulator SACOL2325 and the ABC transporter SACOL0718-720 (vraFG) were induced by milk. In addition, the putative exotoxin SACOL0442 seemed to require the intramammary environment for expression. Gene-disrupted mutants for SACOL0720 and SACOL0442 showed no growth defect in vitro but were attenuated during bovine IMI, causing infections with significant reductions in bacterial and somatic cell counts. The milk from the mammary quarters infected with these mutants also showed better appearance and composition than milk from quarters infected with the wild type. In conclusion, we have identified genes that are most likely important for S. aureus IMI. These represent novel candidates to include in a vaccine.

Mammary gene expression and activity of antioxidant enzymes and concentration of the mammalian lignan enterolactone in milk and plasma of dairy cows fed flax lignans and infused with flax oil in the abomasum.

The objectives of the study were to investigate the effects of dietary supplementation of flax hulls and/or flax oil on the activity of antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX)) in plasma and the mammary gland and the relative mRNA abundance of antioxidant genes in the mammary gland of dairy cows. A total of eight dairy cows were used in a replicated 4 × 4 Latin square design. There were four treatments: control with no flax hulls (CONT), 9·88% flax hulls in the DM (HULL), control with 500 g flax oil/d infused in the abomasum (COFO), 9·88% flax hulls in the DM and 500 g flax oil/d infused in the abomasum (HUFO). Plasma GPX activity tended to decrease with flax oil supplementation. Cows fed HULL had higher levels of CAT, GPX1 and SOD1 mRNA in the mammary gland and lower mRNA abundance of GPX3, SOD2 and SOD3 compared with those fed CONT. Abundance of CAT, GPX1, GPX3, SOD2 and SOD3 mRNA was down-regulated in the mammary gland of cows fed HUFO compared to those fed CONT. The mRNA abundance of CAT, GPX1, GPX3 and SOD3 was lower in the mammary gland of cows fed COFO than in the mammary gland of cows fed CONT. The present study demonstrates that flax hulls contribute to increasing the abundance of some antioxidant genes, which can contribute to protecting against oxidative stress damage occurring in the mammary gland and other tissues of dairy cows.

Evaluation of some Staphylococcus aureus iron-regulated proteins as vaccine targets.

Staphylococcus aureus is an important pathogen that is responsible for a wide range of infections, including bovine mastitis. Previously, 54 genes from S. aureus that were up-regulated in an iron-restricted medium and in mice were identified. Seven of those genes were selected from five iron-acquisition systems (isd, feo, sir, sst, and fhu), and the proteins were evaluated as potential vaccine targets to prevent bovine mastitis. The antigenicity of the recombinant proteins obtained with each studied gene was evaluated in rabbits and/or cattle. Immune sera were used to test the bacterial accessibility of the native proteins. All the proteins were immunogenic in rabbits or cattle. IsdH, IsdB, FeoB and SstD were expressed on the bacterial surface, with IsdB and IsdH more expressed in an iron-restricted environment. The capacity of antibodies to prevent infection was measured in a mouse mastitis model. Preincubation of S. aureus with serum against IsdH or with the pool of sera against IsdB, SstD and FeoB led to decreased colonization of the mouse mammary glands. Lastly, cattle immunization with IsdH induced a strong and long-lasting immune response with IgG2 production. The protein IsdH appears to be a good vaccine candidate to prevent S. aureus bovine mastitis.

Lack of protection of mice against Staphylococcus aureus despite a significant immune response to immunization with a DNA vaccine encoding collagen-binding protein.

Collagen-binding protein (CNA) is the major Staphylococcus aureus adhesin responsible for high affinity binding to collagen and is assumed to be a major virulence factor in infection and disease. Mutants lacking the cna gene are less virulent than the parent strain in models of septic arthritis, osteomyelitis, keratinitis, and endocarditis. In order to investigate the immunological and protective properties of a CNA-based DNA vaccine, a eukaryotic expression vector pCNA was constructed which expressed the collagen-binding domain of this adhesin in transfected cells. Three groups of 11 Balb/c mice received three injection of either pCNA, the empty expression vector (pCI) or PBS. Those injected with pCNA showed hi titre (64000) antibody and evidence of a cell-mediated immune response (CMI). The anti-CNA antibodies recognized the intact bacteria and prevented binding to collagen in vitro. However, the vaccination did not protect against bacterial challenge using the intra-peritoneal route of infection. Moreover, S. aureus that had been treated with sera from vaccinated mice caused a more severe infection than bacteria treated with sera from non-vaccinated mice. In summary, DNA vaccination against CNA produced a strong antibody and cellular response in mice but failed to protect from i.p. infection by S. aureus.

Effects of nitric oxide on bovine polymorphonuclear functions.

The effects of nitric oxide (NO) on the functionality of polymorphonuclear neutrophils (PMNs) in bovine milk or blood were investigated. In 2 experiments, mastitis was induced by infusing both hind quarters with saline containing Escherichia coli endotoxins. In addition, the left hind quarter was infused with aminoguanidine, an inhibitor of the inducible form of NO synthase (iNOS). At various times after infusion, somatic cells were isolated from milk samples, and superoxide (O2-) production induced by phorbol myristate acetate was evaluated. In both experiments, the addition of aminoguanidine had no inhibitory effect on the number of milk somatic cells or on their O2- production. The effect of NO and iNOS inhibitors on the functionality of bovine PMNs isolated from blood was investigated in vitro. The neutrophils did not produce NO. A neutrophil:monocyte co-culture system was used to study the effect of NO derived from monocytes on O2- production by bovine neutrophils. Neither NO derived from activated monocytes nor the iNOS inhibitors aminoguanidine and L-N6-(1-iminoethyl)lysine had an effect on the ability of bovine neutrophils to release O2-. Moreover, aminoguanidine did not affect the ability of bovine neutrophils to phagocytose bacteria. These results suggest that inhibition of NO release during inflammation does not interfere with the migration of immune cells to the site of infection or the ability of these cells to destroy pathogens. Thus, NO does not appear to play a major role in the control of the functions of bovine neutrophils.

Protective immune responses to a multi-gene DNA vaccine against Staphylococcus aureus.

To investigate the strategy of using a multivalent polyprotein DNA vaccine against Staphylococcus aureus, a series of plasmids was used to immunize mice followed by infectious challenge. The plasmid vaccines expressed Clumping factor A (Clfa), fibronectin binding protein A (FnBPA) and the enzyme Sortase (Srt) as single proteins or combined as a polyprotein. All animals produced a mixed Th1 and Th2 response including functional antigen-specific, mostly IgG2a antibodies, sustained production of IFN-gamma and a predominantly CD8+ T-cell response. Upon challenge with a virulent S. aureus isolate (Sa042), after 21 days, 55% of the multi-gene vaccinated mice survived infection compared to only 15% of the control groups. Vaccinated mice showed no signs of arthritis when challenged with the less virulent "Newman" strain that caused reactive arthritis in the controls. The results suggest that a multi-gene polyprotein-expressing nucleic acid vaccine alone produces a combined Th1 and Th2 response that can contribute to protection against the complex pathogenesis of S. aureus.

Transcriptional modulation of some Staphylococcus aureus iron-regulated genes during growth in vitro and in a tissue cage model in vivo.

Staphylococcus aureus can proliferate in iron-limited environments such as the mammalian host. The transcriptional profiles of 460 genes (iron-regulated, putative Fur-regulated, membrane transport, pathogenesis) obtained for S. aureus grown in iron-restricted environments in vitro and in vivo were compared in order to identify new iron-regulated genes and to evaluate their potential as possible therapeutic targets in vivo. Iron deprivation was created in vitro by 2,2-dipyridyl, and in vivo, S. aureus was grown in tissue cages implanted in mice. Bacterial RNA was obtained from each growth condition and cDNA probes were co-hybridized on DNA arrays. Thirty-six upregulated and 11 downregulated genes were commonly modulated in animals and in the low-iron medium. Real-time PCR confirmed the iron-dependent modulation of four novel genes (SACOL0161, 2170, 2369, 2431) with a Fur box motif. Some genes expressed in the dipyridyl medium were not expressed in vivo (e.g., copA, frpA, SACOL1045). Downregulated genes included an iron-storage protein gene and genes of the succinate dehydrogenase complex, reminiscent of a small RNA-dependent regulation thus far only demonstrated in Gram-negative bacteria. The expression of iron-regulated genes in distinct low-iron environments provided insight into their relative importance in vitro and in vivo and their usefulness for vaccine and drug development.

In vivo expression of the antimicrobial defensin and lactoferrin proteins allowed by the strategic insertion of introns adequately spliced.

A major limitation of conventional shuttle expression system, when cloning a bactericidal gene, is the basal expression level in bacteria, which is lethal. Although the expression level is low, the bactericidal feature inherent to the molecule leads to subsequent failure to recover intact transformants when the related gene is cloned into a conventional expression vector. Contrary to popular belief, the human cytomegalovirus immediate-early region 1 promoter (CMV), which is to date one of the most powerful promoters for eukaryotic expression, is active in bacteria. In this study, bactericidal genes were cloned into a conventional shuttle eukaryote expression vector harbouring the CMV promoter, but were interrupted with a sequence independent splicing element (SISE), thus inhibiting lethal gene expression in bacteria. The insertion strategy of the intron uses a universal restriction site-free cloning approach, which has been developed to insert a DNA fragment into a specific location of a gene, through a PCR-based cloning technique. We have found that one intervening sequence, which derives from an adenovirus, can be spliced in a mammalian system without respect to its location, thus the bactericidal protein is synthesized only when transfected into mammalian cells. Therein, lactoferrin and defensin proteins were produced in vivo without the necessity of complex expression systems. By introducing the adeno SISE within the coding sequence of the bactericidal genes, such genes can be easily synthesized in vitro through cloning into bacteria and still are able to express biologically active proteins when introduced into mammalian cells.

DNA immunization of dairy cows with the clumping factor A of Staphylococcus aureus.

Blocking the primary stages of Staphylococcus aureus infection, specifically the bacterial adhesion to cell and the colonization of the mucosal surface, may be the most effective strategy for preventing infections. Clumping factor A (ClfA) is considered to be one of the most important adhesions factors of S. aureus to host cells. The present study describes the immune response of dairy cattle to a DNA vaccine against ClfA and evaluates the ability of specific genetic adjuvants, targeting sequences (granulocyte macrophage colony-stimulating factor and cytotoxic T lymphocyte antigen-4) and transporter molecules (chitosan and copolymer) to modify the immune response of cows. The results show that vaccination of cows with fibrinogen-binding region A induced a strong and specific antibody response to ClfA in comparison with a control group injected with the pCI vector alone. Although the co-expression of both genetic adjuvants and the addition copolymer transporter did not augment the overall antibody response, these approaches decreased the number of non-responsive cows. Chitosan was the only factor that did not enhance the immune response. Three months after the last DNA immunization, three cows from each of the pGM-CSF, internal ribosomal entry site (IRES), pCTLA and pCI groups were injected with 200 microg of recombinant ClfA protein in incomplete Freund's adjuvant. A strong humoral response was observed in all groups following this protein boost, with the response occurring slightly earlier in DNA-primed protein boost cows. Sera and milk samples taken from cows after the second DNA injection or after the protein boost (sera only) were analyzed for their ability to block adherence and increase phagocytosis. Pre-incubation of S. aureus with sera or milk from vaccinated cows significantly reduced the pathogen's ability to adhere to MAC-T cells relative to the sera and milk samples from the pCI-injected control cows. Similarly, pools of sera and milk from vaccinated cows increased phagocytosis of S. aureus by neutrophils. After the protein boost, sera were more efficient promoters of phagocytosis, reflecting the higher anti-ClfA antibody level of these sera. DNA-prime/protein boost regimes combined with molecular adjuvants appeared to be effective in generating a strong immune response to S. aureus antigens in cattle.