Evaluation of nanoparticle-encapsulated outer membrane proteins for the control of Campylobacter jejuni colonization in chickens.

Numerous vaccination strategies have been evaluated to develop effective vaccines against Campylobacter jejuni colonization in poultry but with limited success. The following experiments were conducted to investigate the effect of biodegradable and biocompatible poly (lactide-co-glycolide) nanoparticle (NP) encapsulated outer membrane proteins (OMP) of C. jejuni. Chickens were vaccinated with different routes [subcutaneous (s/c) or oral] and doses (25, 125, or 250 µg) of candidate nanoparticle vaccine with appropriate control groups. Serum and cloacal fecal samples were taken at regular intervals of time, and the birds were euthanized 7 d postchallenge with C. jejuni. The results were interpreted based on anti-OMP immunoglobulin response in chicken and intestinal colonization of C. jejuni. The C. jejuni colonization in cecal and cloacal contents at 7 d postchallenge was below the detection limit in the s/c vaccinated groups, but the other groups demonstrated varying degrees of colonization. The serum IgA was higher in the group vaccinated s/c with OMP only compared with the rest of the groups. The serum- and fecal-IgY titers were consistently higher in the s/c vaccinated groups (with or without NP) than the rest of the groups. Elevated levels of OMP specific serum antibodies correlated with below the limit of detection levels of Campylobacter colonization in broiler chickens receiving 125 µg of OMP alone and the OMP+NP vaccine s/c. In conclusion, the s/c route of vaccination with or without NP encapsulated OMP of C. jejuni may serve as a candidate vaccine for control of C. jejuni colonization in chickens.

Effects of in ovo interleukin-4-plasmid injection on anticoccidia immune response in a coccidia infection model of chickens.

Two experiments were conducted to study the effects of an in ovo interleukin (IL)-4 plasmid injection in a coccidia infection model. In experiment I, chicks were hatched from eggs that had been injected in ovo with an empty vector or with 10 or 15 µg of IL-4 plasmid, and then challenged posthatch with coccidia. In experiment II, chicks were hatched from eggs that had been vaccinated with coccidia and injected in ovo with an empty vector or with 10 or 15 µg of IL-4 plasmid, and then challenged posthatch with coccidia. In experiment II, the BW gain of birds hatched from eggs vaccinated with live oocysts plus 15 µg of IL-4 plasmid was 25% higher than the BW gain of birds hatched from eggs vaccinated with live oocysts plus empty plasmid. In both experiments I and II, a 15-µg IL-4-plasmid injection decreased fecal oocyst shedding, decreased the number of CD8(+) cells in the cecal tonsils, and decreased cecal tonsil lymphocyte cell proliferation postcoccidia challenge. In experiment I, splenic macrophages of chicks hatched from eggs injected with 15 µg of IL-4 plasmid had higher nitric oxide production than those of chicks hatched from eggs injected with the empty plasmid. In experiment II, a 15-µg IL-4-plasmid injection increased serum anticoccidia IgG postcoccidia challenge. It could be concluded that 15 µg of IL-4 plasmid improved anticoccidia immune responses synergistically with in ovo coccidia vaccination in chickens.

Chemical constituents and biological studies of Origanum vulgare Linn.

Bioassay-guided isolation of methanolic extract of the leaves of Origanum vulgare Linn., yielded two protocatechuic acid ester derivatives, origanol A (1) and origanol B (2) along with ursolic acid (3), oleanolic acid (4), ß-sitosterol (5), and triacontanol (6). Structures of the compound were established based on physical and spectral data (UV, IR, (1)H and (13)C NMR and mass). Origanol A (1) showed significant mushroom tyrosinase inhibition activity.

Chemokine receptor CCR7 and CXCR5 mRNA in chickens following inflammation or vaccination.

The CCR7 and CXCR5 chemokine receptor mRNA contents of different immune organs were studied in normal, healthy birds and in birds treated with either lipopolysaccharide (LPS) as a systemic inflammatory challenge or coccidial vaccine (Coccivac B; Intervet/Schering-Plough Animal Health Corp., Millsboro, DE) as an enteric vaccination challenge. The CCR7 mRNA content of the spleen of normal, healthy birds was approximately 150-fold higher than CCR7 mRNA content of any other organs studied. The CXCR5 mRNA content of the bursa of normal, healthy birds was approximately 80-fold higher than the CXCR5 mRNA content of any other organs studied. The LPS injection decreased the splenic CCR7 mRNA content by approximately 100 times and the bursal CXCR5 mRNA content by approximately 5-fold at 24 h post-LPS injection (P < 0.01). The LPS injection increased the CXCR5 content of cecal tonsils by approximately 3-fold at 24 h post-LPS injection (P < 0.05). At 10 d postvaccination, CCR7 mRNA content was approximately 15-fold higher and CXCR5 mRNA content was approximately 12-fold higher in cecal tonsils of the vaccinated group than in the control group (P < 0.01). In conclusion, CCR7 and CXCR5 mRNA levels were dependent on the immune organs and the inflammatory status of the organs in chickens.

Chicken chemokine receptors in T cells isolated from lymphoid organs and in splenocytes cultured with concanavalin A.

Chemokine receptors guide immune cells to specific organs during health and disease. The mRNA content of the chemokine receptors CCR2, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CXCR4, CXCR5, and CX3CR1 in CD4(+) cells (T-helper cells) isolated from blood, bursa, cecal tonsil, spleen, and thymus and in CD8(+) cells (T-cytotoxic cells) isolated from blood, cecal tonsil, spleen, and thymus were investigated. The CD4(+) cells isolated from thymus had the highest amount of CCR7 and CCR8 mRNA. The CD4(+) cells isolated from bursa, cecal tonsil, and thymus had the highest amount of CCR5 mRNA. The CD4(+) cells isolated from cecal tonsils had the highest amount of CCR9 mRNA. The CD4(+) cells isolated from bursa and thymus had the highest amount of CXCR5 mRNA. The CD8(+) cells isolated from cecal tonsil had the highest mRNA amount of all receptors studied except CCR9 and CX3CR1. The CD4(+) cells treated with concanavalin A had increased CCR2, CCR4, CCR7, CCR8, and CXCR5 mRNA amounts at 24 h of stimulation. The CD8(+) cells treated with concanavalin A had increased CCR4 mRNA at 72 h, increased CCR6 mRNA at 24 h, and decreased CCR8 and CXCR4 mRNA at 24 h of stimulation.

Effect of erythorbate, storage and high-oxygen packaging on premature browning in ground beef.

Premature browning (PMB) was investigated in ground beef patties with (0.04%, w/w) and without erythorbate. In Experiment 1, patties were stored at 4 °C for 48 h; at -18 °C for 21 days; or at -18 °C for 21 days, thawed at 4 °C for 24 h; and cooked. Bulk ground beef was stored at -18 °C for 24 days, thawed for 24 h at 4 °C, and patties prepared and cooked immediately. In Experiment 2, fresh patties were overwrapped with oxygen-permeable film or packaged in 80% O(2)/20% N(2) (MAP), and stored for 48 h at 4 °C, or at -18 °C for 21 days, and cooked. Total reducing activity and color (L*, a* and b* values) were measured immediately prior to cooking. Patties were cooked to internal temperatures of 60, 66, 71 and 77 °C and internal cooked color was measured. Total reducing activity was higher for the erythorbate treatment than controls for all storage conditions (P<0.05). a* Values of cooked patties were higher for erythorbate than control treatments under all storage and packaging conditions at 60 and 66 °C (P<0.05). The presence of erythorbate in ground beef patties appeared to maintain red color at cooked internal temperatures of 60 and 66 °C. Frozen bulk storage appeared to increase the susceptibility of ground beef to PMB when compared to fresh and frozen patties. Patties cooked directly from frozen state appeared less susceptible to PMB than frozen-thawed and bulk storage. Ground beef appeared predisposed to PMB when stored in high-oxygen MAP at 4 °C for 48 h.

Effect of muscle source on premature browning in ground beef.

Premature browning (PMB) describes cooked beef that may appear done before reaching 71 °C. Ground beef from paired Longissimus lumborum (LL) and Psoas major (PM) muscles was formed into patties. Patties were cooked immediately, and after 48 and 96 h storage at 4 °C. Total reducing activity (TRA) and external color were measured immediately prior to cooking. Patties were cooked to internal end point temperatures of 60, 66, 71 or 77 °C and internal cooked color (L(*), a(*) and b(*) values) was measured. Raw PM patties had greater L(*) values and lesser a(*) values than those from LL (P<0.05). For LL and PM, raw a(*) and b(*) values decreased with storage from 0 h to 96 h (P<0.05). At 0 and 48 h storage, cooked patties prepared from PM had greater a(*) values than those prepared from LL at all internal endpoint temperatures (P<0.05). Internal cooked a(*) values of patties from PM decreased with storage of raw patties, whereas it was stable for LL patties (P<0.05).

Inactivation of Escherichia coli O157: H7 and Listeria monocytogenes by PR-26, a synthetic antibacterial peptide.

This study reports the antibacterial effect of PR-26, a synthetic peptide derived from the first 26 amino acid sequence of PR-39, an antimicrobial peptide isolated from porcine neutrophils. A three-strain mixture of Escherichia coli O157:H7 or Listeria monocytogenes of approximately 10(8) CFU was inoculated to a final concentration of 10(7) CFU/ml in 1% peptone water (pH 7.0), containing 50 or 75 microg/ml of PR-26, and incubated at 37 degrees C for 0, 6, 12, and 24 h; at 24 degrees C for 0, 12, 24, and 36 h; or at 10 or 4 degrees C for 0, 24, 72, and 120 h. Control samples included 1% peptone water inoculated with each pathogen mixture but containing no PR-26. The surviving population of each pathogen at each sampling time was determined by plating on tryptic soy agar with incubation at 37 degrees C for 24 h. At 37 degrees C, PR-26 decreased E. coli O157:H7 and L. monocytogenes populations by >5.0 log CFU/ml at 12 h, with complete inactivation at 24 h. At 24 degrees C, PR-26 reduced E. coli O157:H7 and L. monocytogenes by approximately 3.5, 4.0, and 4.5 log CFU/ml at the end of 12-, 24-, and 36-h incubations, respectively. At 4 and 10 degrees C, the inhibitory effect of PR-26 on E. coli O157:H7 and L. monocytogenes was significantly lower (P < 0.05) than that at 37 and 24 degrees C: a 2- to 3-log CFU/ml reduction was observed at 120-h incubation. Results indicate that PR-26 could potentially be used as an antimicrobial agent, but applications in appropriate foods need to be validated.

Discovery of MC-02,331, a new cephalosporin exhibiting potent activity against methicillin-resistant Staphylococcus aureus.

A systematic approach toward building activity against methicillin-resistant staphylococci into the cephalosporin class of beta-lactam antibiotics is described. Initial work focused on finding the optimal linkage between the cephem nucleus and a biphenyl pharmacophore, which established that a thio linkage afforded potent activity in vitro. Efforts to optimize this activity by altering substitution on the pharmacophore afforded iodophenylthio analog MC-02,002, which although highly potent against MRSA, was also highly bound to serum proteins. Further work to decrease serum protein binding showed that replacement of the iodo substituent by the positively-charged isothiouronium group afforded potent activity and reduced serum binding, but insufficient aqueous solubility. Solubility was enhanced by incorporation of a second positively-charged group into the 7-acyl substituent. Such derivatives (MC-02,171 and MC-02,306) lacked sufficient stability to staphylococcal beta-lactamase enzymes. The second positive charge was incorporated into the cephem 3-substituent in order to utilize the beta-lactamase-stable aminothiazolyl(oximino)acetyl class of 7-substituents. These efforts culminated with the discovery of bis(isothiouroniummethyl)phenylthio analog MC-02,331, whose profile is acceptable with respect to potency against MRSA, serum binding, aqueous solubility, and beta-lactamase stability.