Cathelicidins (CATHLs) are small, cationic antimicrobial peptides that establish an early innate immune defense against infections in mammals. Beyond their wide spectrum of antimicrobial activity, these peptides play important roles in wound repair, chemotactic activity, and apoptosis. Thus, comprehensive characterizing of bovine CATHLs could potentially identify underlying inherited differences in innate immunity and disease resistance in cattle. The purpose of the present study was to verify the placement of the CATHL cluster at the distal end of bovine chromosome 22 (BTA22), identify any single nucleotide polymorphisms (SNPs) and insertion-deletion (indel) polymorphisms within the gene family, explore copy number variation, and investigate the functional impact any of these variants may have in overall bovine innate immunity. A framework radiation hybrid map was constructed with 7 markers screened against the bovine 12,000 rad whole genome RH (12K WG-RH) panel, which when compared to the current genome assembly (Btau_4.0) confirmed current gene order. Comparative sequence analysis for 10 domestic cattle breeds representing both Bos taurus taurus and Bos taurus indicus revealed 60 SNPs, 7 of which were nonsynonymous, and 5 indel mutations. Data from array comparative genomic hybridization (aCGH) between four Angus and four Nelore animals showed a 2-fold increase in copy number of the CATHL4 locus, which was verified by quantitative PCR (qPCR) of genomic DNA. Nelore animals showed an approximate 2-fold increase in the CATHL4 gene. Subsequently, the expression of CATHL4 in Nelore neutrophils exhibited a range of 2- to 5-fold increases in CATHL4 gene expression. Finally, a colorimetric bactericidal assay was performed on the neutrophils of the same Angus and Nelore animals previously genotyped for copy number variations (CNVs). After in vitro challenges to Staphylococcus aureus, Salmonella typhimurium, Mannheimia haemolytica, and Pasteurella multocida, the killing capacity of Nelore neutrophils was approximately 20 percent greater than Angus neutrophils for M. haemolytica and 10 percent greater for P. multocida. Characterization of this antimicrobial gene family is central to developing a firm understanding regarding the effects CATHL variation has with respect to bovine innate immunity.

Despite the success of modern medicine in treating infections, infectious diseases remain a major source of morbidity and mortality worldwide. The evolution of antibiotic resistant strains of bacteria means that new innovations in therapeutics must be pursued to combat this emerging threat. A novel approach is to utilize the anti-infective properties of endogenous host defense peptides by creating smaller synthetic peptides with enhanced protective activities. Some of these peptides directly kill bacteria and many display varied immunomodulatory activities, enhancing the host innate immune response to more effectively clear an infection. Here I examined the efficacy of several synthetic peptides in a murine model of invasive bacterial infection, induced by the Gram positive bacterium Staphylococcus aureus. Several peptides were able to significantly reduce peritoneal bacterial load in vivo by up to 4-logs relative to the controls, either through direct antibacterial killing or immunomodulatory activity. The latter class was studied in more detail; in particular, the peptides IDR-1 and 1002 displayed significant immunomodulatory effects in vivo. Both peptides were able to significantly induce the proinflammatory chemokines MCP-1, RANTES and KC, as well as increased recruitment of neutrophils and monocytes to the site of infection. These effects were not dependent on live bacteria, as heat inactivated S. aureus was also able to induce chemokines and cell migration. Mice that had been depleted of macrophages did not respond to peptide treatment, indicating that macrophages are an important effector cells through which immunomodulatory peptides counter infections. These results suggest that synthetic peptides have the potential to become a viable treatment option for bacterial infections.

Understanding how antimicrobial growth promoters (AGPs) affect small intestinal microbiota may help to discover effective alternatives. The impact of dietary supplementation with tylosin phosphate on small intestinal microbial profiles was investigated in growing pigs, and compared with the microbial profile of pigs fed flaxseed or its fractions. Eighteen ileal-cannulated barrows (33.1 +/- 2.4 kg) received either the control diet (C, wheat, peas and soybean meal), or C plus 22 mg/kg tylosin phosphate (T), 20% whole flaxseed (WF), 18% hot-water extracted flaxseed (HWE), 4% flaxseed hulls (H), or 8% flaxseed oil (O) during three 21-d periods in a change over design. Ileal digesta (100 mL) was collected on d 16 and 17 of each period. Two chaperonin-60 universal target (cpn60 UT) libraries were constructed from pooled ileal digesta DNA extracted from the C and T diets. A total of 1634 nucleotide sequences were determined, and 117 different cpn60 UT sequences identified. Microbial diversity was greatest in the C library compared to T. Taxonomic composition between libraries differed, and included Lactobacillales (94% of C and 86% of T sequences), Enterobacteriaceae (3% of C and 13% of T), Clostridiales, Bacillales and Bifidobacterium taxa. T had a reduced ratio of Lactobacillales: Enterobacteriaceae sequences (6:1) compared to C (35:1). Lactobacilli: enterobacteria plate count ratios were highest in WF compared to C or T diets. Lactobacillus johnsonii genomes detected by qPCR were increased by 17.2 and 12%, in T and WF diets, respectively, compared to C. Numbers of L. amylovorus genomes were 25% lower in the H diet compared to C. Numbers of Escherichia coli and Streptococcus alactolyticus genomes were unaffected by dietary treatment, despite differences in library clone frequency for these species. Increased L. johnsonii colonization with tylosin suggests possible probiotic properties of this bacterium. Only inclusion of whole flaxseed resulted in a similar increase in L. johnsonii. Overall, ileal microbial profiles of growing pigs were similar and remained mostly unaffected by dietary tylosin or flaxseed inclusion.

This study describes the pharmacokinetic profile of procaine penicillin G after intraperitoneal (IP) administration in 8 lactating dairy cows. Procaine pencillin G (PPG, 21,000 IU/kg) was deposited into the abdominal cavity of each cow following an incision in the right paralumbar fossa. Blood and milk samples were taken over the following 10 days, at which point the cows were euthanized. Plasma, milk, muscle, liver, and kidney penicillin concentrations were determined by HPLC, with a limit of detection (LOD) of 5 ppb for plasma and milk samples. Noncompartmental methods were used to analyze plasma kinetics. The mean pharmacokinetic parameters (} s.d.) were: Cmax, 5.5 } 2.6 Êg/mL; Tmax, 0.75 } 0.27 h; AUC0-, 10.8 } 4.9 Êg*h/mL; MRT, 2.2 } 0.9 h. All milk from treated cows contained penicillin residues for a minimum of 3 milkings (31 h) and maximum of 5 milkings (52 h) after administration. Concentrations of penicillin G in all muscle, liver, and kidney samples taken 10 days post-administration were below the limit of detection. Necropsy examinations revealed foci of hemorrhage on the rumenal omentum of most cows but peritonitis was not observed. Systemic inflammation as determined by altered leukograms and fibrinogen was noted in one cow. The results of this study demonstrate that IP procaine penicillin G is absorbed and eliminated rapidly in lactating dairy cows.

Listeria monocytogenes is a foodborne pathogenic bacterium responsible for ~500 deaths and a financial burden of ~$2.3 billion each year in the United States. Though a zero tolerance policy is enforced with regard to its detection in cooked ready-to-eat foods, additional preemptive control alternatives are required for certain products. Among these alternatives are strategies permitting the usage of food antimicrobial combinations to control the pathogen. Research on antimicrobial combinations can provide insight into more efficient control of the pathogen, but is currently lacking. The purpose of this study was to evaluate the in vitro inhibition of L. monocytogenes exposed to the antimicrobials e-Poly-L-Lysine (EPL), lauric arginate ester (LAE), and sodium lactate (SL) at pH 7.3, octanoic acid (OCT) at pH 5.0, and nisin (NIS) and acidic calcium sulfate (ACS) at both pH 5.0 and 7.3. A broth dilution assay was used to determine single antimicrobial minimum inhibitory and bactericidal concentrations for L. monocytogenes Scott A, 310, NADC 2783, and NADC 2045. Optical density differences (delta<0.05 at 630 nm) were used to denote inhibition. Concentrations producing population decreases of greater than or equal to 3.0 log10 CFU/ml after incubation were considered bactericidal. Inhibition resulting from combinations of antimicrobials (NIS+ACS, EPL+ACS, SL+ACS, NIS+LAE, OCT+ACS, and OCT+NIS) was assessed using a checkerboard assay, and fractional inhibitory concentrations (FIC) were determined. FIC values were plotted on isobolograms and were used to create FIC indices (FICI). Isobologram curvature was used to classify combinations as synergistic, additive, or antagonistic. From FIC indices, interactions were defined as antagonistic (FICI >1.0), additive (FICI =1.0), or synergistic (FICI &lt;1.0). Strain-dependent factors had a bearing on MIC and MBC values for NIS and EPL. At pH 7.3, NIS+ACS displayed synergistic inhibition, NIS+LAE and EPL+ACS demonstrated additive-type interactions, and the SL+ACS pairing was unable to be defined. At pH 5.0, interpretation of the OCT+NIS interaction also presented challenges, while the OCT+ACS combination resulted in synergistic behavior. Additional studies are needed to validate in vitro findings on surfaces of ready-to-eat meats. Future in vivo studies should investigate the ability of synergistic combinations (NIS+ACS and OCT+ACS) to control the pathogen. Better characterizations of inhibitory mechanisms should also be performed.

Campylobacter jejuni is a commensal microorganism of the poultry gastrointestinal tract. Broilers, layers, ducks, turkeys, and quails can be colonized by Campylobacter without illness occurring. The vast majority of human Campylobacter infections are recognized as being foodborne. For 2008, preliminary FoodNet data showed that the Campylobacter incidence of infection, 12.68 per 100,000 of the U.S. population, is the second highest, only behind Salmonella at 16.20 per 100,000. To further understand Campylobacter’s role as a foodborne pathogen, analysis at the molecular level is needed. Microbial molecular typing allows for identification and differentiation of bacterial strains beneath the species level. In this study, the “gold standard” method for molecular subtyping, Pulsed Field Gel Electrophoresis (PFGE), along with Diversilab® repetitive element Polymerase Chain Reaction (rep-PCR) and 16S-23S Internal Spacer Region Denaturing Gradient Gel Electrophoresis (ISR DGGE) were used for the molecular typing of Campylobacter jejuni isolates obtained during different stages of commercial broiler production and processing. In addition, the C. jejuni isolates were tested for resistance to antimicrobials commonly used in both veterinary and human medicine. Antimicrobial resistance testing was carried out using a broth dilution system. The majority of recovered isolates came from post-harvest carcass rinsates. Carcass rinses were obtained at post-evisceration, post-chill stages. All isolates (n = 46) were identified by the Polymerase Chain Reaction as Campylobacter jejuni. Three genotypes (n = 44, n = 1, n = 1) were identified by PFGE. The 46 rep-PCR products grouped into seven clusters and two outliers. Clustering of rep-PCR products by sample source was not observed. No relatedness trends were observed for isolates recovered from the same source. The combination of PFGE and Diversilab rep-PCR methods provides highly discriminatory molecular typing results. These results provide practical epidemiological information that shows postevisceration and post-chill stages are still important targets for intervention studies. The very high occurrence of C. jejuni isolates exhibiting genotype A suggests it may differentially express certain gene(s) that enable this strain to more favorably survive under the different harsh environmental conditions encountered during production and processing. In addition, phenotypic testing revealed all of the isolates were not resistant to the antimicrobials azithromycin, ciprofloxacin, erythromycin, gentamycin, tetracycline, florfenicol, nalidixic acid, telithromycin, and clindamycin at any of the concentrations tested. All the C. jejuni isolates exhibited an indistinguishable two-band 16S-23S ISR DGGE profile. Overall, these C. jejuni commercial broiler pre- and post-harvest isolates exhibited an extremely low degree of molecular and phenotypic variability.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2009. / Härtill 5 uppsatser.

Preliminary studies have shown that manuka honey affects the cell cycle of MRSA by impeding cell division, but mode of action was unknown. Cell division depends on the formation of septa and cleavage of peptidoglycan at cytokinesis. This study investigated how manuka honey might alter the cell cycle of EMRSA-15. Physiological and chemical changes in the bacteria exposed to manuka honey were determined using time to kill studies, confocal and electron microscopy. Data indicated that honey had a bactericidal effect on MRSA, inhibiting the cell cycle cytokinesis. Increased septum formation was noted in honey treated cells by transmission electron microscopy. Cell division components including FtsZ and Endo-B-N-Acetylglucosaminidase were investigated using cell wall turbidity assays, zymography, immunofluorescence and immuno gold labelling. Manuka honey treated MRSA cells showed a marked reduction in hydrolase activity after 12 hours compared to untreated cells. The immunofluorescence indicated an initial increase in FtsZ production followed by a significant decrease by 24 hours. PCR of FtsZ showed a 10% increase in production after 1 and 4 hours. Localization by gold labelling gave inconclusive results. Immunofluorescence of Endo-B-N-Acetylglucosaminidase showed a decrease in the amount of enzyme over 24 hours and localization by gold labelling indicated altered distribution of this enzyme. PCR showed no significant difference in expression. 2-D electrophoresis showed a differing proteomic profile between control cells and those treated with honey, with a potential target protein being identified. Methylglyoxal (an antibacterial component of manuka honey) was investigated after a report named this as potentially the active component of manuka honey. Results showed it has an effect but is not wholly responsible for the effects induced by manuka honey. It was concluded that increased numbers of cells with septa were formed and alteration in production of proteins and enzymes resulted in MRSA cells exposed to bactericidal concentrations of manuka honey. The work was also carried out with artificial honey controls, indicating that effects seen were not due to sugar content within honey or methylglyoxal content.

Despite the success of modern medicine in treating infections, infectious diseases remain a major source of morbidity and mortality worldwide. The evolution of antibiotic resistant strains of bacteria means that new innovations in therapeutics must be pursued to combat this emerging threat. A novel approach is to utilize the anti-infective properties of endogenous host defense peptides by creating smaller synthetic peptides with enhanced protective activities. Some of these peptides directly kill bacteria and many display varied immunomodulatory activities, enhancing the host innate immune response to more effectively clear an infection. Here I examined the efficacy of several synthetic peptides in a murine model of invasive bacterial infection, induced by the Gram positive bacterium Staphylococcus aureus. Several peptides were able to significantly reduce peritoneal bacterial load in vivo by up to 4-logs relative to the controls, either through direct antibacterial killing or immunomodulatory activity. The latter class was studied in more detail; in particular, the peptides IDR-1 and 1002 displayed significant immunomodulatory effects in vivo. Both peptides were able to significantly induce the proinflammatory chemokines MCP-1, RANTES and KC, as well as increased recruitment of neutrophils and monocytes to the site of infection. These effects were not dependent on live bacteria, as heat inactivated S. aureus was also able to induce chemokines and cell migration. Mice that had been depleted of macrophages did not respond to peptide treatment, indicating that macrophages are an important effector cells through which immunomodulatory peptides counter infections. These results suggest that synthetic peptides have the potential to become a viable treatment option for bacterial infections.

Despite their beneficial effects, concerns have been raised about the role of antimicrobial growth promoters (AGP) in the emergence of antibiotic resistant bacteria. This study evaluated the effects of approved AGP on the emergence of antibiotic resistance in commensal E. coli and foodborne pathogen Salmonella. A survey of antibiotic resistance levels in commercial broiler chicken farms in the Fraser Valley (B.C.) and an experimental feeding trial were conducted from May 2004 to February 2005 and May to November 2005, respectively. The latter examined the effects of ten AGP formulations (bambermycin, penicillin, salinomycin, bacitracin, combination of salinomycin and bacitracin, chlortetracycline, virginiamycin 11ppm, virginiamycin 22ppm, monensin and narasin) on bird performance as well. Multiple antibiotic resistant commensal E. coli and Salmonella carrying virulence genes were found at commercial broiler chicken farms and therefore may serve as reservoirs for these genes. There was no significant difference between feed formulations on the phenotypic or genotypic characteristics of the isolates, except for tetracycline resistance gene tet(B). In the experimental feeding trial, broiler chickens were fed a diet including or excluding AGP. Birds were sampled prior to and weekly during feeding of the control and the AGPP containing diets. Although not detected on day 0, E. coli increased after day 7 to more than 9.9 log10 CFU/g in ceca. Multi-drug resistant E. coli were isolated from birds fed the ten AGP containing diets as well as the control diet. Except for penicillin, none of the AGP containing diets significantly improved bird performance compared to the control diet (P>0.05). Good management practices can significantly improve broiler chickens performance and decrease the mortality rate.