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Multi-drug-resistant (MDR) bacteria in food animals are a potential problem in both animal and human health. In this study, MDR commensal Escherichia coli isolates from poultry were examined. Thirty-two E. coli isolates from broiler carcass rinses were selected based on their resistance to aminoglycosides, β-lactams, chloramphenicols, tetracyclines, and sulfonamide antimicrobials. Microarray analysis for the presence of antimicrobial resistance and plasmid genes identified aminoglycoside [aac(6), aac(3), aadA, aph, strA, and strB], β-lactam (bla(AmpC), bla(TEM), bla(CMY), and bla(PSE-1)), chloramphenicol (cat, flo, and cmlA), sulfamethoxazole (sulI and sulII), tetracycline [tet(A), tet(C), tet(D), and tetR], and trimethoprim (dfrA) resistance genes. IncA/C plasmid core genes were detected in 27 isolates, while IncHI1 plasmid genes were detected in one isolate, indicating the likely presence of these plasmids. PCR assays for 18 plasmid replicon types often associated with MDR in Enterobacteriaceae also detected one or more replicon types in all 32 isolates. Class I integrons were investigated by PCR amplification of the integrase I gene, intI1, and the cassette region flanked by conserved sequences. Twenty-five isolates were positive for the intI1 gene, and class I integrons ranging in size from ~1,000 to 3,300 bp were identified in 19 of them. The presence of class I integrons, IncA/C plasmid genes, and MDR-associated plasmid replicons in the isolates indicates the importance of these genetic elements in the accumulation and potential spread of antimicrobial resistance genes in the microbial community associated with poultry.

Multidrug-resistant (MDR) Salmonella enterica serovar Typhimurium is the most prevalent penta-resistant serovar isolated from animals by the U.S. National Antimicrobial Resistance Monitoring System. Penta-resistant isolates are often resistant to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline. To investigate MDR in Salmonella Typhimurium (including variant 5-), one isolate each from cattle, poultry, and swine with at least the ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline phenotype were selected for each year from 1997 to 2007 (n = 33) for microarray analysis of antimicrobial resistance, incompatibility IncA/C, and HI1 plasmid genes. Cluster analysis based on these data separated 31 of the isolates into two groups A and B (15 and 16 isolates, respectively). Isolates in group A were phage type DT104 or U302 and were mostly swine isolates (7/15). Genes detected included intI1, bla(PSE-1), floR, aadA, sulI, tet(G), and tetR, which are often found in Salmonella Genomic Island I. Isolates in group B had numerous IncA/C plasmid genes detected and were mostly cattle isolates (9/16). Genes detected included bla(CMY-2), floR, aac(3), aadA, aphA1, strA, strB, sulI, sulII, dfrA, dhf, tet(A)(B)(C)(D), and tetR, which are often found on MDR-AmpC IncA/C plasmids. The IncA/C replicon was also detected in all group B isolates. The two remaining isolates did not cluster with any others and both had many HI1 plasmid genes detected. Linkage disequilibrium analysis detected significant associations between plasmid replicon type, phage type, and animal source. These data suggest that MDR in Salmonella Typhimurium is associated with DT104/Salmonella Genomic Island I or IncA/C MDR-AmpC encoding plasmids and these genetic elements have persisted throughout the study period.

A potential factor leading to the spread of antimicrobial resistance (AR) in bacteria is the horizontal transfer of resistance genes between bacteria in animals or their environment. To investigate this, swine fecal samples were collected on-farm and cultured for Escherichia coli, Salmonella enterica, Campylobacter spp., and Enterococcus spp. which are all commonly found in swine. Forty-nine of the samples from which all four bacteria were recovered were selected yielding a total of 196 isolates for analysis. Isolates were tested for antimicrobial susceptibility followed by hybridization to a DNA microarray designed to detect 775 AR-related genes. E. coli and Salmonella isolated from the same fecal sample had the most AR genes in common among the four bacteria. Genes detected encoded resistance to aminoglycosides (aac(3), aadA1, aadB, and strAB), β-lactams (ampC, ampR, and bla(TEM)), chloramphenicols (cat and floR), sulfanillic acid (sul1/sulI), tetracyclines (tet(A), tet(D), tet(C), tet(G), and tet(R)), and trimethoprim (dfrA1 and dfh). Campylobacter coli and Enterococcus isolated from the same sample frequently had tet(O) and aphA-3 genes detected in common. Almost half (47%) of E. coli and Salmonella isolated from the same fecal sample shared resistance genes at a significant level (χ², p

To understand the mechanisms and epidemiology of antimicrobial resistance (AR), the genetic elements responsible must be identified. Due to the myriad of possible genes, a high-density genotyping technique is needed for initial screening. To achieve this, AR genes in the National Center for Biotechnology Information GenBank database were identified by their annotations and compiled into a nonredundant list of 775 genes. A DNA microarray was constructed of 70mer oligonucelotide probes designed to detect these genes encoding resistances to aminoglycosides, beta-lactams, chloramphenicols, glycopeptides, heavy metals, lincosamides, macrolides, metronidazoles, polyketides, quaternary ammonium compounds, streptogramins, sulfonamides, tetracyclines, and trimethoprims as well as resistance transfer genes. The microarray was validated with two fully sequenced control strains of Salmonella enterica: Typhimurium LT2 (sensitive) and Typhi CT18 (multidrug resistance [MDR]). All resistance genes encoded on the MDR plasmid, pHCM1, harbored by CT18 were detected in that strain, whereas no resistance genes were detected in LT2. The microarray was also tested with a variety of bacteria, including MDR Salmonella enterica serovars, Escherichia coli, Campylobacter spp., Enterococcus spp., methicillin-resistant Staphylococcus aureus, Listeria spp., and Clostridium difficile. The results presented here demonstrate that a microarray can be designed to detect virtually all AR genes found in the National Center for Biotechnology Information database, thus reducing the subsequent assays necessary to identify specific resistance gene alleles.

The use of antimicrobials in food animal production, particularly those commonly used to treat infections in humans, has become a source of debate in recent years. However, limited data are available regarding the development of resistance following the subtherapeutic or therapeutic administration of antimicrobials in animal production. The objective of this study was to evaluate the effect of the administration of therapeutic and subtherapeutic concentrations of tylosin on the erythromycin susceptibility of Campylobacter jejuni and Campylobacter coli isolated from the ceca of treated broilers. In three replicated studies, day-of-hatch chicks were exposed to macrolide-susceptible C. jejuni or C. coli. At 2 weeks of age, tylosin was administered at subtherapeutic (22 ppm, continuously in the diet) or therapeutic concentrations (529 ppm, in the drinking water for 5 days). Broilers were sacrificed weekly. Total and erythromycin-resistant Campylobacter spp. were enumerated from individual ceca plus cecal contents. Overall erythromycin resistance was observed at a higher frequency (P < 0.01) among C. coli isolates (70.8%) than among C. jejuni isolates (36.8%) following tylosin administration. Across Campylobacter species, erythromycin resistance was observed at a higher frequency (P < 0.001) when tylosin was administered at subtherapeutic (62.7%) than at therapeutic (11.4%) concentrations. Subtherapeutic administration resulted in the recovery of 83.3 and 56.1% erythromycin-resistant isolates compared with only 33.3 and 7.9% of the isolates expressing erythromycin resistance following the administration of therapeutic concentrations for C. coli and C. jejuni, respectively. Further studies are needed to determine the factors involved in the apparent difference in the acquisition of macrolide resistance in C. coli compared with C. jejuni.

The antimicrobial resistance of Arcobacter (n=174) and Campylobacter (n=215) isolated from broiler carcasses in a US poultry processing plant was examined. For Arcobacter, 93.7% (n=163) were resistant to one or more antimicrobials and 71.8% (n=125) were resistant to two or more antimicrobials. For Campylobacter, 99.5% (n=214) were resistant to one or more antimicrobials and 28.4% (n=61) were resistant to two or more antimicrobials. Arcobacter butzleri isolates were particularly resistant to clindamycin (90%; n=126), azithromycin (81.4%; n=114) and nalidixic acid (23.6%; n=33). Resistance to tetracycline was very high in Campylobacter jejuni (99.5%) and Campylobacter coli (96.3%). Our results demonstrate substantial resistance in Arcobacter and Campylobacter to common antimicrobial agents.

Broiler carcasses (n=325) were sampled at three sites along the processing line (prescalding, prechilling, and postchilling) in a commercial poultry processing plant during five plant visits from August to October 2004. Pulsed-field gel electrophoresis (PFGE) was used to determine the genomic fingerprints of Camospylobacter coli (n=27), Campylobacter jejuni (n=188), Arcobacter butzleri (n=138), Arcobacter cryaerophilus 1A (n=4), and A. cryaerophilus 1B (n=31) with the restriction enzymes SmaI and KpnI for Campylobacter and Arcobacter, respectively. Campylobacter species were subtyped by the Centers for Disease Control and Prevention PulseNet 24-h standardized protocol for C. jejuni. A modification of this protocol with a different restriction endonuclease (KpnI) and different electrophoresis running conditions produced the best separation of restriction fragment patterns for Arcobacter species. Both unique and common PFGE types of Arcobacter and Campylobacter strains were identified. A total of 32.8% (57 of 174) of the Arcobacter isolates had unique PFGE profiles, whereas only 2.3% (5 of 215) of the Campylobacter isolates belonged to this category. The remaining Arcobacter strains were distributed among 25 common PFGE types; only eight common Campylobacter PFGE types were observed. Cluster analysis showed no associations among the common PFGE types for either genus. Each of the eight common Campylobacter types consisted entirely of isolates from one sampling day, whereas more than half of the common Arcobacter types contained isolates from different sampling days. Our results demonstrate far greater genetic diversity for Arcobacter than for Campylobacter and suggest that the Campylobacter types are specific to individual flocks of birds processed on each sampling day.

High throughput genotyping is essential for studying the spread of multiple antimicrobial resistance. A test oligonucleotide microarray designed to detect 94 antimicrobial resistance genes was constructed and successfully used to identify antimicrobial resistance genes in control strains. The microarray was then used to assay 51 distantly related bacteria, including Gram-negative and Gram-positive isolates, resulting in the identification of 61 different antimicrobial resistance genes in these bacteria. These results were consistent with their known gene content and resistance phenotypes. Microarray results were confirmed by polymerase chain reaction and Southern blot analysis. These results demonstrate that this approach could be used to construct a microarray to detect all sequenced antimicrobial resistance genes in nearly all bacteria.

As part of a study identifying plasmids in Campylobacter, we isolated and sequenced two novel cryptic plasmids from an agricultural isolate of Campylobacter coli. The larger of the two plasmids, p3384, is 3316 bp in length and has a G+C content of 31.18%. A typical origin of replication consisting of five iterons was observed directly upstream of the first of three putative ORFs. The smaller plasmid, p3386, is 2426 bp in length and has a G+C content of 26.22%. Of the three putative ORFs detected on p3386, one shared homology with a putative protein from Campylobacter upsaliensis. The unique sequence of p3386 makes it attractive for further study concerning the evolutionary relationship of this plasmid to other Campylobacter plasmids, and to other Campylobacter isolates.

IncA/C plasmids are broad-host-range plasmids enabling multidrug resistance that have emerged worldwide among bacterial pathogens of humans and animals. Although antibiotic usage is suspected to be a driving force in the emergence of such strains, few studies have examined the impact of different types of antibiotic administration on the selection of plasmid-containing multidrug resistant isolates. In this study, chlortetracycline treatment at different concentrations in pig feed was examined for its impact on selection and dissemination of an IncA/C plasmid introduced orally via a commensal Escherichia coli host. Continuous low-dose administration of chlortetracycline at 50 g per ton had no observable impact on the proportions of IncA/C plasmid-containing E. coli from pig feces over the course of 35 days. In contrast, high-dose administration of chlortetracycline at 350 g per ton significantly increased IncA/C plasmid-containing E. coli in pig feces ( P < 0.001) and increased movement of the IncA/C plasmid to other indigenous E. coli hosts. There was no evidence of conjugal transfer of the IncA/C plasmid to bacterial species other than E. coli . In vitro competition assays demonstrated that bacterial host background substantially impacted the cost of IncA/C plasmid carriage in E. coli and Salmonella . In vitro transfer and selection experiments demonstrated that tetracycline at 32 μg/ml was necessary to enhance IncA/C plasmid conjugative transfer, while subinhibitory concentrations of tetracycline in vitro strongly selected for IncA/C plasmid-containing E. coli . Together, these experiments improve our knowledge on the impact of differing concentrations of tetracycline on the selection of IncA/C-type plasmids.

Campylobacter is a leading cause of foodborne illness in humans, and improving our understanding of the epidemiology of this organism is essential. The objective of this study was to identify the genes that discriminate isolates of C. jejuni by analysis with whole-genome DNA microarrays. Statistical analyses of whole-genome data from 95 geographically diverse cattle, chicken, and human C. jejuni isolates identified 142 most significant variable genes. Of this total, 125 (88%) belonged to genomic prophage and hypervariable regions. The significance of genomic prophage and hypervariable regions in determining C. jejuni isolate genomic diversity is emphasized by these results. These genes will be useful as biomarkers and components of genotyping systems for C. jejuni to improve our understanding of the epidemiology and population genetics of this major foodborne pathogen.

The objective of this study was to examine the distribution of multidrug resistance in Escherichia coli in relation to plasmid replicon types, animal sources, and genotypes. E. coli isolates (n = 35) from seven different animal sources were selected and tested for susceptibility to 15 antimicrobials; pulsed-field gel electrophoresis was used to determine genetic relationships among the E. coli isolates. Plasmid types based on their incompatibility (Inc) replicon types were determined, and linkage disequilibrium analysis was performed for antimicrobial resistance profiles, replicon types, and animal source. A high degree of genotypic diversity was observed: 34 different pulsed-field gel electrophoresis types among the 35 isolates examined. Twelve different plasmid Inc types were detected, and all isolates carried at least one replicon type. IncF (n = 25; 71.4%) and IncFIB (n = 19; 54.3%) were the most common replicon types identified. Chloramphenicol resistance was significantly linked with four Inc types (A/C, FIIA, F, and Y), and amoxicillin/clavulanic acid was linked with three Inc types (B/O, P and Y). Resistance to any other antimicrobial was linked to two or fewer replicon types. The isolate source was linked with resistance to seven antimicrobials and IncI1. We conclude that commensal E. coli from animal sources are highly variable genotypically and are reservoirs of a diverse array of plasmids carrying antimicrobial resistance.

Traditional plating methods are reliable means for Campylobacter identification from poultry samples but automated gene-based detection systems now available can reduce assay time, data collection and analysis. Bio-Rad and DuPont Qualicon recently introduced Campylobacter assays for their real-time polymerase chain reaction (PCR) instruments. We evaluated the utility of these assays compared with standard plating and enumeration methods routinely used in our laboratory. Two replicates of 40 broiler carcass rinses collected before and after defeathering at a commercial processing plant were tested. All samples were positive for Campylobacter by direct plating of rinses: log10 cfu values ranged from 0.24 to 4.61. In contrast, the Bio-Rad iQ-Check assay returned 60–72.5% positives on direct rinses; the Qualicon BAX Q7 test produced 60–85% positives with direct rinse samples. Using aliquots of 24 h enrichment broth from rinses in the real-time assays significantly improved detection: the Bio-Rad test had 95–100% positive while the Qualicon assay found 90–95% positive. PRACTICAL APPLICATIONS Traditional detection methods for Campylobacter are time-consuming, requiring as much as 5 days to complete. The recent introduction of commercial real-time PCR instruments for the identification of Campylobacter offer more rapid, simplified alternatives to standard culture-based techniques.

The embryonic stress hypothesis of teratogenesis suggests that a proportion of all human congenital defects is due to a failure in essential gene transcription along with translational pre-emption by the heat shock response (HSR). We sought to determine the potential usefulness of the murine HSR to screen agents suspected of being human teratogens. The teratogenic potential of a selected group of known teratogenic (hyperthermia, insulin, retinoic acid and valproic acid) or non-teratogenic (cycloheximide, dinitrophenol and tetracycline) agents were administered to pregnant SWV mice at critical periods of neural tube closure. Following exposure to either teratogenic doses or at the highest dose possible that did not induce maternal toxicity for those compounds that were not teratogenic, the induction of heat shock protein (hsp) synthesis and changes in total protein synthesis were determined in lymphocytes isolated from murine spleens. The varied results obtained in these studies cast doubt on the value of the murine HSR to screen teratogens.

Comparative genome indexing (CGI) using whole-genome DNA microarrays was evaluated as a means of genotyping Campylobacter jejuni relative to two standard methods, pulsed-field gel electrophoresis (PFGE) and flaA short variable region sequencing (flaA SVR typing). Thirty-six geographically diverse C. jejuni isolates were selected from a collection of cattle and chicken isolates. The BioNumerics software program was used for cluster analysis of the data from all 36 isolates for each of the three typing methods. Comparative genome indexing assigned a unique type to each isolate while PFGE and flaA SVR distinguished 29 and 35 different types, respectively. The four common types identified by PFGE were also closely related by CGI, and the overall similarity of the CGI results to those for PFGE indicates the value of CGI as a more informative alternative to PFGE. While flaA SVR was very discriminative, the isolates were all highly similar (78%) resulting in finer distinctions between isolates and fewer genotypic relations to CGI or PFGE. Campylobacter jejuni is one of the most common causative agents of bacterial gastroenteritis in the world. The development of CGI as a molecular typing tool for C. jejuni offers a highly effective and informative means of further understanding the epidemiology of this ubiquitous pathogen.

The genetic basis of macrolide resistance in Campylobacter coli (n = 17) and C. jejuni (n = 35) isolates previously subjected to in vivo selective pressure was investigated to determine if the number of copies of 23S rRNA genes with macrolide-associated mutations affects the minimum inhibitory concentration (MIC) of macrolides. Sequence data for domain V of the 23S rRNA gene revealed that two macrolide-resistant C. coli isolates had adenine-->guanine transitions at position 2059 (A2059G, Escherichia coli numbering). One of the two isolates had the A2059G transition in only two of the three gene copies. Among the macrolide-resistant C. jejuni isolates (n = 9), two different point mutations within domain V were observed. Three macrolide-resistant C. jejuni isolates had A2059G transitions. One of these three C. jejuni isolates had the A2059G transition in only two of the three gene copies. Six macrolide-resistant C. jejuni isolates had an adenine-->cytosine transversion at position 2058 (A2058C, E. coli numbering) in all three copies of the 23S rRNA gene. Campylobacter jejuni isolates with the A2058C transversion had higher erythromycin MICs (>256 microg/mL) compared to C. jejuni isolates with A2059G transitions (64-128 microg/mL). In addition, the C. jejuni and C. coli isolates with only two copies of the 23S rRNA gene having A2059G substitutions had lower macrolide MICs compared to isolates with all three copies of the gene mutated. No isolates were observed having only one copy of the 23S rRNA gene with a mutation. Sequence analysis of ribosomal proteins L4 (rplD) and L22 (rplV) indicated that ribosomal protein modifications did not contribute to macrolide resistance among the collection of Campylobacter examined.

Bovine alveolar macrophage metabolism of arachidonic acid (AA) via cyclooxygenase (CO) and lipoxygenase (LO) pathways is stimulus specific. Under standard conditions with RPMI-1640 media containing 0.4 mM Ca2+, the calcium ionophore A23187 stimulates the release of both CO and LO products, whereas opsonized zymosan and phorbol myristate acetate (PMA) selectively stimulate the CO pathway in these cells. We have examined the effect of varying the extracellular concentration of calcium (0–2.4 mM) on the profiles of AA metabolites secreted with differing stimuli. All stimuli caused the release of CO products, even in the absence of calcium in the media. The magnitude of release was correlated with increasing extracellular calcium concentrations, indicating some dependence of phospholipase activation on extracellular calcium. However, there were notable differences between stimuli regarding the magnitude of CO product formation and dependence on extracellular Ca2+. No 5-LO products were demonstrable with either zymosan or PMA at any concentration of extracellular calcium tested, and inhibition of CO by indomethacin did not result in 5-LO product formation for these stimuli. The production of 5-LO products in bovine alveolar macrophages by A23187 required extracellular calcium, demonstrating an absolute dependence for activation of the 5-LO pathway on an influx of extracellular calcium. Our results indicate that intracellular and extracellular Ca2+ have differing roles in the metabolism of AA down CO and LO pathways in bovine alveolar macrophages depending on the stimulus used. This regulation suggests that the pools of calcium required for activation of phospholipase A2 (PLA2) are not necessarily available for the 5-LO enzyme.

Aims: To obtain an overview of the prevalence and antimicrobial resistance of Campylobacter in faeces of US dairy cows in 2002. Methods and Results: Faeces from 1435 cows, representing 96 dairy operations in 21 US states, were collected for the culture of Campylobacter. A total of 735 Campylobacter strains were isolated (51·2% positive samples) with 94 operations positive (97·9%) for Campylobacter. From this collection, 532 isolates (473 Campylobacter jejuni and 59 Campylobacter coli) were randomly selected for susceptibility testing to eight antimicrobials: azithromycin, chloramphenicol, ciprofloxacin, clindamycin, erythromycin, gentamicin, nalidixic acid and tetracycline. The C. jejuni isolates exhibited resistance to tetracycline (47·4%), nalidixic acid (4·0%) and ciprofloxacin (2·5%), while the C. coli strains exhibited some resistance to all antimicrobials except chloramphenicol and ciprofloxacin. Only 3·6% of the C. jejuni isolates were resistant to two or more antimicrobials but 20·3% of the C. coli strains were multiresistant. Conclusions: On most operations, at least one cow was positive for Campylobacter and more than half of the cows sampled were shedding Campylobacter. The C. coli isolates had significantly higher levels of resistance to macrolides and to tetracycline compared with the C. jejuni strains, but were susceptible to ciprofloxacin. Significance and Impact of the Study: This study demonstrated a high prevalence of Campylobacter on US dairy operations; however, US dairy cattle have not been recognized as a major source of human infection compared with poultry. Campylobacter coli appears to develop antimicrobial resistance more readily than C. jejuni from the same environment.

Broiler carcasses ( n = 325) were sampled in a U.S. commercial poultry processing plant for the prevalence of Arcobacter and Campylobacter at three sites along the processing line: pre-scald, pre-chill and post-chill. Samples (75–125 broilers per site) were collected during five plant visits from August to October of 2004. Arcobacter was recovered from pre-scald carcasses more frequently (96.8%) than from pre-chill (61.3%) and post-chill carcasses (9.6%). Campylobacter was isolated from 92% of pre-scald carcasses, 100% of pre-chill carcasses, and 52% of post-chill carcasses. In total, Arcobacter was isolated from 55.1% (179 of 325), while Campylobacter was isolated from 78.5% (255 of 325) of the carcasses from the three collection sites. For Arcobacter identification, a species-specific multiplex PCR showed that A. butzleri was the most prevalent species (79.1%) followed by A. cryaerophilus 1B (18.6%). A. cryaerophilus 1A was found at low levels (2.3%). PCR identified the most common Campylobacter species as C. jejuni (87.6%) followed by C. coli (12.4%). Overall, significant contamination of broiler carcasses by Arcobacter was observed, although less than that found for Campylobacter . From pre-scald to post-chill, a far greater reduction in Arcobacter numbers was observed than for Campylobacter . Our results for Arcobacter , obtained from the same environment as the closely related pathogen Campylobacter , will aid in the development of control measures for this emerging pathogen.

Campylobacter coli is a food-borne pathogen associated increasingly with human gastroenteritis. C. coli has a high prevalence in swine, but is isolated also from cattle and poultry. Multilocus sequence typing (MLST) systems have been developed to differentiate C. coli strains. Although substantial allelic diversity was identified across all seven C. coli MLST loci, no correlations were made in two previous studies between allele or sequence type (ST) and the source of the organism. However, this may be due to either the relatively small number or the low diversity of C. coli strains used to validate both MLST studies. This study describes the typing of 488 C. coli strains from 4 different food animal sources (cattle, chickens, swine and turkeys), collected at different times over a 6 year period from different USA geographical locations. A total of 149 STs were identified. The 185 swine strains were the most diverse, possessing 82 STs. The cattle strains were the most clonal; 52/63 (83 %) strains possessed a single ST (ST-1068). A subpopulation of C. coli strains, collected primarily from turkeys, was identified, containing both C. coli- and Campylobacter jejuni-associated MLST alleles, specifically the C. jejuni allele aspA103. The majority of STs and alleles were host associated, i.e. found primarily in strains from a single food-animal source. Only 12/149 (8 %) STs were found in multiple sources. Additionally, the majority (34/46, 74 %) of major (n>5) alleles were more prevalent in certain hosts (swine, poultry). The presence of host-associated C. coli MLST alleles could lead potentially to more efficient source tracking in this species, especially in the trace-back of both sporadic and outbreak human clinical C. coli strains to animal sources.

M.D. ENGLEN, P.J. FEDORKA-CRAY, S.R. LADELY AND D.A. DARGATZ. 2005. Aims: This study examined 448 Campylobacter strains isolated in 1999 and 2000 from US feedlot cattle for resistance to 12 antimicrobials. Methods and Results: Isolates were tested for antimicrobial susceptibility using the E-test method. Approximately 60% (n ¼ 267) were resistant to one or more antimicrobials, and 19AE6% (n ¼ 88) were resistant to two or more antimicrobials. Of the Campylobacter jejuni isolates, 49AE1% (n ¼ 187) were resistant to tetracycline, 10AE2% (n ¼ 39) were resistant to nalidixic acid, 8AE4% were resistant to trimethoprim/sulfamethoxazole, and 1AE8% (n ¼ 7) were resistant to ciprofloxacin. Resistance to any of the other eight antimicrobials was 1AE3% or less, but 14AE4% (n ¼ 55) were resistant to two or more antimicrobials. In the Campylobacter coli group, 65AE7% (n ¼ 44) were resistant to tetracycline, 52AE2% (n ¼ 35) were resistant to trimethoprim/sulfamethoxazole, 22AE4% (n ¼ 15) were resistant to nalidixic acid, and 9AE0% (n ¼ 6) were resistant to ciprofloxacin. Resistance to any of the remaining eight antimicrobials was 3AE0% or less, although 49AE3% (n ¼ 33) were resistant to two or more antimicrobials. Conclusions: Although antimicrobials are widely used in US feedlot cattle production, our results demonstrate generally low levels of resistance to a broad range of commonly used antimicrobials relative to other recent studies. Significance and Impact of the Study: Resistance data on Campylobacter isolated from this major US livestock commodity is lacking. This overview enhances current knowledge and provides a basis for further studies.

Use of antimicrobials has increased in both human and veterinary medicine and the emergence of resistance to antimicrobials has become a global problem. This is due, in part, to the widespread availability of antimicrobials and the efficacy they impart in control of certain infectious diseases. Antimicrobial resistance (AR) can diminish the effectiveness or render an antimicrobic ineffective as a therapeutic. Although use may result in bacteria (both food borne and commensal) that are resistant, the exact fate of these populations in terms of persistence and transmission has been difficult to determine. Use patterns in veterinary medicine (therapeutic vs. subtherapeutic use) and agriculture further complicates the picture. Additionally, while transmission of resistant bacteria from animals to humans occurs, it has been difficult to assess the extent to which this occurs and the impact transmission has on actually disseminating resistant populations among humans.

Modification of the silica surface has been shown to reduce its cytotoxicity in vitro and its fibrogenic activity in vivo. We have shown silica to be a potent stimulator of arachidonic acid (AA) metabolism in bovine alveolar macrophages (BAM). To determine the effect of surface-modified silica on AA metabolism in BAM, we exposed BAM in vitro to silica treated with aluminum lactate or polyvinylpyridine-N-oxide (PVPNO). BAM were prelabeled with [3H]AA and incubated with 3 and 5 mg of silica. Unmodified silica at these doses elicited maximal AA metabolite release from BAM. AA metabolites were analyzed by high performance liquid chromatography. Lactate dehydrogenase release was quantitated to determine the cytotoxicity of treated and untreated silica on BAM. Treating silica with aluminum lactate or PVPNO significantly (P less than or equal to 0.05) reduced 5-lipoxygenase metabolite release and significantly (P less than or equal to 0.05) increased cyclooxygenase metabolite release. These changes in AA metabolite release were accompanied by a significant (P less than or equal to 0.05) reduction in the cytotoxicities of the treated silicas compared with untreated silica. Our results suggest that the reduced inflammatory and fibrogenic activity of surface-modified silica may in part be due to reduced AA metabolite release from exposed macrophages.

Lymphocytes isolated from two inbred mouse strains that differed in their genetically determined sensitivity to heat-induced exencephaly were used to compare the in vitro kinetics of heat shock protein synthesis in the two strains following hyperthermic exposure. Differences in protein synthesis were determined by densitometric analysis of autoradiograms of SDS-PAGE gels. The findings were consistent with those observed in vivo in that there was an immediate and prolonged synthesis of heat-shock proteins by lymphocytes from the heat sensitive SWV/SD strain, compared to the response observed in lymphocytes from the heat-resistent DBA/2J strain. These results indicate that an in vitro lymphocyte assay of the heat-shock response may be a useful tool for screening suspected teratogenic agents.

Neural tube defects are common congenital anomalies affecting approximately 1–2 per 1000 liveborn infants (Nakano 1973; Richards et al. 1972). Empiric risk figures, along with numerous clinical studies, indicate that neural tube defects are of a multifactorial origin, having both a genetic and an environmental component to their development (Martin et al. 1983; Holmes et al. 1976; Campbell et al. 1986). Many environmental factors have been implicated as inducers of neural tube defects in humans, including maternal vitamin and folate deficiencies (Smithells 1982), hyperthermia (Edwards et al. 1981, 1986; Fisher and Smith 1981), valproic acid (Bjerkedal et al. 1982; Mastroiacovo et al. 1983; Lindhout and Schmidt 1986) and retinoids (Keitzmann et al. 1986). Although several genetic hypotheses have been put forth to explain the inheritance of neural defects, no single hypothesis fits all of the data currently available.

Bovine alveolar macrophages (BAM) prelabeled with 3H-arachidonic acid (AA) were exposed in vitro to different doses of DQ-12, Minusil-5, and Sigma silicas, or carbonyl iron beads. Arachidonic acid metabolites released into the culture medium by BAM were identified and quantitated using high performance liquid chromatography (HPLC). Cytotoxicity was assessed by the release of lactate dehydrogenase (LDH). At doses of 0.1 or 0.25 mg of DQ-12 silica and of 0.25 or 0.5 mg of Minusil-5 and Sigma silica, the release of cyclooxygenase metabolites (TXB2, PGE2, PGF2, and HHT) comprised greater than 95% of the total released AA metabolites. Silica doses above 0.5 mg led to 5-lipoxygenase metabolite release (LTB4, its two nonenzymatic isomers, and 5-HETE). This shift to 5-lipoxygenase metabolite release paralleled increased cellular cytotoxicity and was observed for each of the silicas. In contrast to silica stimulation, carbonyl iron beads elicited only small quantities of cyclooxygenase metabolites, no 5-lipoxygenase metabolites, and showed little cytotoxicity toward BAM. The relative potency of each particulate for stimulating the release of AA metabolites and LDH was calculated with DQ-12 greater than Minusil-5 greater than Sigma much greater than carbonyl iron beads. Our results indicate that the cytotoxic and presumed fibrogenic potential of a silica may be correlated with the potency to stimulate the release of 5-lipoxygenase metabolites from AM.

The in vitro release of arachidonic acid (AA) metabolites from caprine alveolar macrophages (CAM) stimulated with the calcium ionophore A23187 or opsonized zymosan was examined. Leukotriene B4 [5(S),12(R)-6,14-cis-8,10-trans-dihydroxyeicosatetraenoic acid] was the major AA metabolite elicited with either agonist; smaller amounts of 12- and 5-mono-hydroxyeicosatetraenoic acid (HETE), and 12-hydroxyheptadecatrienoic acid (HHT) were also detected. Zymosan stimulation also caused the release of very small quantities of prostaglandins E2 and F2 alpha, and thromboxane B2. Our report is the first to describe arachidonic acid metabolism in CAM.

Alveolar macrophages (AMs) are capable of producing a variety of inflammatory mediators including those derived from arachidonic acid, the prostaglandins (PGs), leukotrienes (LTs) and hydroxyeicosatetraenoic acids (HETEs). Inflammation associated with release of arachidonate-derived mediators is a result of the combined actions of all of these mediators. Thus, it is critical to determine the entire spectrum of arachidonate-derived metabolites that AMs are capable of producing. In this study bovine AMs were prelabeled with [3H]arachidonic acid prior to stimulation with serum-treated zymosan, phorbol myristate acetate (PMA), or the calcium ionophore A23187. The total release of arachidonate metabolites into the culture media was measured by reverse-phase HPLC with on-line radiometric detection. All stimuli used induced production of metabolites of the cyclooxygenase pathway with thromboxane B2 and HHT being the major metabolites. Lesser amounts of PGF2 alpha, PGE2, and PGD2 were produced. Only stimulation with A23187 resulted in production of LTB4 and 5-HETE, products of the 5-lipoxygenase pathway. This latter result indicates that the two major pathways of arachidonate metabolism in AMs may be selectively stimulated. Such an effect could have important consequences in the development of pulmonary inflammation. Furthermore, the spectrum of arachidonic acid metabolites produced by bovine AMs closely resembles that of human AMs, in contrast to rodent AMs.