Transcriptional Regulation of icaADBC by both IcaR and TcaR in Staphylococcus epidermidis.

S. epidermidis is a primary cause of biofilm-mediated infections in humans due to adherence to foreign bodies. A major staphylococcal biofilm accumulation molecule is polysaccharide intracellular adhesin (PIA), which is synthesized by enzymes encoded by the icaADBC operon. Expression of PIA is highly variable among clinical isolates, suggesting that PIA expression levels are selected in certain niches of the host. However, the mechanisms that govern enhanced icaADBC transcription and PIA synthesis in these isolates are not known. We hypothesized that enhanced PIA synthesis in these isolates was due to function of IcaR and/or TcaR. Thus, two S. epidermidis isolates (1457 and CSF41498) with different icaADBC transcription and PIA expression levels were studied. Constitutive expression of both icaR and tcaR demonstrated that both repressors are functional and can completely repress icaADBC transcription in both 1457 and CSF41498. However, it was found that IcaR was the primary repressor for CSF41498 and TcaR was the primary repressor for 1457. Further analysis demonstrated that icaR transcription was repressed in 1457 in comparison to CSF41498, suggesting that TcaR functions as a repressor only in the absence of IcaR. Indeed, DNase I footprinting suggests IcaR and TcaR may bind to the same site within the icaR-icaA intergenic region. Lastly, we found mutants expressing variable amounts of PIA could rapidly be selected from both 1457 and CSF41498. Collectively, we propose that strains producing enhanced PIA synthesis are selected within certain niches of the host through several genetic mechanisms that function to repress icaR transcription, thus increasing PIA synthesis.IMPORTANCEStaphylococcus epidermidis is a commensal bacterium that resides on our skin. As a commensal, it protects humans from bacterial pathogens through a variety of mechanisms. However, it is also a significant cause of biofilm infections due to its ability to bind to plastic. Polysaccharide intercellular adhesin is a significant component of biofilm, and we propose that the expression of this polysaccharide is beneficial in certain host niches, such as providing extra strength when the bacterium is colonizing the lumen of a catheter, and detrimental in others, such as colonization of the skin surface. We show here that fine-tuning of icaADBC transcription, and thus PIA synthesis, is mediated via two transcriptional repressors, IcaR and TcaR.

Efficacy of oral meloxicam suspension for prevention of pain and inflammation following band and surgical castration in calves.

BACKGROUND: Castration is one of the most common procedures performed on beef and dairy cattle. The objective of the study was to determine the efficacy of meloxicam oral suspension in reducing pain and inflammation in calves following band or surgical castration. METHODS: Two identical trials with the exception of the method of castration (Band Castration Study 1 and Surgical Castration Study 2) were conducted. Sixty (60) healthy Holstein calves 4 to 5 months of age (138-202 Kg) were used. Animals received either Meloxicam Oral Suspension at a dose of 1 mg/kg BW (n = 15 Study 1 and 15 Study 2) or Saline (n = 15 Study 1 and 15 Study 2) 2 h before castration. Physiological (Heart Rate, Plasma Cortisol and Plasma Substance P) and Behavioral (Visual Analog Scale (VAS), Accelerometers and tail Pedometers) evaluations were conducted before (day -1) and after Castration (Day 0, 1, 2, 3). Inflammation was evaluated daily by providing an individual animal score (Study1) or with a measurement of scrotal thickness (Study 2). RESULTS: Heart rates were significantly greater in control animals following band and surgical castration. Plasma cortisol and substance P were significantly reduced in animals receiving Meloxicam Oral Suspension. Control animals had significantly greater VAS scores. Accelerometers showed that meloxicam treated animals had a significantly greater motion index and number of steps as well as less % time lying and number of lying bouts. The scrotal inflammation (based on scrotal swelling) was significantly decreased in the meloxicam treated animals compared to the control animals on day 1, day 2 and 3. CONCLUSION: Meloxicam Oral Suspension was able to significantly reduce the display of painful behaviors and physiological responses to pain in band castrated and surgical castrated calves for up to 72 h following a single oral treatment of 1 mg/kg body weight. Meloxicam Oral Suspension was able to significantly reduce scrotal inflammation in band castrated and surgical castrated calves.

World Association for the Advancement of Veterinary Parasitology (WAAVP): Guideline for the evaluation of drug efficacy against non-coccidial gastrointestinal protozoa in livestock and companion animals.

The current guideline was written to aid in the design, implementation and interpretation of studies for the assessment of drug efficacy against non-coccidial gastrointestinal protozoan parasites, with Giardia spp. as the leading example. The information provided in this guideline deals with aspects of how to conduct controlled studies using experimental infection models (dose determination and dose confirmation) and efficacy studies in commercial facilities (field effectiveness studies). Furthermore, the selection of suitable animals, housing, infection procedure, choice of diagnostic technique and data analysis are discussed. This guideline is intended to assist investigators in conducting specific studies, to provide specific information for registration authorities involved in the decision-making process, to assist in the approval and registration of new drugs and to facilitate the worldwide adoption of uniform procedures. The primary parameter for drug efficacy is the reduction in either parasite excretion or parasite counts and a minimum efficacy of 90% is required against non-coccidial gastrointestinal protozoa. A supporting efficacy parameter is a significant difference in the proportion of infected animals between treated and non-treated groups. Persistent efficacy is considered as an additional claim to therapeutic efficacy.

Ruggedness and reproducibility of the MBEC biofilm disinfectant efficacy test.

The MBEC™ Physiology & Genetics Assay recently became the first approved ASTM standardized biofilm disinfectant efficacy test method. This report summarizes the results of the standardization process using Pseudomonas aeruginosa biofilms. Initial ruggedness testing of the MBEC method suggests that the assay is rugged (i.e., insensitive) to small changes to the protocol with respect to 4 factors: incubation time of the bacteria (when varied from 16 to 18h), treatment temperature (20-24°C), sonication duration (25-35min), and sonication power (130-480W). In order to assess the repeatability of MBEC results across multiple tests in the same laboratory and the reproducibility across multiple labs, an 8-lab study was conducted in which 8 concentrations of each of 3 disinfectants (a non-chlorine oxidizer, a phenolic, and a quaternary ammonium compound) were applied to biofilms using the MBEC method. The repeatability and reproducibility of the untreated control biofilms were acceptable, as indicated by small repeatability and reproducibility standard deviations (SD) (0.33 and 0.67 log10(CFU/mm(2)), respectively). The repeatability SDs of the biofilm log reductions after application of the 24 concentration and disinfectant combinations ranged from 0.22 to 1.61, and the reproducibility SDs ranged from 0.27 to 1.70. In addition, for each of the 3 disinfectant types considered, the assay was statistically significantly responsive to the increasing treatment concentrations.

Arginine deiminase in Staphylococcus epidermidis functions to augment biofilm maturation through pH homeostasis.

Allelic replacement mutants were constructed within arginine deiminase (arcA1 and arcA2) to assess the function of the arginine deiminase (ADI) pathway in organic acid resistance and biofilm formation of Staphylococcus epidermidis 1457. A growth-dependent acidification assay (pH ∼5.0 to ∼5.2) determined that strain 1457 devoid of arginine deiminase activity (1457 ΔADI) was significantly less viable than the wild type following depletion of glucose and in the presence of arginine. However, no difference in viability was noted for individual 1457 ΔarcA1 (native) or ΔarcA2 (arginine catabolic mobile element [ACME]-derived) mutants, suggesting that the native and ACME-derived ADIs are compensatory in S. epidermidis. Furthermore, flow cytometry and electron paramagnetic resonance spectroscopy results suggested that organic acid stress resulted in oxidative stress that could be partially rescued by the iron chelator dipyridyl. Collectively, these results suggest that formation of hydroxyl radicals is partially responsible for cell death via organic acid stress and that ADI-derived ammonia functions to counteract this acid stress. Finally, static biofilm assays determined that viability, ammonia synthesis, and pH were reduced in strain 1457 ΔADI following 120 h of growth in comparison to strain 1457 and the arcA1 and arcA2 single mutants. It is hypothesized that ammonia synthesis via the ADI pathway is important to reduce pH stress in specific microniches that contain high concentrations of organic acids.

Cryptosporidium andersoni in Western Australian feedlot cattle.

Cryptosporidium andersoni has not been previously reported in feedlot beef cattle in Western Australia. Faecal samples were collected from 10 groups of cattle ranging in age from 11 to 36 months in five different feedlots in Western Australia. The incidence of C. andersoni ranged from 0% to 26%. There were no clinical signs associated with C. andersoni infection, but there was a significant reduction in rate of gain of 0.44 kg in infected animals compared with negative pen mates. Cryptosporidium andersoni is characterised by large oocysts (7.4 x 5.5 µm) and was confirmed by 18S sequencing.

Giardia and Cryptosporidium in harp and hooded seals from the Gulf of St. Lawrence, Canada.

Giardia and Cryptosporidium are protozoan parasites known to cause enteric disease in terrestrial wildlife species (mammals, reptiles and birds). Few surveys for Giardia and Cryptosporidium in marine wildlife species, such as pinnipeds, have been reported. The objective of this study was to determine the prevalence and genotype of Giardia and Cryptosporidium in two species of pinnipeds, harp seal (Phoca groenlandica) and hooded seal (Cystophora cristata), from the Gulf of St. Lawrence, Canada. Faecal samples were collected from pup and adult seals and examined for the presence of cysts of Giardia and oocysts of Cryptosporidium using microscopy and immunofluorescent staining. Tissues from the small intestine of adult seals were also collected and examined for infections using the polymerase chain reaction (PCR) technique. Giardia cysts were found in the faeces of 42% (16/38) of adult harp seals, but in none of the harp seal pups (0/20). Although Giardia cysts were not detected in faeces of adult hooded seals (0/10) using microscopy, 80% tested positive for Giardia using PCR of intestinal tissue indicative of a true replicating infection. Both harp and hooded seals harboured infections with the zoonotic strain, Giardia duodenalis Assemblage A, as determined using a nested-PCR technique to amplify a small subunit ribosomal (SSU-rRNA) gene of Giardia. Cryptosporidium was not detected by microscopy, nor using the PCR technique on intestinal tissues from any of the 68 seals examined.

Effect of subtherapeutic administration of antibiotics on the prevalence of antibiotic-resistant Escherichia coli bacteria in feedlot cattle.

Antibiotic-resistant Escherichia coli in 300 feedlot steers receiving subtherapeutic levels of antibiotics was investigated through the collection of 3,300 fecal samples over a 314-day period. Antibiotics were selected based on the commonality of use in the industry and included chlortetracycline plus sulfamethazine (TET-SUL), chlortetracycline (TET), virginiamycin, monensin, tylosin, or no antibiotic supplementation (control). Steers were initially fed a barley silage-based diet, followed by transition to a barley grain-based diet. Despite not being administered antibiotics prior to arrival at the feedlot, the prevalences of steers shedding TET- and ampicillin (AMP)-resistant E. coli were >40 and <30%, respectively. Inclusion of TET-SUL in the diet increased the prevalence of steers shedding TET- and AMP-resistant E. coli and the percentage of TET- and AMP-resistant E. coli in the total generic E. coli population. Irrespective of treatment, the prevalence of steers shedding TET-resistant E. coli was higher in animals fed grain-based compared to silage-based diets. All steers shed TET-resistant E. coli at least once during the experiment. A total of 7,184 isolates were analyzed for MIC of antibiotics. Across antibiotic treatments, 1,009 (13.9%), 7 (0.1%), and 3,413 (47.1%) E. coli isolates were resistant to AMP, gentamicin, or TET, respectively. In addition, 131 (1.8%) and 143 (2.0%) isolates exhibited potential resistance to extended-spectrum beta-lactamases, as indicated by either ceftazidime or cefpodoxime resistance. No isolates were resistant to ciprofloxacin. The findings of the present study indicated that subtherapeutic administration of tetracycline in combination with sulfamethazine increased the prevalence of tetracycline- and AMP-resistant E. coli in cattle. However, resistance to antibiotics may be related to additional environmental factors such as diet.

Staphylococcus epidermidis saeR is an effector of anaerobic growth and a mediator of acute inflammation.

The saeRS two-component regulatory system regulates transcription of multiple virulence factors in Staphylococcus aureus. In the present study, we demonstrated that the saePQRS region in Staphylococcus epidermidis is transcriptionally regulated in a temporal manner and is arranged in a manner similar to that previously described for S. aureus. Studies using a mouse foreign body infection model demonstrated that the virulence of strain 1457 and the virulence of a mutant, strain 1457 saeR, were statistically equivalent. However, histological analyses suggested that the polymorphonuclear neutrophil response at 2 days postinfection was significantly greater in 1457-infected mice than in 1457 saeR-infected mice, demonstrating that SaeR influences the early, acute phases of infection. Microarray analysis demonstrated that a saeR mutation affected the transcription of 65 genes (37 genes were upregulated and 28 genes were downregulated); in particular, 8 genes that facilitate growth under anaerobic conditions were downregulated in 1457 saeR. Analysis of growth under anaerobic conditions demonstrated that 1457 saeR had a decreased growth rate compared to 1457. Further metabolic experiments demonstrated that 1457 saeR had a reduced capacity to utilize nitrate as a terminal electron acceptor and exhibited increased production of lactic acid in comparison to 1457. These data suggest that in S. epidermidis SaeR functions to regulate the transition between aerobic growth and anaerobic growth. In addition, when grown anaerobically, 1457 saeR appeared to compensate for the redox imbalance created by the lack of electron transport-mediated oxidation of NADH to NAD+ by increasing lactate dehydrogenase activity and the subsequent oxidation of NADH.

Molecular analysis of Francisella tularensis subspecies tularensis and holarctica.

Rapid methods are needed for public health and military applications to specifically identify Francisella tularensis, the causative agent of tularemia in humans. A comparative analysis of the capabilities of multiple technologies was performed using a well-defined set of organisms to determine which approach would provide the most information in the shortest time. High-resolution molecular techniques, including pulsed-field gel electrophoresis, amplified fragment length polymorphism, and ribotyping, provided subspecies level identification within approximately 24 hours after obtaining an isolate, whereas multilocus variable number tandem repeat analysis with 8 or 25 targets provided strain level discrimination within about 12 hours. In contrast, Raman spectroscopy provided species level identification in 10 minutes but could not differentiate between subspecies tularensis and holarctica.

Genomic deletion marking an emerging subclone of Francisella tularensis subsp. holarctica in France and the Iberian Peninsula.

Francisella tularensis subsp. holarctica is widely disseminated in North America and the boreal and temperate regions of the Eurasian continent. Comparative genomic analyses identified a 1.59-kb genomic deletion specific to F. tularensis subsp. holarctica isolates from Spain and France. Phylogenetic analysis of strains carrying this deletion by multiple-locus variable-number tandem repeat analysis showed that the strains comprise a highly related set of genotypes, implying that these strains were recently introduced or recently emerged by clonal expansion in France and the Iberian Peninsula.

SigmaB and SarA independently regulate polysaccharide intercellular adhesin production in Staphylococcus epidermidis.

The production of polysaccharide intercellular adhesin (PIA) is an essential process in foreign body infections mediated by Staphylococcus epidermidis. Transcriptional regulation of the icaADBC operon, the genes responsible for production of enzymes that synthesize PIA, is multi-factorial and involves at least SarA and sigmaB. Transcriptional and promoter fusion studies revealed that the decreased transcription of the icaADBC operon observed in a S. epidermidis 1457 sigB mutant is not mediated through a direct interaction of sigmaB-RNA polymerase at the icaADBC promoter region but instead through the upregulation of IcaR, a known repressor of icaADBC transcription. Transcriptional analysis of a 1457 sigB-icaR double mutant confirmed that the decreased icaADBC transcript in 1457 sigB is IcaR dependent. Furthermore, primer extension studies suggest that the icaR promoter appears to be sigmaA dependent, suggesting that sigmaB indirectly controls icaR transcription through an unknown pathway. In addition, it was confirmed that the loss of SarA results in the loss of icaADBC transcription and PIA production in S. epidermidis. It was further demonstrated, through the over-production of SarA in 1457 sigB, that the loss of sarP1 promoter activity in 1457 sigB has little or no effect on the loss of PIA production in this mutant. Finally, it was demonstrated that PIA production could be restored in both 1457 sigB and 1457 sarA by complementing these mutants with a full-length icaADBC operon controlled by a cadmium-inducible noncognate promoter. It is concluded that sigmaB and SarA operate independently of each other to regulate PIA production and biofilm development in S. epidermidis.

Adherence of Staphylococcus epidermidis to biomaterials is augmented by PIA.

Staphylococcus epidermidis is the most common cause of orthopaedic prosthetic device infections. Polysaccharide intercellular adhesin (PIA) is important in the pathogenesis of intravascular catheter-associated infection, and has an essential role in cellular aggregation and biofilm formation. However, the role of PIA in orthopaedic infections is less well understood. We used genetically defined strains of S. epidermidis in an in vitro adherence assay to assess the importance of PIA in the adherence to various orthopaedic biomaterials. On all biomaterials tested (zirconia, ultra-high molecular weight polyethylene, polymethylmethacrylate, cobalt chromium, titanium, stainless steel, and silastic), PIA-positive S. epidermidis 1457 exhibited greater levels of adherence thanS. epidermidis 1457 M10, an isogenic icaA Tn917 mutant. PIA appears to play a critical role in the adherence of S. epidermidis to orthopaedic biomaterials, and may serve as an important virulence determinant in orthopaedic prosthetic device infections.

Temporal prevalence of antimicrobial resistance in Campylobacter spp. from beef cattle in Alberta feedlots.

Antimicrobial resistance (AMR) was temporally assessed in campylobacters isolated from beef cattle (7,738 fecal samples from 2,622 animals) in four commercial feedlots in Alberta. All calves were administered chlortetracycline and oxytetracycline in feed, and a majority of the animals (93%) were injected with long-acting oxytetracycline upon arrival at the feedlot. Fecal samples from individual animals were collected upon arrival (i.e., entry sample), 69 days (standard deviation [SD] = 3 days) after arrival (i.e., interim sample), and 189 days (SD = 33 days) after arrival (i.e., exit sample) at the feedlot. In total, 1,586 Campylobacter isolates consisting of Campylobacter coli (n = 154), Campylobacter fetus (n = 994), Campylobacter jejuni (n = 431), Campylobacter hyointestinalis (n = 4), and Campylobacter lanienae (n = 3) were recovered and characterized. The administration of antimicrobials did not decrease carriage rates of campylobacters, and minimal resistance (< or =4%) to azithromycin, ciprofloxacin, enrofloxacin, gentamicin, and meropenem was observed. In contrast, substantive increases in the prevalence of isolates resistant to tetracycline and doxycycline (56 to 89%) for C. coli, C. fetus, and C. jejuni, as well as in the number of animals (7 to 42%) from which resistant isolates were recovered, were observed during the feedlot period. Increased resistance to erythromycin (total isolates and carriages rates) was also observed in isolates of C. coli over the three isolation times. The majority of C. fetus isolates recovered were resistant to nalidixic acid, but this was independent of when they were isolated. A relatively limited number of multidrug-resistant isolates were recovered and consisted primarily of C. coli resistant to tetracyclines and erythromycin (10% of isolates). Over the course of the feedlot period, considerable increases in antimicrobial resistance were observed in C. coli, C. fetus, and C. jejuni, but with the exception of erythromycin resistance in C. coli, the administration of antimicrobial agents to beef cattle was found to have a minimal impact on resistance to macrolides and fluoroquinolones, the two classes of antimicrobials used to treat campylobacteriosis in humans. However, the widespread use of antimicrobial agents in beef production and the possible horizontal transfer of mobile genetic elements with antimicrobial resistance determinants among Campylobacter and other bacterial taxa emphasize the need to monitor AMR development in bacteria from beef cattle.

Skin wound healing in the first generation (F1) offspring of Yorkshire and red Duroc pigs: evidence for genetic inheritance of wound phenotype.

Fibroproliferative scars in humans often demonstrate familial inheritance patterns, and genetics may contribute to healing and scarring. Genetic factors may also influence the scarring phenotype in a porcine model. Healing of full thickness excisional skin wounds in Yorkshire pigs closely resembles normal healing in humans, while identical wounds in red Duroc pigs form hypercontracted, hyperpigmented scars. The present study has evaluated the healing process in the first generation cross (F1) of red Duroc and Yorkshire pigs. Gross and histologic analysis revealed that the F1 animals exhibit an intermediate healing phenotype, with some features of each parent breed. F1 full thickness wounds were significantly hypercontracted and fibrotic, but apigmented. Analysis of mRNA expression patterns for a panel of relevant molecules (N=32) in the F1 animals revealed some similarities to each parent breed, as well as unique patterns for other molecules. Furthermore, a depth dependency to the healing response was observed at the gross, histologic, and molecular levels, with deep dermal wounds healing similar to Yorkshire wounds. These findings suggest that the genetic contribution to scar phenotype in this animal model is complex. However, the results indicate that further understanding in this model may provide insights into risk factors for hypertrophic scarring in human burn patients.

Molecular and morphological characterization of Echinococcus in cervids from North America.

Many issues concerning the taxonomy of Echinococcus have been resolved in recent years with the application of molecular tools. However, the status of Echinococcus maintained in transmission cycles involving cervid intermediate hosts remains to be determined. The recent characterization of the parasite from cervids in Finland has highlighted the paucity of data available, particularly that from North America. In this study, we have characterized a large number of Echinococcus isolates from cervids from Western Canada on the basis of morphology and molecular genetic techniques. Our results support earlier studies suggesting that Echinococcus of cervid origin is phenotypically and genetically distinct to Echinococcus maintained in domestic host assemblages, and also confirms that Echinococcus of cervid origin does not constitute a genetically homogeneous group. However, our data do not support the existence of 2 distinct genotypes (strains/subspecies) with separate geographical distributions. Our data appear to support the existence of only 1 species in cervids, but additional isolates from cervids and wolves in other endemic regions should be characterized before a final decision is made on the taxonomic status of Echinococcus in cervids.

Cryptosporidium and cryptosporidiosis.

Cryptosporidium is one of the most common enteric protozoan parasites of vertebrates with a wide host range that includes humans and domestic animals. It is a significant cause of diarrhoeal disease and an ubiquitous contaminant of water which serves as an excellent vehicle for transmission. A better understanding of the development and life cycle of Cryptosporidium, and new insights into its phylogenetic relationships, have illustrated the need to re-evaluate many aspects of the biology of Cryptosporidium. This has been reinforced by information obtained from the recent successful Cryptosporidium genome sequencing project, which has emphasised the uniqueness of this organism in terms of its parasite life style and evolutionary biology. This chapter provides an up to date review of the biology, biochemistry and host parasite relationships of Cryptosporidium.

Effects of subtherapeutic administration of antimicrobial agents to beef cattle on the prevalence of antimicrobial resistance in Campylobacter jejuni and Campylobacter hyointestinalis.

The influence of antimicrobial agents on the development of antimicrobial resistance (AMR) in Campylobacter isolates recovered from 300 beef cattle maintained in an experimental feedlot was monitored over a 315-day period (11 sample times). Groups of calves were assigned to one of the following antimicrobial treatments: chlortetracycline and sulfamethazine (CS), chlortetracycline alone (Ct), virginiamycin, monensin, tylosin phosphate, and no antimicrobial agent (i.e., control treatment). In total, 3,283 fecal samples were processed for campylobacters over the course of the experiment. Of the 2,052 bacterial isolates recovered, 92% were Campylobacter (1,518 were Campylobacter hyointestinalis and 380 were C. jejuni). None of the antimicrobial treatments decreased the isolation frequency of C. jejuni relative to the control treatment. In contrast, C. hyointestinalis was isolated less frequently from animals treated with CS and to a lesser extent from animals treated with Ct. The majority (> or =94%) of C. jejuni isolates were sensitive to ampicillin, erythromycin, and ciprofloxacin, but more isolates with resistance to tetracycline were recovered from animals fed Ct. All of the 1,500 isolates of C. hyointestinalis examined were sensitive to ciprofloxacin. In contrast, 11%, 10%, and 1% of these isolates were resistant to tetracycline, erythromycin, and ampicillin, respectively. The number of animals from which C. hyointestinalis isolates with resistance to erythromycin and tetracycline were recovered differed among the antimicrobial treatments. Only Ct administration increased the carriage rates of erythromycin-resistant isolates of C. hyointestinalis, and the inclusion of CS in the diet increased the number of animals from which tetracycline-resistant isolates were recovered. The majority of C. hyointestinalis isolates with resistance to tetracycline were obtained from cohorts within a single pen, and most of these isolates were recovered from cattle during feeding of a forage-based diet as opposed to a grain-based diet. The findings of this study show that the subtherapeutic administration of tetracycline, alone and in combination with sulfamethazine, to feedlot cattle can select for the carriage of resistant strains of Campylobacter species. Considering the widespread use of in-feed antimicrobial agents and the high frequency of beef cattle that shed campylobacters, the development of AMR should be monitored as part of an on-going surveillance program.

Individual intake and antiparasitic efficacy of free choice mineral containing fenbendazole for grazing steers.

A 95-day study (June 25-September 27, 2001) was conducted using 120 steers (311.9+/-2.4 kg) randomly allocated to two treatments: (1) mineral containing 0.55% fenbendazole (FBZ) and (2) control, no FBZ in the mineral. Animals in the FBZ group were individually identified by an electronic tag that was read each time an animal attended the mineral feeder. The feeder was equipped with load cells that enabled individual mineral intakes to be estimated. The FBZ group was provided with non-medicated mineral during a 14-day adaptation period (July 23-August 5) and an 8-day post-medication period (September 17-24). The intake of FBZ was monitored for 14 days during each of the two treatment periods; August 6-19 and September 3-16, separated by a 14-day non-medicated period, August 20-September 2. Control animals had access to non-medicated mineral for the entire 95-day study period. All steers were grazed on alfalfa-grass pasture for the duration of the study and had free access to flocculated, filtered and chlorinated water via an automatic waterer. Fecal samples were collected from steers three times during the experiment July 23, August 27 and September 27, and analyzed for nematode eggs and Giardia sp. cysts. Seventy-five and 83% of the steers in the FBZ group visited the mineral feeder during the first and second treatment periods, respectively. Individual daily mineral and FBZ intake for the first and second treatment periods was 52.9+/-6.6g per day and 10.1+/-1.2mg/kg BW; 72.3+/-8.4 g per day and 11.8+/-1.4 mg/kg BW, respectively. FBZ animals were separated into three groups during each treatment period based on the recommended dose (RD) of FBZ (5 mg/kg/BW), those that received > the RD, those that received < RD but > 50% RD and those that received < 50% of RD. Nematode egg counts and the number of animals infected with nematodes was reduced (p < 0.05) in all cattle that consumed FBZ as compared to control animals. In contrast to nematode eggs, numbers of Giardia cysts was not reduced (p > 0.05) by FBZ as compared to controls in either treatment period. These results may be a reflection of Giardia re-infection occurring following treatment and highlight the need for variation in treatment regimes specifically targeted at the parasite of interest.

DNA sequence analysis of regions surrounding blaCMY-2 from multiple Salmonella plasmid backbones.

The emergence in the United States of resistance to expanded-spectrum cephalosporin (e.g., ceftriaxone) within the salmonellae has been associated primarily with three large (>100-kb) plasmids (designated types A, B, and C) and one 10.1-kb plasmid (type D) that carry the blaCMY-2 gene. In the present study, the distribution of these four known blaCMY-2-carrying plasmids among 35 ceftriaxone-resistant Salmonella isolates obtained from 1998 to 2001 was examined. Twenty-three of these isolates were Salmonella enterica serotype Newport, 10 were Salmonella enterica serotype Typhimurium, 1 was Salmonella enterica serotype Agona, and 1 was Salmonella enterica serotype Reading. All 23 serotype Newport isolates carried a type C plasmid, and 5, 4, and 1 serovar Typhimurium isolate carried type B, A, and C plasmids, respectively. Both the serotype Agona and serotype Reading isolates carried type A plasmids. None of the isolates carried a type D plasmid. Hybridization data suggested that plasmid types A and C were highly related replicons. DNA sequencing revealed that the region surrounding blaCMY-2 was highly conserved in all three plasmid types analyzed (types B, C, and D) and was related to a region surrounding blaCMY-5 from the Klebsiella oxytoca plasmid pTKH11. These findings are consistent with a model in which blaCMY-2 has been disseminated primarily through plasmid transfer, and not by mobilization of the gene itself, to multiple Salmonella chromosomal backbones.