Effect of Exogenous Fibrolytic Enzyme Application on the Microbial Attachment and Digestion of Barley Straw In vitro.

The effects of exogenous fibrolytic enzymes (EFE; a mixture of two preparations from Trichoderma spp., with predominant xylanase and ß-glucanase activities, respectively) on colonization and digestion of ground barley straw and alfalfa hay by Fibrobacter succinogenes S85 and Ruminococcus flavefaciens FD1 were studied in vitro. The two levels (28 and 280 µg/ml) of EFE tested and both bacteria were effective at digesting NDF of hay and straw. With both substrates, more NDF hydrolysis (p<0.01) was achieved with EFE alone at 280 than at 28 µg/ml. A synergistic effect (p<0.01) of F. succinogenes S85 and EFE on straw digestion was observed at 28 but not 280 µg/ml of EFE. Strain R. flavefaciens FD1 digested more (p<0.01) hay and straw with higher EFE than with lower or no EFE, but the effect was additive rather than synergistic. Included in the incubation medium, EFE showed potential to improve fibre digestion by cellulolytic ruminal bacteria. In a second batch culture experiment using mixed rumen microbes, DM disappearance (DMD), gas production and incorporation of (15)N into particle-associated microbial N ((15)N-PAMN) were higher (p<0.001) with ammoniated (5% w/w; AS) than with native (S) ground barley straw. Application of EFE to the straws increased (p<0.001) DMD and gas production at 4 and 12 h, but not at 48 h of the incubation. EFE applied onto S increased (p<0.01) (15)N-PAMN at 4 h only, but EFE on AS increased (p<0.001) (15)N-PAMN at all time points. Prehydrolysis increased (p<0.01) DMD from both S and AS at 4 and 12 h, but reduced (p<0.01) (15)N-PAMN in the early stage (4 h) of the incubation, as compared to non-prehydrolyzed samples. Application of EFE to barley straw increased rumen bacterial colonization of the substrate, but excessive hydrolytic action of EFE prior to incubation decreased it.

Cutinolytic esterase activity of bacteria isolated from mixed-plant compost and characterization of a cutinase gene from Pseudomonas pseudoalcaligenes.

The objective of the current study was to examine cutinolytic esterase (i.e., cutinase) activity by pseudomonads and bacteria isolated from mixed-plant compost. Approximately 400 isolates representing 52 taxa recovered from mixed-plant compost using cuticle baits, along with 117 pseudomonad isolates obtained from a culture collection (i.e., non-compost habitats), were evaluated. The ability of isolates to degrade the synthetic cutin polycaprolactone (PCL) was initially measured. Isolates from 23 taxa recovered from the compost degraded PCL. As well, isolates from 13 taxa of pseudomonads cleared PCL. Secondary screening measured esterase activity induced by the presence of apple cuticle using the chromogenic substrate p-nitrophenyl butyrate. Eighteen isolates representing four taxa (Alcaligenes faecalis , Bacillus licheniformis , Bacillus pumilus , and Pseudomonas pseudoalcaligenes) recovered from compost exhibited substantial esterase activity when grown with cuticle. In contrast, none of the pseudomonad isolates from the culture collection produced appreciable esterase activity. Although degradation of PCL was not correlated with esterase activity, isolates that were unable to degrade PCL failed to produce measureable esterase activities. Zymogram analysis indicated that the esterases produced by bacteria from compost ranged in size from 29 to 47 kDa. A gene from P. pseudoalcaligenes (cutA) was found to code for a cutin-induced esterase consisting of 302 amino acids and a theoretical protein size of 32 kDa. The enzyme was unique and was most closely related to other bacterial lipases (≤48% similarity).

Isolation and characterization of a ferulic acid esterase (Fae1A) from the rumen fungus Anaeromyces mucronatus.

AIMS: A novel ferulic acid esterase gene from rumen fungus Anaeromyces mucronatus was cloned, heteroexpressed in Escherichia coli and characterized. METHODS AND RESULTS: A total of 30 clones exhibiting activity on α-naphthyl acetate (α-NA) were isolated from an A. mucronatus YE505 cDNA library. Sequence analysis revealed that these clones represented two esterase-coding sequences. The gene, fae1A, showed highest amino acid sequence identity to CE family 1 esterases from anaerobic micro-organisms such as Orpinomyces sp., Ruminococcus albus and Clostridium thermocellum. The gene comprised 828 nucleotides encoding a polypeptide of 275 amino acids. The coding sequence was cloned into the pET30a expression vector and overexpressed in E. coli BL21 (DE3). Gene product Fae1A was found to exhibit activity against a number of substrates including naphthyl fatty acid esters, p-nitrophenyl fatty acid esters and hydroxylcinnamic acid esters. CONCLUSIONS: Fae1A exhibited a lower K(m) and higher catalytic efficiency (k(cat) /K(m) ) on ferulic acid esters than on α-NA or p-nitrophenyl acetate, suggesting that it has a higher affinity for ethyl and methyl ferulate than for the acetyl esters. It releases ferulic acid and p-coumaric acid from barley straw. Activity of Fae1A was inhibited by the serine-specific protease inhibitor, phenylmethylsulfonyl fluoride, indicating that a serine residue plays a role in its activity. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first report of characterization of carbohydrate esterase gene from the genus of Anaeromyces.

Farm-to-fork characterization of Escherichia coli associated with feedlot cattle with a known history of antimicrobial use.

This study investigated antimicrobial-resistant (AR) Escherichia coli isolated from "farm-to-fork" production of cattle fed diets containing the antimicrobial growth promoter (AGP) chlortetracycline plus sulfamethazine (44ppm each, AS700) or no AGP (control). For each treatment, samples included: feces just prior to euthanization; hides after euthanization; intestinal digesta from the lower digestive tract; carcasses immediately after evisceration and after 24h in the chiller; and ground beef stored at 5 degrees C for 1 and 8days. Samples were also collected from the abattoir environment and from air during hide removal. Total, ampicillin (Amp(r))-, and tetracycline (Tet(r))-resistant E. coli were isolated on MacConkey agar or MacConkey agar containing ampicillin or tetracycline, respectively. Amp(r) and Tet(r)E. coli were isolated from the feces and hides of all cattle. Compared to the control, the prevalence of Amp(r) (26.5% vs. 7.9%) and Tet(r) (50.9% vs. 12.6%) E. coli was greater in feces from AS700 treated animals (P<0.05), but was similar between treatments for hide samples (P>0.05). The prevalence of carcass or ground beef contamination with AR E. coli was not different between treatments. Resistant E. coli were isolated from the abattoir environment after processing of both groups of cattle. Susceptibilities to 11 antimicrobials and pulsed-field gel electrophoresis (PFGE) analyses were conducted on 360 Amp(r) and Tet(r)E. coli isolates. Twenty-five antibiogram profiles were detected, with isolates exhibiting resistance to up to 9 antimicrobials. Most (28.2%) Amp(r)E. coli were also resistant to streptomycin and tetracycline, whereas Tet(r)E. coli (53.5%) were mainly resistant to only tetracycline. Thirty one genotypes were detected by PFGE with most isolates from meat and environmental samples having similar genetic profiles to isolates from hides or digesta. These data demonstrate that antimicrobial-resistant E. coli can contaminate meat products during slaughter and enter the food chain regardless of whether or not cattle are administered AGP. The abundance of AR E. coli on the hides of animals is likely a key element for controlling end-product contamination.

Longitudinal characterization of resistant Escherichia coli in fecal deposits from cattle fed subtherapeutic levels of antimicrobials.

Model fecal deposits from cattle fed or not fed antimicrobial growth promoters were examined over 175 days in the field for growth and persistence of total Escherichia coli and numbers and proportions of ampicillin-resistant (Amp(r)) and tetracycline-resistant (Tet(r)) E. coli. In addition, genotypic diversity and the frequency of genetic determinants encoded by Amp(r) and Tet(r) E. coli were investigated. Cattle were fed diets containing chlortetracycline (44 ppm; A44 treatment group), chlortetracycline plus sulfamethazine (both at 44 ppm; AS700 treatment group), or no antibiotics (control). Fecal deposits were sampled 12 times over 175 days. Numbers of Tet(r) E. coli in A44 and AS700 deposits were higher (P < 0.001) than those of controls and represented up to 35.6% and 20.2% of total E. coli, respectively. A time-by-treatment interaction (P < 0.001) was observed for the numbers of Tet(r) and Amp(r) E. coli. Except for Amp(r) E. coli in control deposits, all E. coli numbers increased (P < 0.001) in deposits up to day 56. Even after 175 days, high Tet(r) E. coli numbers were detected in A44 and AS700 deposits [5.9 log(10) CFU (g dry matter)(-1) and 5.4 log(10) CFU (g dry matter)(-1), respectively]. E. coli genotypes, as determined by pulsed-field gel electrophoresis, were diverse and were influenced by the antimicrobial growth promoter and the sampling time. Of the determinants screened, bla(TEM1), tetA, tetB, tetC, sul1, and sul2 were frequently detected. Occurrence of determinants was influenced by the feeding of antimicrobials. Fecal deposits remain a source of resistant E. coli even after a considerable period of environmental exposure.

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.

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.

First Report of the Purple Variant of Curtobacterium flaccumfaciens pv. flaccumfaciens, Causal Agent of Bacterial Wilt of Bean, in Canada.

Bacterial wilt of bean (Phaseolus vulgaris L.) caused by the yellow and orange variants of Curtobacterium flaccumfaciens pv. Flaccumfaciens (Hedges) Collins & Jones was found in western Canada in 2002 (1). A purple variant was found in a pooled sample of discolored cull seeds of great northern bean (cv. US1140) from a crop grown near Bow Island, Alberta, Canada in 2005. Bacterial colonies isolated from purple seed using modified Burkholder's agar (MBA) (3) were convex, glistening, and smooth edged with blue pigment diffusing into the medium. Three isolates (V154, V155, and V254) were identified with conventional tests (2), carbohydrate oxidation (GP Microplates, Biolog Inc., Hayward, CA), and cellular fatty acids (CFA) (MIDI, Inc., Newark, DE). All were grampositive, motile, aerobic rods with yellow colonies producing extracellular blue pigment on MBA when grown at 20 ± 2°C. Bacterial isolates grew at 27°C but grew weakly at 37°C. They were positive for catalase and hydrolysis of hippurate and indoxyl acetate and negative for urease, gelatin liquification, and oxidase. CFA profiles were approximately 48% 15:0 anteiso, 40% 17:0 anteiso, 7% 16:0 iso, and 3% 15:0 iso; with 17:1 anteiso A variable but <1%. Many carbohydrates were oxidized in the Biolog microplates with little acid production. The results match C. flaccumfaciens (2) and the MIDI and Biolog databases, as well as the purple variant of C. flaccumfaciens found in Nebraska, the only previous report of this variant (4). The pathogenicity of the three isolates was tested. Seeds of great northern (cv. US1140) and navy (cv. Morden003) beans were soaked in a bacterial suspension (1 × 108 CFU/ml) or distilled water (control) for 1 h, planted in Cornell mix in root trainers, incubated at 28/22°C (16-h day/8-h night) in a growth cabinet for 14 days, and examined for seedling wilt. The test had three replicates per treatment and 20 seeds per replicate in a completely randomized design. All three isolates were pathogenic to both bean cultivars. The wilt incidences were 51, 57, and 56% on US1140 and 64, 76, and 69% on Morden003 for isolates V154, V155, and V254, respectively. The purple variant of C. flaccumfaciens was reisolated from hypocotyls of wilted seedlings but not from healthy controls. The experiment was repeated using the reisolated bacteria and the results were similar to the first experiment, thus fulfilling Koch's postulates. To our knowledge, this is the first report of the purple variant of C. flaccumfaciens pv. flaccumfaciens in Canada. References: (1) T. F. Hsieh et al. Plant Dis. 86:1275, 2002. (2) K. Komagata et al. Page 1313 in: Bergey's Manual of Systematic Bacteriology. Vol. 2. Williams and Wilkens, Baltimore, MD, 1986. (3) G. A. Nelson and G. Semeniuk. Phytopathology 54:330, 1964. (4) M. L. Schuster et al. Can. J. Microbiol. 14:423, 1968.

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.

Transfer of a rifampicin-resistant Escherichia coli strain among feedlot cattle.

AIMS: To determine the incidence of transfer of a naturally occurring rifampicin-resistant strain of Escherichia coli (RREC) among cattle in a research feedlot. METHODS AND RESULTS: During three separate experiments, steers in three different pens were orally inoculated with RREC originally isolated from bovine faeces. Faecal swabs were performed on all steers in the feedlot at approximately 5 week intervals thereafter. Faecal grab samples were collected from steers in the inoculated and the immediately adjacent pens for up to 4 months. In all three experiments, the inoculated steers and penmates shed RREC within 48 h, and then shed intermittently throughout the sampling periods. Transfer of RREC to steers in an adjacent pen was confirmed only during the first experiment, but never to those in non-adjacent pens. All recovered RREC isolates were compared with the inoculated strain using multiple methods indicating that all RREC isolates were descendants of the original inoculated strain. CONCLUSIONS: Detection of the RREC strain on the pen floor and within the animal handling system, but not in the feed troughs or water bowls, suggests faecal-oral to be the primary mode of transmission among animals. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that in the absence of selective pressure, antibiotic-resistant bacteria may persist in cattle for a short duration but widespread transfer among cattle in a feedlot environment may be an exception rather than the norm. Modifications to feedlot management are discussed.

First Report of Pink Seed of Lentil and Chickpea Caused by Erwinia rhapontici in Canada.

A new disease of lentil (Lens culinaris Medik.) and chickpea (Cicer arietinum L.) caused by Erwinia rhapontici (Millard) Burkh. was found in seed samples from commercial fields in Saskatchewan, Canada in 2002. Infected seeds had a pink or pinkish-brown discoloration of the seed coat. Isolation from surface-sterilized pink seeds resulted in bacterial cultures that produced a water-soluble pink pigment on potato dextrose agar (PDA). Four isolates from different lentil crops, LRC 8265, LRC 8310, LRC 8309, and LRC 8313 and one isolate from a chickpea crop, LRC 8266, were tested as previously described (2). Results of the tests were identical to those for pink bean isolates of E. rhapontici (2) with the following minor exceptions: all were negative for Voges-Proskauer; LRC 8266 was positive for tagatose; LRC 8266, LRC 8309, and LRC 8313 were negative for lactose; and LRC 8266 and LRC 8309 were positive for 5-keto gluconate. For pathogenicity tests, each isolate was inoculated into 30 pods from 6 lentil plants (cv. Laird), 30 pods from 6 desi chickpea plants (cv. Myles), and 30 pods from 6 kabuli chickpea plants (cv. Sanford) by the method described for pink seed of pea (1) and bean (2). Each pod was inoculated with 0.1 ml (0.2 ml for kabuli chickpeas) of bacterial suspension, approximately 108 CFU/ml, by injection through the mid-rib at the basal end. The same number of uninoculated and water-inoculated pods served as controls. Plants were kept in the greenhouse (20 ± 5°C) for 4 weeks, after which isolations of the pathogen were performed as described above. In duplicate experiments, all the isolates caused pink lesions on pods and seeds of lentil, desi chickpea, and kabuli chickpea. The frequency of infected seeds among the five isolates (four lentil and one chickpea) ranged from 50 to 100% on lentil, 73 to 100% on desi chickpea, and 43 to 100% on kabuli chickpea. E. rhapontici was reisolated from seeds with lesions but not from asymptomatic seeds. The study demonstrates that in addition to pea (1) and common bean (2), E. rhapontici is also the causal agent of pink seed of lentil and chickpea. The observation that lentil isolates can infect chickpea and vice versa suggests that host specificity may be lacking in E. rhapontici. To our knowledge, this is the first record of E. rhapontici on lentil and chickpea. References: (1) H. C. Huang et al. Can. J. Plant Pathol. 12:445, 1990. (2) H. C. Huang et al. Plant Dis. 86:921, 2002.

Persistence of Escherichia coli O157:H7 in barley silage: effect of a bacterial inoculant.

AIMS: The effect of a lactic acid producing bacterial (LAB) inoculant on the elimination of Escherichia coli O157:H7 from barley forage was assessed. METHODS AND RESULTS: Triplicate mini-silos were prepared for four treatments and six sampling times (1, 3, 7, 15, 30 and 42 d post-ensiling). The treatments were (i) 10(5) cfu g(-1) Pediococcus pentosaceus and Propionibacterium jenzenii (P2); (ii) 10(5) cfu g(-1) E. coli O157:H7 strain 3081 and 10(5) cfu g(-1) E. coli Biotype 1 strains 719IE10, 719IE14 and 614ME49 (EC); (iii) P2 + EC; and (iv) the control (sterile distilled water). Triplicate mini-silos were opened at each sampling time for pH, volatile fatty acid (VFA) and lactate determinations and E. coli, E. coli O157:H7 and LAB were enumerated. On d 3 and 7, numbers of E. coli O157:H7 in P2 + EC were significantly lower than in EC (P < 0;05). Escherichia coli O157:H7 was not detected in P2 + EC and EC at 7 and 15 d post-ensiling, respectively. On d 15 through 42, E. coli Biotype 1 was not detected in P2 + EC or EC. Populations of LAB were higher in P2 and P2 + EC than in the control and EC on d 3 and 7 (P < 0.05). After 3 d of ensiling, lactate levels were higher (P < 0.05) and pH was lower (P < 0.05) in P2 and P2 + EC as compared to the control and EC. Bacteriocins of P2 were not found to be inhibitory to E. coli O157:H7 using the agar-spot procedure. Escherichia coli O157:H7 inoculated into the control silage at a level of 10(3) cfu g(-1) and exposed to aerobic conditions at 22 degrees C was not detected after 1 d and remained undetectable for the 28 d exposure period. CONCLUSIONS: Silage inoculant P2 increased lactate levels and decreased pH more rapidly during ensiling, which appeared to hasten the elimination of E. coli O157:H7 from the silage. SIGNIFICANCE AND IMPACT OF THE STUDY: Results emphasize the importance of adequate ensiling since E. coli O157:H7 may be maintained and spread among cattle through feed.

First Report of Bacterial Wilt of Common Bean Caused by Curtobacterium flaccumfaciens in Western Canada.

Bacterial wilt of common bean (Phaseolus vulgaris L.) caused by Curtobacterium flaccumfaciens pv. flaccumfaciens (Hedges) Collins & Jones (4) was found in 1947 in Ontario, Canada (3), but not in western Canada. Infected seeds exhibit yellow, orange, or purple discoloration (4). Examination of 36.7 kg of cull beans of crops grown in southern Alberta in 2001 obtained from a processing plant revealed 5.9% yellow and 0.014% orange seeds, each with wrinkled seed coats. Bacteria were isolated on potato dextrose agar. Three strains were identified using conventional tests (2), carbohydrate oxidation (GP Microplates, Biolog Inc., Hayward, CA), and cellular fatty acids (CFA) (MIDI, Inc., Newark, DE). Strains were gram-positive, motile, aerobic rods with yellow (YSB-1, YSB-2) or orange (OSB-3) colonies. Growth occurred at 27 and 37°C. The strains were positive for citrate utilization, catalase, hydrolysis of hippurate, and indoxyl acetate, and negative for urease, gelatin liquification, and oxidase. CFA profiles were ≈48% 15:0 anteiso, 37% 17:0 anteiso, 8% 16:0 iso, 3% 15:0 iso, and 3% 16:0; with17:1 anteiso A sometimes present at <2%. Acid production was weak from carbohydrates, but all oxidized many carbohydrates in the microplates. These results match C. flaccumfaciens pv. flaccumfaciens (2) in MIDI and Biolog databases. Strains were tested for pathogenicity using seed and pod inoculations. Seeds of great northern ('US1140') and navy ('AC Skipper') beans were soaked in bacterial suspension (1 to 3 × 108 CFU/ml) for 1 h, sown in Cornell Peatlite Mix (1) in Root Trainers (Spencer-Lemaire Industries, Edmonton, AB, Canada), incubated at 28°C (16-h day) and 22°C (8-h night), and examined for seedling wilt after 10 days. Seeds soaked in sterile distilled water served as controls. Testing was repeated once with 3 replicates per treatment and 10 seeds per replicate. Experiments were conducted using a complete randomization design. For pod inoculation, a suspension (0.1 ml) of each strain was injected into the midrib at the basal end of each young pod of 'AC Skipper'. Pods inoculated with sterile distilled water, 0.1 ml per pod, were used as controls. After 21 days, pods were harvested and examined. Testing was repeated once with three plants per treatment and five pods per plant. Bacteria were reisolated from hypocotyls of wilted seedlings and diseased pods. Results of seed inoculations showed all strains were pathogenic to both cultivars. Wilt incidence was 38, 35, and 57% for strains YSB-1, YSB-2, and OSB-3, respectively, on 'US1140' and 44, 40, and 63% respectively, on 'AC Skipper'. Results of pod inoculations showed 63% (YSB-1) and 55% (YSB-2) of seeds had wrinkled, yellow seed coats, and 72% (OSB-3) of seeds had wrinkled, orange seed coats. Control seedlings and seeds remained healthy. C. flaccumfaciens pv. flaccumfaciens was reisolated from wilted seedlings and seeds showing yellow or orange discoloration, but not from the controls. To our knowledge, this is the first report of bacterial wilt of bean caused by yellow and orange strains of C. flaccumfaciens pv. flaccumfaciens in western Canada. References: (1) J. W. Boodley and R. Sheldrake Jr. N.Y. State Coll. Agric. Life Sci. Inform. Bull. 43, 1977. (2) K. Komagata et al. Page 1313 in: Bergey's Manual of Systematic Bacteriology, Vol. 2, Williams and Wilkens, Baltimore, MD, 1986. (3) Z. A. Patrick, Can. J. Bot. 32:705, 1954. (4) A. W. Saettler. Bacterial wilt. Page 31 in: Compendium of Bean Diseases. R. Hall, ed. American Phytopathology Society, St. Paul, MN, 1994.

First Report of Pink Seed of Common Bean Caused by Erwinia rhapontici.

In 2001, a new disease of common bean (Phaseolus vulgaris L.) caused by Erwinia rhapontici (Millard) Burkh. was detected in seed samples from southern Alberta, Canada. Infected seeds had pink or pinkish-brown lesions on the seed coat. The disease was found in great northern (cv. US1140), pink (cv. Viva), and pinto (cv. Othello) beans at low (<0.1%) frequencies. Isolation from surface-sterilized pink seeds resulted in bacterial cultures, which produced a water-soluble pink pigment on potato dextrose agar (PDA). Seven isolates were tested for physiological characteristics using conventional tests (1) and API 50CHE test strips (bioMérieux Canada, St. Laurent, Quebec), and tested for cellular fatty acids using the MIDI system (Newark, DE). All isolates were gram-negative, motile, facultative anaerobic rods with mucoid colonies and produced a pink pigment on PDA. They were positive for citrate utilization, catalase, methyl red, and Voges-Proskauer, and negative for arginine dihydrolase, lysine and ornithine decarboxylases, urease, gelatin liquification, indole production, oxidase, and gas production. Fatty acid profiles matched with E. rhapontici (approximately 30% each 16:0 and 16:1 ω7c/15:0 iso 2OH; 12% 18:1 ω7c: 8% each 17:0 cyclo and 14:0 3OH/16:1 iso; 4 to 5% each 12:0 and 14:0). Isolates were positive for acid production from: N-acetyl glucosamine, l-arabinose, amygdalin, arbutin, cellobiose, esculin (hydrolysis), d-fructose, d-fucose, d-galactose, ß-gentiobiose, d-glucose, glycerol, i-myo-inositol, lactose, maltose, d-mannitol, d-mannose, melibiose, d-raffinose, l-rhamnose, ribose, salicin, d-sorbitol, sucrose, trehalose, and d-xylose. These results match published results for E. rhapontici (4). For pathogenicity tests, each isolate was inoculated in 30 pods from six bean plants (cv. US1140) as described for pink seed of peas (2). Each pod was inoculated with 0.1 ml of bacterial suspension, approximately 109 CFU/ml, by injection through the mid-rib at the basal end. The same number of uninoculated and water-inoculated pods served as controls. Plants were kept in the greenhouse (20 ± 5°C) for 4 weeks, after which isolations were done as described above. In duplicate experiments, all isolates caused lesions on pods extending up to 5 cm from the inoculation point with corresponding discoloration of seeds. The frequency of infected seeds varied among isolates, ranging from 20 to 50%. E. rhapontici was reisolated from seeds with lesions, but not asymptomatic seeds. The study concludes that pink seed of common bean is due to E. rhapontici, a pathogen previously reported on peas in Alberta, Canada (2), and Montana (3). References: (1) D. J. Brenner. Bergey's Manual of Systematic Bacteriology, vol.1, Williams and Wilkens, Baltimore, MD, 1984. (2) H. C. Huang et al. Can. J. Plant Pathol. 12:445, 1990. (3) B. K. Schroeder et al. Plant Dis. 86:188, 2002. (4) L. Verdonck et al. Int. J. Syst. Bacteriol. 37:4, 1987.

An improved method to illuminate and record a phytase assay in SDS-PAGE zymogram gels.

Experimentation at the Lethbridge Research Center in Alberta, Canada using cross-polarized transmitted light to photographically record a staining technique on zymograms has proved to be successful with both color and black and white films. It has been possible to obtain the desired visible contrast without compromising the intensity of the enzyme activity bands. Increasing numbers of such PAGE gels are being submitted for photographic recording when it is believed that the image will be used for records, publication, scientific posters or AV presentations.

Growth and survival of immature Heamatobia irritans (Diptera; Muscidae) is influenced by bacterial isolated from cattle manure and conspecific larvae.

Twenty species of bacteria were isolated from cattle manure and seven species were isolated from the gut of larval horn fly Hematobia irritans (L.). Bacteria in manure belonged to the Bacillaceae, Pseudomonadaceae, Micrococcaceae, Corynebacteriaceae, Enterobacteriaceae, Microbacteriaceae, and two unassigned genera. Gut bacteria belonged to the Enterobacteriaceae, Bacillaceae, Neisseriaceae, and Pseudomonadaceae. H. irritans larval survival and growth on the various bacterial species were evaluated by rearing larvae in sterilized cattle manure that was inoculated with single bacterial isolates. H. irritans larvae failed to develop in sterilized, uninoculated manure, indicating that bacteria are necessary for larval development. Survival averaged 74% in nonsterilized manure and ranged from 4 to 53% in manure with individual isolates. Survival was highest when larvae were reared on manure inoculated with Pseudomonadaceae, Corynebacteriaceae, Micrococcaceae, and Bacillaceae and was lowest when reared in manure inoculated with Enterobacteriaceae and Microbacteriaceae. Pupal weights were heaviest when reared on the Flavobacteria, followed by the Pseudomonadaceae and Corynebacteriaceae. Pupae averaged 4.9 +/- 0.08 mg when reared on gram-negative isolates, compared with 3.6 +/- 0.09 mg when reared on gram-positive isolates. Pupal weights were not significantly correlated with larval survival, indicating that bacteria that promote growth do not necessarily promote survival. A reproductive index was used as a measure of fitness and was highest for larvae reared in the nonsterile control, followed most closely by Pseudomonadaceae and Corynebacteriaceae. These groups appeared to best meet the nutritional requirements of larvae and may be used in further experiments to define an artificial rearing media for H. irritans.

Lachnobacterium bovis gen. nov., sp. nov., a novel bacterium isolated from the rumen and faeces of cattle.

Phenotypic and phylogenetic analysis was performed on four strains of a previously undescribed Gram-positive, rod-shaped, anaerobic bacterium isolated from the rumen and faeces of cattle. This bacterium fermented glucose primarily to lactic acid along with minor amounts of acetic and butyric acids. The four strains produced a temperature-sensitive bacteriocin-like inhibitory substance. Comparative 16S rRNA gene sequence analysis indicated that the bacterium was a member of the clostridial XIVa cluster of the low-G+C content Gram-positive bacteria. Based on phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium be assigned to a new genus, Lachnobacterium, as Lachnobacterium bovis gen. nov., sp. nov. The type strain is YZ 87T (= ATCC BAA-151T = DSM 14045T = LRC 5382T). Its G+C content is 33.9 mol %.

Characterization of acute interstitial pneumonia in cattle in southern Alberta feedyards.

Field data were collected over 2 consecutive years to characterize acute interstitial pneumonia (AIP) in feedyard cattle. Thirty-eight cattle with clinical symptoms of AIP were examined following emergency slaughter; 31 (all heifers) were confirmed to have AIP on the basis of gross and histological lung pathology. The 7 without AIP, plus 17 asymptomatic penmates, were used as contemporary controls. Plasma concentrations of 3-methylindole (3MI) metabolites were higher (P < 0.001) in heifers afflicted with AIP than in the control animals, and concentrations of 3MI mercapturates in the urine were lower (P < 0.007) in affected heifers. Concentrations of 3MI adducts in lung tissue and in microsomal protein did not differ (P > 0.05) between the 2 groups, and 3MI was not detected in ruminal fluid from either group. Total ruminal bacterial numbers and populations of lactobacilli and protozoa were similar (P > 0.05) between the AIP-positive and unafflicted groups, but fewer (P < 0.05) cellulolytic bacteria were present in the positive group. Bovine respiratory syncytial virus antigen was not found in lung tissue from any of the heifers confirmed to have AIP. To our knowledge, this study is the first to implicate 3MI metabolites as having a role in feedyard AIP. Further research is required to determine the factors responsible for the elevation in 3MI adducts in plasma and urine of feedyard cattle afflicted with AIP.

Effect of steroidal saponin from Yucca schidigera extract on ruminal microbes.

The effects of steroidal saponins (SAP) isolated from Yucca schidigera extract on ruminal bacteria and fungi were investigated in pure culture studies. Prevotella bryantii, Ruminobacter amylophilus, Selenomonas ruminantium and Streptococcus bovis were cultured through ten 24-h transfers in ruminal fluid medium containing 0 or 25 microg SAP ml-1 (measured as smilagenin equivalents). The four strains, each non-exposed or pre-exposed to SAP, were then inoculated into medium containing 0 or 250 microgram smilagenin equivalents ml-1 and 24-h growth curves were determined. The cellulolytic ruminal bacteria Ruminococcus flavefaciens, Fibrobacter succinogenes and Rc. albus were cultured for 72 h on Whatman no. 1 filter paper in medium containing 0, 9, 90 or 180 microgram SAP ml-1 for the determination of filter paper digestion and endoglucanase activity. The ruminal bacteria differed in their responses to SAP. Steroidal saponins in the medium reduced the growth of Strep. bovis (P < 0.01 at 2, 3, 4, 5, 6 and 8 h), P. bryantii (P < 0.05 at 4, 5, 6, 8, 10 and 24 h) and Rb. amylophilus (P < 0.05 at 14 and 24 h), but the growth of S. ruminantium was enhanced (P < 0.05) at 10, 14 and 24 h. The growth curves of all four non-cellulolytic species were similar (P > 0.05) between pre-exposed and non-exposed cultures and the concentrations of total SAP and soluble (deglycosylated) SAP in the liquid fraction were unchanged (P > 0.05) over time. Steroidal saponins inhibited the digestion of filter paper by all three cellulolytic bacteria, but F. succinogenes was less (P < 0.05) sensitive to SAP and more (P < 0. 05) effective at deglycosylating SAP than were Rc. flavefaciens or Rc. albus. Transmission electron microscopy revealed that SAP altered the cell walls of the SAP-inhibited non-cellulolytic bacteria. The ruminal fungi, Neocallimastix frontalis and Piromyces rhizinflata, were cultured on filter paper in medium containing 0, 0. 45, 2.25 or 4.5 microgram SAP ml-1. Filter paper digestion by both fungi was completely inhibited by 2.25 microgram SAP ml-1. Steroidal saponins from Y. schidigera inhibit cellulolytic ruminal bacteria and fungi, but their effects on amylolytic bacteria are species dependent and similar to the effects of ionophores. As such, SAP may be useful in nutritional applications targeting starch-digesting ruminal micro-organisms.

Localization of phytase in Selenomonas ruminantium and Mitsuokella multiacidus by transmission electron microscopy.

The localization of phytase (myo-inositol-hexaphosphate phosphohydrolase) in the ruminal bacteria, Selenomonas ruminantium JY35 and Mitsuokella multiacidus 46/5(2), was determined with transmission electron microscopy. Phosphate produced from the enzymatic dephosphorylation of the calcium salt of phytic acid is precipitated as calcium phosphate. The calcium is then replaced with lead to produce electron-dense lead phosphate. This deposition of lead phosphate localized phytase in S. ruminantium JY35 and M. multiacidus 46/5(2) to the outer membrane, and confirmed intracellular expression of the enzyme in Escherichia coli pSrP.2, the recombinant clone which possesses the gene (phyA) encoding phytase (phyA) in S. ruminantium.