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Applied and Environmental Microbiology, August 2008, p. 5153-5158, Vol. 74, No. 16
0099-2240/08/$08.00+0     doi:10.1128/AEM.00723-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Characterization of Verotoxin-Encoding Phages from Escherichia coli O103:H2 Strains of Bovine and Human Origins{triangledown}

Musafiri Karama and Carlton L. Gyles*

Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario N1G 2W1, Canada

Received 27 March 2008/ Accepted 3 June 2008


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ABSTRACT
 
The objectives of this study were to induce and characterize verotoxin-encoding phages from a collection of 91 verotoxin-producing Escherichia coli (VTEC) O103:H2 strains of human and bovine origins. All the strains carried the vt1 gene, and two carried the vt2 gene as well. The phages were induced by UV irradiation and characterized by DNA restriction fragment length polymorphism (RFLP), genome size, morphology, and Q and P genes, characteristic of lambdoid phages. A total of 32 vt-positive phages were induced and isolated from 31 VTEC O103:H2 strains. Thirty phages were vt1 positive, and two were vt2 positive. Ten of the 30 vt1-positive phages (33.3%) were from cattle strains, and 20 (66.6%) were from human strains. The two vt2-positive phages were from human strains. Phages belonged to 21 RFLP profiles, of which 17 were single-phage profiles and 4 were multiple-phage profiles. The estimated genome size of the phages ranged from 34 to 84 kb. Two phages that were examined by electron microscopy possessed hexagonal heads with long tails, and one had an elongated head with a long tail. The Q and P genes were amplified in all 32 phages, and the Q-stxA1 gene region yielded an amplicon in 19 phages (59.3%). It is concluded that the VTEC O103:H2 strains of human origin were more readily inducible than those of bovine origin and that the genotypic profiles of verotoxin-encoding phages were highly diverse, as revealed by their RFLP profiles.


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INTRODUCTION
 
Verotoxin-producing Escherichia coli (VTEC), also termed Shiga toxin-producing E. coli, is a food-borne pathogen that has been incriminated in sporadic cases and outbreaks of bacterial enteritis in humans. Cattle are the main reservoir of VTEC (18). Human infections are acquired through food or water contaminated directly or indirectly with cattle feces. Most human infections due to VTEC have been attributed to serotype O157:H7. However, non-O157 VTEC is increasingly being recognized to be of considerable importance, since it is also frequently incriminated in sporadic illness and outbreaks of mild and severe VTEC disease in humans around the world (11). Some of the non-O157 serogroups that are being frequently incriminated in human disease are O26, O111, and O103 (11).

The main virulence factors of VTEC are verotoxins (VTs), which are encoded on highly diverse lambdoid phages (20). Analysis of the sequence data from VTEC O157:H7 strains EDL933 and Sakai (8, 26) and nonpathogenic E. coli K-12 (4) has revealed that the genomes of the E. coli EDL933 and Sakai strains possess 18 and 24 multigenic phage-related regions, respectively, and most of the differences between the VTEC strains and nonpathogenic E. coli are attributable to prophage or prophage-like elements. Temperate phages encoding VT contribute to the horizontal transfer of vt genes among bacteria. Wide dissemination of these phages has resulted in more than 500 serotypes of VTEC (www.microbionet.com.au/vtectable.htm and www.lugo.usc.es/ecoli).

The genetic organization of VT phages is similar to that of lambda and related phages (9), and phage genes regulate toxin production through phage replication and toxin release through lysis of the host bacterial cell (10, 19, 34, 35). Verotoxin genes are located between the Q and S genes of lambdoid phages, which encode a transcriptional antiterminator and a holin protein, respectively (34, 38). Conserved genes that are found upstream of the Q gene are the cI repressor gene, responsible for phage immunity, and the two replication genes O and P (19).

Most of the research on VT phages has focused on VTEC O157:H7 phages and phages that carry the vt2 gene. One objective of this study was to determine the inducibility of phages in a collection of 91 VTEC O103:H2 strains from humans and cattle and from North America and Europe. The second objective was to characterize the induced phages with respect to their diversity as indicated by restriction fragment length polymorphism (RFLP), genome size, morphology, and Q and P genes, which are typical for lambdoid phages.


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MATERIALS AND METHODS
 
Bacterial strains.
A total of 91 VTEC O103:H2 strains from frozen culture collections at the University of Guelph and the Laboratory for Food-borne Zoonoses, Public Health Agency of Canada, in Guelph, Ontario, Canada, were used for this study. Strains originated from three countries in Europe (Germany, Switzerland, and Belgium) and from North America (United States and Canada) and were isolated between 1991 and 2002. The 91 VTEC O103:H2 isolates consisted of 89 strains that were positive for vt1 only and 2 strains that were positive for both vt1 and vt2. Forty-six strains were of human origin, and 45 strains were of bovine origin. All cattle strains were isolated from healthy cattle in Canada. The 46 strains of human origin were all from persons with disease and consisted of 23 strains from North America and 23 from continental Europe.

Induction of cultures.
Phage induction was carried out by UV irradiation of the VTEC O103:H2 strains according to the method of O'Brien et al. (20). A loopful of an overnight broth culture of VTEC O103:H2 was added to a 250-ml baffled flask (Bellco, Vineland, NJ) containing 45 ml of modified Luria-Bertani (LB) broth prepared with 2.5 g/liter of NaCl instead of the usual 5 g and supplemented with 0.01 M CaCl2. Following incubation at 37°C with shaking at 200 rpm for 3 to 4 h (37), the bacteria were pelleted by centrifugation, suspended in 5 ml of 0.01 M CaCl2, and transferred to a 50-ml sterile glass petri dish. The dish was placed on a black surface 40 cm below a UV light (G3OT8; GE) in a biological safety cabinet (NuAire Inc., Plymouth, MN). Initially, phage induction was performed in the dark by irradiating the bacterial suspension for 90 s. For strains that were not induced after 90 s of exposure to the UV light, the duration of exposure was increased to 120 s. The suspension was transferred to a foil-covered 250-ml baffled flask (Bellco) containing 45 ml of modified LB broth and incubated with shaking (200 rpm) for 5 to 6 h at 37°C. The culture was centrifuged at 4,000 x g for 45 min, the supernatant was filtered through a 0.45-µm-pore-size membrane, and a few drops of chloroform were added prior to storage at 4°C.

Isolation of phages.
Phages were isolated by an adaptation of the double-layer agarose method for bacteriophage {lambda} isolation (30). A mixture of 100 µl of filtrate from the induced culture, 0.01 M CaCl2, and an overnight culture of E. coli K-12 strain MC1061 (20) was incubated at 37°C for 30 min, and then it was added to 3 ml of modified LB soft (top) agarose (2 g LB, O.5 g agarose, 0.01 M CaCl2, 100 ml of H2O), gently mixed, and then poured on 1.5% modified LB agarose (15 g agarose, modified LB, 1.2 g MgSO4·7H2O, 1 liter of H2O). The agarose was allowed to solidify, and the plates were incubated overnight at 37°C and monitored for complete lysis or plaque formation for up to 20 h. In cases of complete lysis, serial dilutions of the filtrate were tested for plaque formation as described above. An isolated plaque was aspirated from the agar and suspended in 500 µl 0.01 M CaCl2 solution. The phages were amplified on an overnight culture of E. coli K-12 strain MC1061 in soft agarose. After complete lysis or extensive plaque formation, 5 ml of SM buffer solution (5.8 g NaCl, 0.98 g MgSO4·7H2O, 50 ml of 1 M Tris-Cl [pH 7.5], 5 ml of 2% gelatin, 1 liter of H2O) was poured on the plate, which was then kept overnight at 4°C with intermittent gentle shaking. The phage suspension was harvested, and phages from at least five petri dishes were combined. The phage suspension was centrifuged at 4,000 x g for 30 min, and the supernatant was transferred to a sterile 50-ml Corning plastic tube (Fisher Scientific Ltd., Nepean, Ontario, Canada). A few drops of chloroform were added, and the phage suspension was stored at 4°C.

Extraction of phage DNA.
Phage DNA was prepared with the Qiagen Lambda Midi kit (Qiagen, Mississauga, Ontario, Canada) according to the manufacturer's instructions.

PCR for phage genes.
In order to determine whether the isolated phages were vt positive, PCR was used to amplify vt genes (25). After confirmation of the possession of the vt genes, the phages were tested for the presence of the Q and P genes (10) and for a PCR product following amplification with primers that targeted the Q-stxA region (34). PCR was performed in an Eppendorf Mastercycler (Eppendorf AG, Brinkman Instruments, Inc., Westbury, NY) with 25-µl reaction mixtures, which consisted of 2.5 µl of phage DNA, 2.5 µl of 10x PCR buffer, 1.5 or 2 mM MgCl2, 200 µM of each deoxynucleoside triphosphate, and 2 U of Taq DNA polymerase. The E. coli O157:H7 strain EDL933 was used as a positive control for all the amplifications.

RFLP and genome size estimation.
The DNA concentration was estimated by running 2 µl in a 0.8% agarose gel, staining with ethidium bromide (0.5 µg/ml), and comparing the intensity of the band on the gel with that of known concentrations of DNA. A total of 2 to 4 µl of phage DNA was digested with the NdeI restriction enzyme (New England BioLabs, Pickering, Ontario, Canada) according to the manufacturer's instructions. DNA bands were separated by electrophoresis in a 0.7% agarose gel, and the bands were visualized under UV light after ethidium bromide staining. The RFLP patterns were analyzed for similarity, and a dendrogram was generated by the Bionumerics, version 3.5, software program (Applied Maths, Saint Martens-Latem, Belgium) with Dice similarity indices (complete linkage, optimization, 1.5%; position tolerance, 1.5) and the unweighted pair group method with arithmetic mean. Genome sizes were also estimated with the aid of the Bionumerics software.

Electron microscopy.
Three phages selected from the major RFLP groups were examined by electron microscopy. Phages were purified by the glycerol gradient method (30), negatively stained, and examined by electron microscopy. A 30-ml suspension of phages of approximately 109 PFU/ml was centrifuged in a Beckman XL-90 ultracentrifuge (SW 28 rotor, 25,000 rpm) for 1 h, and the pellet was resuspended in sterile 0.1 M HEPES buffer (Boehringer Mannheim, Montreal, Quebec, Canada). A drop of the pellet containing the phages was deposited on a 200-mesh Formvar-coated copper grid, negatively stained with 1% uranyl acetate, and examined with a Philips CM10 transmission electron microscope operated at 80 kV (Guelph Regional STEM facility, University of Guelph, Guelph, Ontario, Canada).

Statistical analysis.
The chi-square test was used to determine whether there were significant differences in rates of recovery of VT phages from VTEC O103:H2 of bovine and human origins and of North American and European origins.


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RESULTS
 
Phage induction and isolation.
VT-positive phages were detected in 31 (35.4%) of the 91 VTEC O103:H2 strains after UV induction. All the phages that were tested possessed vt genes. The sources and geographic origins of the 31 strains from which phages were recovered are listed in Table 1. Twenty-nine of the strains were positive for vt1 only, and two were positive for both vt1 and vt2. A total of 32 phages, consisting of 30 VT1-encoding phages and two VT2-encoding phages, were recovered. The vt1-positive phages were induced from 29 strains that carried vt1 only and one strain that carried both vt1 and vt2. The VT2 phages were recovered from the two strains that were positive for both vt1 and vt2.


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  		TABLE 1. RFLP subtypes and presence of Q, P, and Q-stxA1 PCR products for 32 phages from O103:H2 VTEC

Of the 30 vt1-positive phages, 10 (33.3%) were from cattle strains and 20 (66.6%) were from human strains. The 20 phages from human strains consisted of 9 from the 23 North American strains and 11 from the 23 European strains. The proportion of phages induced and recovered from human VTEC O103:H2 strains (20/46 or 43.4%) was significantly higher than that from cattle strains (10/45 or 22.2%) (P = 0.02). Significant differences were also detected between the proportions of North American cattle and human strains that yielded phages (P = 0.01), while the difference between proportions of North American and European strains of human origin that yielded phages was not significant (P = 0.06).

RFLP and genome sizes.
Phage DNA was digested with the NdeI restriction enzyme (Fig. 1), and a dendrogram depicting the relationships among phages based on their RFLP profiles was produced (Fig. 2). Analysis of the RFLP data revealed a total of 21 subtypes (Table 1). Seventeen subtypes represented 20 phages (18 vt1 phages plus 2 vt2 phages) recovered from 19 human strains; 3 subtypes (subtypes 1, 4, and 6) represented 6 phages isolated from 6 cattle strains, and one subtype (subtype 10) consisted of 4 phages isolated from cattle strains and 2 from human strains (Table 1). The 21 profiles showed a Dice similarity index of 44.92%. Seventeen subtypes were single-phage profiles, and four profiles consisted of more than one phage. Profile 1 represented four phages recovered from four cattle strains that were isolated in 1992 in the province of Ontario. Profile 7 was obtained with three phages from strains of human origin; two were VT1 phages from strains isolated in two Canadian provinces (Alberta and Ontario) in 1999 and 2000, and the third was a VT2 phage from a strain isolated in Germany in 1996. Profile 10 comprised six phages: a VT1 phage from a human strain isolated in 1996 in Germany, a VT1 phage from a Canadian strain isolated in Quebec in 1998, and four phages from cattle strains isolated in Ontario in 1991, 1994 (2), and 1997. Profile 19 consisted of two phages isolated from two human strains in Ontario, Canada, in 1997.


Figure 1
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  		FIG. 1. Electrophoresis in a 0.8% agarose gel of NdeI restriction enzyme digests of DNA of VT phages isolated from VTEC O103:H2 strains. M, molecular size markers. The sizes of the molecular size markers in kb are shown at the right of the figure.


Figure 2
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  		FIG. 2. Dendrogram showing relationships among VT phages isolated from O103:H2 VTEC strains, based on RFLP.

The estimated genome sizes of the 32 phages ranged from 34 kb to 84 kb on the basis of sums of the restriction endonucleases fragments.

Phage morphology and dimensions.
Two of the three phages selected for electron microscopy belonged to subtypes 1 (28P) and 10 (65P), in which four and five phages were clustered, respectively, and one phage (6P) belonged to subtype 15, which was a single-phage subtype. Two morphological types (Fig. 3) were identified: two phages showed typical hexagonal heads with long tails, and one phage had an elongated head with a long tail. It was not possible to identify the presence of base-plate-like structures. The three phages had the following dimensions (nm): 6P, head, 48 by 48, and tail, 133 by 10; 28P, head, 72 by 67, and tail, 130 by 11; 65P, head, 99 by 57, and tail, 163 by 11.


Figure 3
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  		FIG. 3. Electron micrographs of three VT phages. Phages 6P and 28P have hexagonal heads, whereas phage 65P has an elongated head. All three phages have long tails. Bar = 50 nm.

Detection of phage-encoded genes by PCR.
The phages, the sources and geographic origins of the host strains, vt status, and results of amplification of the Q and P genes and the Q-stxA1 region are shown in Table 1. All the phages yielded an amplicon for the Q and P genes, and 19/32 (59.4%) produced an amplicon for the Q-stxA1 region.


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DISCUSSION
 
UV mutagenesis induced phages in approximately one-third of the O103:H2 strains. The decision to use UV mutagenesis in this study was influenced by the fact that several earlier attempts to induce these strains with the commonly used mitomycin C were unsuccessful (data not shown). Failure to induce phage and lack of plaque formation after lysis with mitomycin C treatment have been reported in other studies (7, 10, 28, 38). It is difficult to compare our results with those of others, since in most investigations in which phages were isolated from VTEC strains, only a few VTEC isolates were investigated (20, 27) and phages were detected after spontaneous release from wild-type VTEC (33) or after induction or spontaneous release from E. coli strains which had been lysogenized in vitro with phage obtained after induction or spontaneous release from a small collection of VTEC (3). In addition, studies which have induced phages from vt1-positive strains are infrequent (3, 15, 24). This contrasts with several studies in which VT2 phages have been induced (3, 10, 13, 15-17, 24, 28, 39).

The proportion of strains from which phages were isolated in this study was higher than that obtained by Muniesa et al. (16), who isolated VT2 phages by mitomycin C induction in 18% of strains (30/168) belonging to a VTEC collection of 98 O157:H7 VTEC strains and 70 non-O157 VTEC strains from cows, calves, beef, and sheep. Allison et al. (3) also isolated vt2-encoding phages from 18% (8/48) of VTEC strains, while in another study the same group of researchers induced and isolated VT phages from 22% of 463 VTEC O157:H7 strains (32). Muniesa et al. (17) reported a VT2 phage induction/isolation rate of 100% (49/49); however, the O157:H7 VTEC collection had originated with a single human outbreak. Osawa et al. (24) obtained 12 vt1-positive phages and 37 vt2-converting phages from 27 O157:H7 VTEC strains. In studies by Osawa et al. (24) and Allison et al. (3), mitomycin C and UV light were used for induction. Our rate of inducible phages is higher than most of those obtained in the above-mentioned studies (except for that of Osawa et al. [24]), and we suggest that induction of phage by UV irradiation might be a better method for vt1-positive phage induction than mitomycin C induction. Factors other than the method of induction might have contributed to the failure of induction and isolation for the 64.6% of VTEC O103:H2 strains which did not yield phages. In some cases the propagation strain may have been killed in the presence of colicin-producing VTEC O103:H2 strains, thereby precluding plaque formation (3). The same agents that induce phages also induce colicins, and among the 35 colicin-producing strains in our collection (M. Karama, R. P. Johnson, R. Holtslander, and C. L. Gyles, unpublished data), there were only 3 that yielded VT phages. Unsuccessful phage induction might also be ascribed to the presence of defective phages that do not allow the switch from lysogeny to the lytic mode.

Strains of human origin yielded significantly more VT phages than did strains of bovine origin. In fact, the proportion of strains of human origin that yielded phages was two times that of strains of bovine origin. To our knowledge, this is the first study that has compared vt-converting phage inducibility between human strains and cattle strains of VTEC O103:H2. The few above-mentioned studies that examined large numbers of VTEC carried out phage induction in collections of exclusively bovine or human strains (3, 16, 17, 24, 32). Although it is not known to what extent in vitro phage induction reflects in vivo phage induction in VTEC strains, our results suggest that VTEC O103:H2 strains of human origin may represent a subset of strains that carry phages that are more readily induced in the human gastrointestinal tract. However, additional tests on a larger number of strains, including European strains from cattle, are needed to reach a firm conclusion.

Phage induction, which is coregulated with toxin production, is a crucial event in disease pathogenesis. Previous studies have demonstrated that phage induction followed by bacterial lysis enhances bacterial toxin production and release and that both events are concomitant (19, 35). Recently Robinson et al. (29) showed that VT2 not only exacerbated disease by its cytotoxicity but also enhanced bacterial colonization in the mouse intestine by recruiting nucleolin, a host cell receptor for intimin (31). Muniesa et al. (17) observed that there was a high correlation between VTEC disease severity in human patients and in vitro VT production in strains of VTEC O157:H7. Also, Ritchie and coworkers (28) showed that both basal and mitomycin C-induced levels of VT2 were higher in VTEC from hemolytic-uremic syndrome patients than in VTEC from cattle. There is evidence, however, that the type of VT also plays a major role in severity of disease (23).

Epidemiological studies have shown a correlation between antibiotic therapy and an increased risk of severe disease, including hemolytic-uremic syndrome (36). The use of antibiotics in VTEC disease has therefore become controversial, and there is a high probability that this risk is a consequence of the high inducibility of strains of human origin. In addition to toxin amplification, phage induction and release allow for phage survival by offering them the opportunity to lysogenize E. coli or other bacterial hosts, thereby increasing the pool of VT-producing bacteria in the intestine. In this way VT phages also enlarge their host range, thereby diversifying their genomes (2, 21, 22).

Restriction enzyme digestion analysis revealed 21 patterns for the 32 phages, reflecting appreciable diversity among vt-encoding phages isolated from VTEC O103:H2. The variability was also demonstrated by the low Dice similarity index of 44.92%. This is not surprising, since the VTEC O103:H2 parent hosts from which these phages had been isolated were also highly heterogeneous in their pulsed-field gel electrophoresis and plasmid patterns (Karama et al., unpublished data). Lambdoid phages are very diverse and consist of genetically diverse DNA segments that can be interchanged during recombination. Our results with O103:H2 VTEC are similar to those for O157:H7 reported by Osawa et al. (24), who used phage RFLP subtyping and detected high diversity among VT phages isolated from VTEC O157. They reported a Dice similarity index of 44% for 37 vt2-positive phages and 55% for 12 vt1-encoding phages. Johansen et al. (10) also reported diversity in their collection of VT2 phages induced from VTEC O157 strains.

The considerable diversity among RFLP profiles of VT phages has been attributed to multiple recombination events which occur when phages elude bacterial immunity and restriction modification systems, resulting in chromosomal integration and interruptions in the bacterial genome (5). Multiple inducible phages or prophage elements can infect and recombine with or within a single bacterial host strain (6). Recombination is mainly facilitated by the {lambda}-encoded Red recombinase system, which is present in many VT phages (2). This system controls recombination events in the bacterial lysogen or between incoming and resident phages more efficiently than the regular E. coli recombination system (3).

The morphologies of the phages in this study are in agreement with those in other studies (17, 20, 27), which have shown that most VT phages have either an elongated hexagonal head and a long, thin, flexible tail or a regular hexagonal head and a short, thin tail. The genome sizes of the phages in this study (34 to 84 kb) were highly variable but were generally in the range for lambdoid or VT phages reported elsewhere (16, 17, 38).

The Q and P genes were amplified in all phages, while the Q-stxA region was amplified in only 19 of the 32 phages. The frequency of the Q gene obtained in this study is consistent with findings of previous studies, which found that Q is conserved in most VT-encoding inducible phages while the vt flanking regions are very diverse (10, 34). The P gene region in the population of VTEC O103:H2 phages tested was also homogeneous. Previously Lejeune et al. (14) detected the Q gene of bacteriophage 933W (Q933) in 90% of human disease-origin E. coli O157:H7 isolates and in 44.5% of bovine strains. However, Ahmad and Zurek (1) examined 262 VTEC O157:H7 strains from cattle and flies and found that only 3.4% (9/262) of strains carried the Q933 gene while 161 (61.5%) were positive for the Q21 gene alone and 92 (35.1%) carried both Q variants. This shows that results obtained for the Q gene depend on which Q variants are targeted.

In conclusion, induction by exposure to UV irradiation resulted in isolation of vt-positive phages from 36.3% of 91 VTEC O103:H2 strains of human and bovine origins. The rate of induction of phages from VTEC O103:H2 strains of human origin was more than twice that for strains of bovine origin. The relatively high rate of phage induction in strains of VTEC O103:H2 of human origin suggests that these strains may belong to a subpopulation of VTEC O103:H2 strains that are more virulent than a larger cattle strain population, as has been suggested previously by Kim et al. (12). RFLP typing of phage DNA showed that there was considerable diversity (21 subtypes for 32 phages) among the VTEC O103:H2 phages, a reflection of the mosaic structure of phages facilitated by a high rate of recombinational events.


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ACKNOWLEDGMENTS
 
This work was supported by a grant from the Natural Sciences and Engineering Research Council (NSERC) of Canada and the Ontario Ministry of Agriculture, Food and Rural Affairs.

We are grateful to Bob Harris for assistance in electron microscopy and to Ro Osawa, Department of Bioscience, Graduate School of Science and Technology, Kobe University, for valuable advice on phage induction with UV irradiation. We also thank D. Pierard, L. Beutin, S. Alesic, A. Burnens, W. Johnson, and P. Tarr, who kindly donated VTEC O103:H2 strains.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, ON N1G 2W1, Canada. Phone: (519) 824-4120, ext. 54657. Fax: (519) 824-5930. E-mail: cgyles{at}uoguelph.ca Back

{triangledown} Published ahead of print on 13 June 2008. Back


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Applied and Environmental Microbiology, August 2008, p. 5153-5158, Vol. 74, No. 16
0099-2240/08/$08.00+0     doi:10.1128/AEM.00723-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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