Received 3 May 2006/Returned for modification 23 October 2006/Accepted 4 December 2006

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 2007, p Vol. 45, No /07/$ doi: /jcm Copyright 2007, American Society for Microbiology. All Rights Reserved. Geno- and Phenotypic Diversity of Avian Isolates of Streptococcus gallolyticus subsp. gallolyticus (Streptococcus bovis) and Associated Diagnostic Problems M. S. Chadfield, 1 J. P. Christensen, 1 A. Decostere, 2 H. Christensen, 1 * and M. Bisgaard 1 The Royal Veterinary and Agricultural University, Department of Veterinary Pathobiology, Stigbøjlen 4, DK-1870 Frederiksberg C, Denmark, 1 and Laboratory of Veterinary Bacteriology and Mycology, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium 2 Received 3 May 2006/Returned for modification 23 October 2006/Accepted 4 December 2006 Recently, strains of Streptococcus bovis were reclassified as Streptococcus gallolyticus. In the present study we describe for the first time an outbreak of S. gallolyticus in a broiler flock. Mortality during the first week was normal (<1%), with a final total mortality at the end of production reaching 4.3%. Specific symptoms were not observed. Postmortem pathology demonstrated enlarged and light spleens and livers accompanied by multifocal irregular necroses surrounded by a hemorrhagic zone. In addition, these birds suffered from arthritis and osteomyelitis. Strains isolated from liver and spleen lesions showed clonality as demonstrated by pulsed-field gel electrophoresis. Compared to strains representing previously derived phylogeny, including the S. bovis-s. equinus complex, the 16S rrna-derived phylogeny of the strains investigated in this study demonstrated a paraphyletic group (S. gallolyticus) well separated from two monophyletic groups: (i) S. equinus-s. bovis plus S. infantarius and (ii) S. alactolyticus plus S. intestinalis. According to information in GenBank, none of the strains included from the two monophyletic groups have been isolated from birds. Further biochemical analyses, including tannase activity, identified for the first time avian isolates belonging to S. gallolyticus subsp. gallolyticus. However, these investigations also demonstrated a clear heterogeneity with pigeon isolates. The classification and identification of Streptococcus bovis has been confusing and problematic for a long time. Although listed as separate species (20), S. bovis and S. equinus were reported to be subjective synonyms by Farrow et al. (18). Based upon phenotypic diversity among strains of S. bovis, three biovars (I, II/1, and II/2) have been reported (9, 27, 28). Five additional biovars and two sub-biovars, in addition to five serovars, were reported among 60 strains of S. bovis from healthy and septicemic pigeons by De Herdt et al. (11), who concluded that S. bovis from pigeons is different from S. bovis from humans. Recently, strains of S. bovis were reclassified as S. caprinus (6) and S. gallolyticus (25). The synonymy of S. caprinus and S. gallolyticus has subsequently been reported, S. gallolyticus having nomenclatural priority (32). Based upon DNA hybridizations, human isolates of S. bovis biovars I and II/2 belonged to S. gallolyticus (25). Another species, S. infantarius, that is closely related to the S. bovis complex and includes two subspecies was suggested by Schlegel et al. (29). Strains of S. bovis biovar II/1 were distributed among both subspecies. In addition, Poyart et al. (26) have suggested that the strains identified as S. bovis biovar II/2 should be renamed S. pasteurianus and the strains identified as S. infantarius subsp. coli be named S. lutetiensis. Based upon biochemical traits, DNA-DNA relatedness and divergence in 16S rrna sequences of the S. bovis-s. equinus complex and related species, Schlegel et al. (30) finally suggested S. gallolyticus to include three subspecies: gallolyticus, macedonicus, and pasteurianus. In addition, S. waius and S. intestinalis were indistinguishable from S. gallolyticus subsp. macedonicus and S. alactolyticus, respectively. In contrast to human medicine, S. bovis has been considered relatively unimportant in veterinary medicine (34). However, in pigeons S. bovis, subsequently suggested to be renamed as S. gallolyticus (14), has been reported as a major pathogen associated with septicemia (13, 10, 12), significant lesions including extensive areas of multifocal necrosis in different organs, arthritis, and endocarditis. Based upon growth characteristics and biochemical reactions, serotype 4 strains were subsequently identified as S. bovis (3). Increased mortality in turkeys of 1 to 3 weeks of age on three independent turkey facilities in California was reported by Droual et al. (15). However, none of the avian isolates have been characterized genotypically thus far, and it remains to be investigated to which taxa avian isolates of S. gallolyticus and S. bovis belong and whether avian isolates represent a new potential zoonosis. For the same reasons the aims of the present investigation were to characterize isolates from a recent outbreak in broilers, avian reference strains from the United States and available serovars and biovars associated with pigeon septicemia in Belgium and Denmark to investigate their phenotypic and genetic relationship and outline possible characteristics for phenotypic identification. * Corresponding author. Mailing address: Department of Veterinary Pathobiology, Veterinary Medicine, Life Science, Copenhagen University, Dyrlaegevej 88, Room 014A, 1870 Fredriksberg, Denmark. Phone: Fax: hech@life.ku.dk. Published ahead of print on 13 December MATERIALS AND METHODS Flock data. Of two broiler houses with an average flock size of approximately 63,000, one was affected. Mortality during the first week was normal ( 1%), with a final total mortality at the end of production (6 weeks) reaching 4.3% in the affected house compared to 2.7% in the unaffected house. Specific symptoms 822

2 VOL. 45, 2007 S. GALLOLYTICUS SUBSP. GALLOLYTICUS DIVERSITY IN BIRDS 823 TABLE 1. Origin of strains investigated Strain(s) Origin Source or reference Country S. bovis ATCC T ; CCUG T Cow feces 31 England S. gallolyticus subsp. gallolyticus CCUG T Koala feces 25 Australia S. gallolyticus subsp. macedonicus CCUG T Greek Kasseri cheese 35 Greece S. gallolyticus subsp. pasteurianus CIP T Human meningitis 26 France CCUG T MV1 (serovar 2; biovar 4) Pigeon Belgium 329 (serovar 2; biovar 1) Pigeon Belgium 669 (serovar 5; biovar 1) Pigeon Belgium 598 (serovar 3; biovar 2b) Pigeon Belgium 827 (serovar 1; biovar 2b) Pigeon Belgium C13466 ( ) Broilers, septicemia Denmark C16883 ( ) Pigeon squabs, septicemia Denmark Layers, endocarditis NCSU a United States a NCSU, J. Barnes, North Carolina State University, College of Veterinary Medicine. were not observed, but the feed conversion rate of the affected flock increased to 2.36 compared to 1.93 for the unaffected flock. An increased mortality was observed in the affected house during the last 2 weeks of rearing, and 10 dead chickens were received for postmortem examinations shortly before slaughter of the affected flock at the age of 6 weeks. Gross lesions from seven of the chickens included hepato- and splenomegaly accompanied by general vascular disturbances and discoloration of subperitoneal and subepicardial fat tissues. Multiple, focal, grayish 1- to 2-mm large necroses without a sharp demarcation or bleedings from the surrounding tissue were observed in five of these chickens. Two of the seven chickens which were emaciated had large multifocal irregular necroses surrounded by a hemorrhagic zone in the liver and spleen. In addition, these birds suffered from arthritis and osteomyelitis. One of these also had grayish irregular necroses in the myocardium. With the exception of a more or less pronounced tibial dyschondroplasia which was observed in all 10 chickens, the remaining three broilers did not show specific lesions. Histopathology, through hematoxylin-eosin staining of liver and spleen samples, confirmed the existence of multifocal necrosis accompanied by heterophil granulocyte infiltration and fibrinoid degeneration of sheathed capillaries in the spleen. Small colonies of cocci were observed in relation to the necrotic foci. The condemnation of birds in the affected flock was over twice that of the unaffected flock at 1.9% compared to 0.8%, respectively, and the reasons in- Downloaded from on April 27, 2018 by guest FIG. 1. Dendrogram derived from the unweighted-pair-group-average linkages of correlation coefficients (expressed for convenience as percentages of value) between PFGE profiles of all strains examined.

3 824 CHADFIELD ET AL. J. CLIN. MICROBIOL. cluded emaciation (1.0%), liver disorders and or ascites (0.6%), skin disorders (0.2%), and arthritis (0.1%). Bacteriology. Bacteriological cultures were made from livers and/or spleens on blood agar (Blood Agar Base CM 55; Oxoid, Basingstoke, United Kingdom), containing 5% sterile bovine blood. A single colony from each blood plate showing pure culture or a majority of one colony type was subcultured onto blood agar and reincubated at 37 C overnight. A total of 10 clinical isolates were isolated in pure culture after postmortem examination from the livers and spleens of the seven broilers showing typical lesions of septicemia (Table 1). The isolates were subsequently harvested in brain heart infusion broth (Difco, Heidelberg, Germany) with 30% (vol/vol) glycerol (KVL Pharmacy, Copenhagen, Denmark) and stored at 80 C until use. For comparison with the clinical outbreak strains, reference type strains of relevant streptococcal species, including S. bovis and S. gallolyticus and its three subspecies (gallolyticus, macedonicus, and pasteurianus), five pigeon strains (Belgium) representing reference biovars and/or serovars of S. gallolyticus and two further pigeon field isolates (Denmark), and three reference field isolates of S. bovis isolated from layers of 56 weeks of age in the United States, were included (Table 1). Differentiation between Enterococcus and Streptococcus species was verified by the ability to enzymatically hydrolyze a 1-pyrrolidonyl- -naphthalamide substrate (36). Isolates were investigated for the presence of Lancefield group D antigen (16) by using a latex bead agglutination reagent after enzymatic lysis at 37 C for 30 min (Oxoid). Enzymatic reactions and fermentation of carbohydrates were determined by using API 20 STREP system (17) according to the manufacturer s instructions (biomérieux, La Balme les Grottes, France), in addition to other conventional methods for characterization (5). Growth and colonial appearance were tested on Slantez agar (Oxoid) and Edwards agar (Oxoid) with 5% bovine blood. Clotting in litmus milk (Oxoid) was also tested. Tannase production was determined by a modification of the method used by Osawa et al. (25), where 0.3% filter-sterilized tannic acid was incorporated into BHI agar (Difco, Heidelberg, Germany) with 0.5% yeast extract, followed by inoculation and culture for up to 72 h anaerobically. Genotypic characterization. (i) Purification of chromosomal DNA. Strains were cultured in 10 ml of BHI broth (Difco) and incubated overnight at 37 C with shaking. A total of 200 l of the bacterial broth was added to 800 l ofse buffer (75 mm NaCl [Merck, Darmstadt, Germany], 25 mm EDTA [ph 7.4; Merck]), and the optical density at 578 nm was measured. A 3-ml portion of the bacterial suspension was centrifuged at 4,000 rpm for 10 min; the supernatant was then discarded, and the pellet was resuspended in 4.5 ml of SE buffer. The cell suspension was then centrifuged as described above. After the supernatant was discarded, SE buffer was added to the cell suspension. A 500- l portion of a solution containing 1 g of chromosomal-grade agarose (Bio-Rad, Richmond, CA) and 50 ml of SE buffer was mixed with 500 l of cell suspension for each sample and formed into an agarose block. The blocks containing the cells were lysed under incubation at 37 C overnight in lysozyme (Boehringer-Mannheim, Germany). The blocks were then washed for 30 min in TE buffer (10 mm Tris [Sigma, Steinheim, Germany], 10 mm EDTA [ph 7.4]) and incubated overnight at 56 C in a solution of proteinase K (Roche). The blocks were washed once in cold (4 C) TE buffer and then kept at room temperature for 1 h. The blocks containing purified DNA were stored in TE buffer at 4 C until use. The purified DNA was washed in EN buffer 2 (B7002S; BioLaps) and digested with the restriction endonucleases SmaI (R0141S; BioLaps) for pulsed-field gel electrophoresis (PFGE). (ii) PFGE. PFGE was carried out as described by Ojeniyi et al. (23) with digestion of DNA of 15 clinical isolates and 9 reference strains by SmaI. The gels were prepared with 1% agarose (Seakem GTG Agarose; FMC Bioproducts) in 0.5 TB buffer (Tris/boric acid [Merck], 0.5 M EDTA [ph 8.2]) and evaluated using the CHEF-DR III system (Bio-Rad) for 20 h at 14 C in 0.5 TB buffer. The conditions for running included the following: for block 1, initial time of 1 s, a final time of 15 s, 5.6 V/cm, included angle of 120, and an actual current of 120 V. The gel was placed in a solution of ethidium bromide (200 l; 10 mg of ethidium bromide [Sigma]/ml in 1 liter of deionized water) for 15 min and kept for 15 min in deionized water and then photographed under UV light. PFGE profiles produced from the clinical isolates were analyzed by the unweightedpair-group method, and a comparison was made by using the DICE similarity coefficient. (iii) Ribotyping. Isolation of DNA and digestion with HindIII (Boehringer- Mannheim) and subsequent electrophoretic separation of the DNA fragments using agarose gel electrophoresis was performed as previously described (7). DNA was transferred to nylon hybridization membranes (Hybond-N; Amersham, United Kingdom) by vacuum blotting and fixed to the membrane by incubation at 80 C for 1 to 3 h. rrna (16S and 23S) from Escherichia coli (Sigma) was labeled with digoxigenin by using reverse transcriptase (Boehringer- FIG. 2. Phylogenetic analysis of sequenced strains C (chicken isolate, DK) and 283 (chicken isolate, US), five reference strains (MV1, 669, 329, 598, and 827 [pigeon isolates, Belgium]), and 46 additional sequences from GenBank (Table 2). The maximumlikelihood analysis showed two monophyletic groups and one paraphyletic group of strains, with the type strain of S. dysgalactiae located as an outgroup. The avian strain sequences were located in the paraphyletic group between the monophyletic groups; however, no particular subgroups could be identified in this group, including type strains of the three subspecies of S. gallolyticus. (The boxed strain numbers indicate the sequenced strains from the present study). Mannheim) as previously reported (7). The probes were hybridized with membrane-fixed DNA under the conditions described by Christensen et al. (7). Sequencing of 16S rrna genes. 16S rrna gene sequencing was performed as previously reported (2, 8). In all, seven strains were subjected to 16S rrna gene sequencing: a single outbreak strain (C ), representing the clonal outbreak in broilers (Denmark); an isolate from layer birds in the United States (isolate 283); and the five pigeon reference strains (Belgium). Searches for sequence data in GenBank (4) was performed by BLAST (1). Pairwise comparisons for similarity were performed by Bestfit (Wisconsin Sequence Analysis Package Genetics Computer Group, Madison, WI). Multiple alignments were performed by using CLUSTALX (33). The region from positions 107 to 1327 of the E. coli rrnb gene was compared. Maximum-likelihood analysis was performed by fastdnaml, including bootstrap analysis (24) run on a Linux 7.2 compatible server. The transition/transversion ratio was set to 1.5. Parsimony, neighbor-joining, and consensus comparisons were computed by PHYLIP (19). RESULTS AND DISCUSSION Genotypic analysis. Ribotyping (HindIII) demonstrated the same pattern of only two bands for all 10 chicken iso-

4 VOL. 45, 2007 S. GALLOLYTICUS SUBSP. GALLOLYTICUS DIVERSITY IN BIRDS 825 TABLE 2. Type strains of streptococci included for 16S rrna sequenced comparative investigations Reference strain Origin Strain Accession no. Primary strains S. equinus Bovine HDP89506 T AF S. infantarius subsp. coli Unknown HDP90246 T AF S. infantarius subsp. infantarius Human HDP90056 T AF S. gallolyticus subsp. gallotyticus Koala ACM3611 T X94337 S. gallolyticus subsp. macedonicus CCUG T Z94012 S. gallolyticus subsp. pasteurianus Human CIP T AF S. alactolyticus Porcine ATCC T AF Heterotypic synonyms published after the species above S. bovis (S. equinus) Bovine ATCC T AB S. intestinalis (S. alactolyticus) Porcine ATCC T AB S. waius (S. gallolyticus subsp. macedonicus) Dairy NZRCC20100 T AF Outgroup S. dysgalactiae subsp. dysgalactiae Pig CCUG27301 T AB Other sequences included in the group S. bovis Pig ATCC AF Human and ruminants JB1 AF Bovine H2-2 AF Kangaroo YE49 AY Bovine C14b1 AF Ruminants NCFB2476 AF Ruminants 1276 AF Human NCTC11436 AJ Camel MPR5 AY Sheep SbR1 AY Bovine Sb5 AY Ruminant B315 AF Camel and deer RD11 AY Camel MPR4 AY Camel MPR2 AY Camel MPR1 AY Camel and deer RD09 AY S. caprinus Goat ACM3968 Y10869 S. gallolyticus Goat ACM3970 Y10867 S. equinus Horse ATCC 9812 AJ S. gallolyticus Human and ruminants ATCC AF Shea cake DSM13808 AF S. infantarius Human/food HDP90104 AF S. luteciae Unknown NEM782 AJ Unknown NEM1603 AJ S. luteciae Unknown NEM760 AJ S. macedonicus Human AF S. pasteuri Unknown NEM1205 AJ S. pasteuri Unknown NEM1202 AJ Streptococcus sp. Bovine LRC0255 AF Bovine LRC0476 AF Ruminant LRC0253 AF Sheep ES11 AF Goat KN3 AF lates (from Denmark) and the two Danish pigeon isolates, whereas the type strain of S. bovis demonstrated several bands only one of which was of the same size as observed for all other isolates (data not shown). Further discrimination was afforded by using PFGE (SmaI), where the three origins of the strains demonstrated separate clonal structures (Fig. 1). The chicken isolates (identified as DK) (C13466, 115 to -122) exhibited the same profile with the exception of three isolates (115, 116, and 121), which differed by a single band. The chicken isolates from the United States (283 to 285) demonstrated an identical pattern but different from the chicken isolates from Denmark (Fig. 1). Similarly, the two pigeon isolates from Denmark (223 and 224) were identical, demonstrating yet another profile distinct from the other two clones. Dendrogram analysis showed these three clones to have 40% similarity (Fig. 1) and 30% similarity with the reference strain of S. gallolyticus subsp. gallolyticus (CCUG T ). It was clear from the study that PFGE

5 826 CHADFIELD ET AL. J. CLIN. MICROBIOL. TABLE 3. Phenotypic characters separating the former Streptococcus bovis-s. equinus complex Characteristic Finding on clinical isolates (chickens) a Finding on reference strain S. bovis T S. gallolyticus subsp.: gallolyticus T macedonicus T pasteurianus T Browns and blackens on Edwards agar Color of colonies on Pink White Pink Pink Pink Slanetz agar Clotting of litmus milk Voges-Proskauer test Tannase -Glucosidase -Glucuronidase Produces acid from: L( )-Arabinose ( ) D( )-Mannitol Trehalose Inulin /( ) Esculin D( )-Glycogen a Obtained from Denmark and the United States. might represent a more discriminative typing method, the potential of which remains to be investigated. Phylogenetic analysis. The 16S rrna sequences of the pigeon reference strains (Belgium) STR 329, STR 669 and the chicken isolate (United States) 283 were identical with each other, as were the sequences of PDH 827 and strain STR 598 (Belgium pigeon reference strains) (Fig. 2). The lowest similarity between the seven strains sequenced was 99.4% between strains MV1 and strains STR 283, STR 329, and STR 669. Phylogenetic analysis was performed with the strains sequenced in the present investigation and 46 additional sequences from GenBank, including the 16S rrna gene sequence of the type strain of S. dysgalactiae as an outgroup (Fig. 2 and Table 2). The maximum-likelihood analysis showed two monophyletic groups and one paraphyletic group of strains, with the type strain of S. dysgalactiae located as an outgroup. The three groups were confirmed by neighbor-joining and maximum-parsimony analysis, and the three groups were supported by bootstrap values of 74% or higher (Fig. 2). The avian strains sequenced in the present study were all located in the paraphyletic group between the monophyletic groups. Due to low sequence variation, no particular subgroups could be identified in this group, including type strains of the three subspecies of S. gallolyticus and strains classified as S. pasteuri, S. caprinus, S. gallolyticus, S. bovis, and Streptococcus sp. The highest similarities of the seven avian strains were found to the type strains of S. gallolyticus subsp. pasteurianus (99.8 to 99.4%), the type strain of S. gallolyticus subsp. macedonicus (99.7 to 98.8%), the type strain of S. waius (99.7 to 98.9%), and S. gallolyticus subsp. gallolyticus (98.4 to 97.8%). The largest monophyletic group showing a bootstrap value of 78%, referred to below as S. bovis-s. equinus plus S. infantarius, included the type strains of S. bovis, S. infantarius, and S. equinus and strains labeled as S. bovis, S. luteciae, and Streptococcus sp. The avian strains showed between 98.3 and 98.9% similarity to strains within this group. The other monophyletic group included the type strains of S. alactolyticus and S. intestinalis. The avian strains showed between 96.3 to 98.1% similarity to strains within this group. Phenotypic analysis. The colony appearance on Slanetz agar, together with browning and blackening on esculin-containing Edwards agar and clotting of litmus milk, effectively distinguished between S. bovis (positive) and S. gallolyticus (negative) (Table 3). Separation between subspecies of S. gallolyticus was achieved, demonstrating tannase and -glucuronidase activity and the production of acid from D( )-mannitol, trehalose, inulin, esculin, and glycogen (Table 3). Clinical isolates from chickens in Denmark and the United States all shared the same phenotype, which was identical to the type strain of S. gallolyticus subsp. gallolyticus, although the genotypes were different. We observed 99.9% 16S rrna similarity between the Danish and American isolates, both of which showed 98.4% 16S rrna similarity with the type strain of S. gallolyticus subsp. gallolyticus. Three reference strains from pigeons MV1 (serovar 2; biovar 4), STR 329 (serovar 2; biovar 1), and PDH 827 (serovar 1; biovar 2b) were all -glucosidase, tannase, glycogen, and esculin positive and -glucuronidase negative. Although differences from the type strain of S. gallolyticus ssp. gallolyticus were observed in L( )-arabinose (MV1 positive), D( )-mannitol (PDH 827 negative), trehalose (PDH 827 negative), and inulin (STR 329 negative), these strains were classified as S. gallolyticus subsp. gallolyticus. All of these strains demonstrated 16S rrna similarities of 99.8%, whereas similarities to the type strain of S. gallolyticus subsp. gallolyticus varied between 97.8% (MV1) and 98.4% (STR 329). Four pigeon strains were tannase negative (STR 598, STR 669, 223, and 224), and two of these showed differences from the type strain of S. gallolyticus subsp. gallolyticus in D( )-mannitol (STR 598 negative), trehalose (STR 598 negative), and inulin (STR 669 negative). STR 598 only showed 97.2% 16S rrna similarity to the type strain of S. gallolyticus subsp. gallolyticus, whereas 97.6% similarity was observed with STR 669. Three to five phenotypic characteristics separated these isolates from S. gallolyticus subsp. macedonicus and pasteurianus; however, 99.7 and 99.5% and 99.0 and 99.6% 16S rrna similarities were observed with STR 669 and STR 598, respec-

6 VOL. 45, 2007 S. GALLOLYTICUS SUBSP. GALLOLYTICUS DIVERSITY IN BIRDS 827 tively, underlining the present uncertainty of using phenotypic criteria for identification of subspecies of S. gallolyticus. Isolates belonging to the same serovar (MV1 and STR 329) were genetically very different, showing 20% similarity according to the PFGE band patterns (Fig. 1). Similar differences may exist for other serovars, explaining differences in supernatant phenotypes and the lack of serovar-specific antigens to protect against S. gallolyticus septicemia in pigeons (22). Hoshino et al. (21) recently evaluated phenotypic and molecular methods for the identification of nonhemolytic streptococci. The rate of correct identification of the strains by both commercial identification systems using the associated databases was below 50% but varied significantly between species. Identification based on multilocus sequence analysis was found to be optimal but laborious. Using these methods, S. gallolyticus and its three subspecies clustered together, branching deeply from S. bovis T, S. lutetiensis, and S. infantarius in accordance with our findings. In conclusion, the present study documents S. gallolyticus subsp. gallolyticus as a pathogen for chickens and demonstrates that strains associated with disease in pigeons might represent different taxa. Finally, problems associated with the correct identification of streptococci were underlined, raising a question as to the unambiguousness of some of the previous publications on S. gallolyticus. ACKNOWLEDGMENTS We acknowledge the kind contribution of the pigeon reference strains from L. Devriese at the Faculty of Veterinary Medicine, University of Ghent, Merelbeke, Belgium, and the clinical isolates associated with layer birds from J. Barnes, College of Veterinary Medicine, North Carolina State University, Raleigh. REFERENCES 1. Altschul, S. F., W. Gish, E. W. Myers, and D. J. 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