Meat Science Publications Meat Science

12-1998 Prevalence and Genetic Variability of Species in Mechanically Separated Turkey Tamara R. Manke Iowa State University

Irene V. Wesley United States Department of Agriculture

James S. Dickson Iowa State University, [email protected]

Karen Harmon United States Department of Agriculture

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Recommended Citation Manke, Tamara R.; Wesley, Irene V.; Dickson, James S.; and Harmon, Karen, "Prevalence and Genetic Variability of Arcobacter Species in Mechanically Separated Turkey" (1998). Meat Science Publications. 5. http://lib.dr.iastate.edu/meatscience_pubs/5

This Article is brought to you for free and open access by the Meat Science at Iowa State University Digital Repository. It has been accepted for inclusion in Meat Science Publications by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Prevalence and Genetic Variability of Arcobacter Species in Mechanically Separated Turkey

Abstract A survey for Arcobacter spp. and Arcobacter butzleri in mechanically separated turkey was conducted during the winter of 1995 and summer and fall of 1996. Arcobacter spp. and A. butzleri were identified by polymerase chain reaction and species-specific lo igonucleotide probes. Arcobacter spp. were isolated from 77% (303 out of 395) of the mechanically separated turkey samples with 74% (223 out of 303) of these samples positive for A. butzleri. Of the 121 A. butz/eri isolates tested, 86 different patterns were evident following amplification of enterobacterial repetitive intergenic consensus sequences. The extent of genetic polymorphism indicated multiple sources of contamination.

Keywords DNA bacterial analysis, food handling, genetic variation, gram-negative

Disciplines Bacteriology | Food Microbiology | Meat Science | Poultry or Avian Science

Comments This article is from Journal of Food Protection 61 (1998): 1623.

Rights Works produced by employees of the U.S. Government as part of their official duties are not copyrighted within the U.S. The onc tent of this document is not copyrighted.

This article is available at Iowa State University Digital Repository: http://lib.dr.iastate.edu/meatscience_pubs/5 1623

Journal of Food Protection, Vol. 61, No. 12, 1998, Pages 1623-1628 Copyright ©, International Association of Milk, Food and Environmental Sanitarians

Prevalence and Genetic Variability of Arcobacter Species in Mechanically Separated Turkeyt:!:

TAMARA R. MANKE,I IRENE V. WESLEY? JAMES S. DICKSON,l* AND KAREN M. HARMON2

lIowa State University, Department of Microbiology, Immunolog); and Preventive Medicine, Iowa State University, Ames, Iowa 50011,. and 2Enteric Diseases and Food Safety Research Unit, National Animal Disease Center, USDA, Agricultural Research Sen'ice, Ames, Iowa 50010, USA

MS 97-186: Received 6 August 1997/Accepted 15 March 1998

ABSTRACT

A survey for Arcobacter spp. and Arcobacter butzleri in mechanically separated turkey was conducted during the winter of 1995 and summer and fall of 1996. Arcobacter spp. and A. butzleri were identified by polymerase chain reaction and species-specific oligonucleotide probes. Arcobacter spp. were isolated from 77% (303 out of 395) of the mechanically separated turkey samples with 74% (223 out of 303) of these samples positive for A. butzleri. Of the 121 A. butz/eri isolates tested, 86 different patterns were evident following amplification of enterobacterial repetitive intergenic consensus sequences. The extent of genetic polymorphism indicated multiple sources of contamination.

Aerotolerant, vibrio-like organisms were first isolated survey and 90.0% of the pork samples (n = 30) tested from aborted bovine and porcine fetuses (8, 9, 22) and during the second survey (4) from a single pork-processing classified as Campylobaeter eryaerophila (21). After morpho- establishment. In contrast, only 5% ofthe samples (n = 120) logical (21), biochemical (21), and phenotypic (14) charac- obtained from four other pork-processing establishments terizations, as well as DNA-DNA (14) and DNA-rRNA yielded Areobaeter spp. hybridizations (28), it was proposed that C. eryaerophila be The Arcobacter Selective Broth and the Arcobacter placed into the new genus, Areobaeter (28). The four species Selective Medium developed by de Boer et al. have been of Areobaeter include A. eryaerophila (subgroups lA and used to enrich for Areobaeter spp. in poultry, beef, and pork IB), A. butzleri, A. skirrowii, andA. nitrofigilis (31). (5). Using this protocol, Areobaeter spp. were isolated from The epidemiology of Areobaeter spp. is not fully 24.1 % of the poultry samples (n = 220) tested with lower understood. A. eryaerophilus organisms have been isolated recoveries, 4.9% and 0.5% for beef and pork, respectively from aborted as well as healthy livestock (23) and from (5). Lammerding used a modified Rosef broth to enrich for human stool samples (14, 26). A. butzleri has been cultured Areobaeter spp. in poultry products (16). Using this method, from animals (1, 19, 22, 32-34) and from humans with A. butzleri was isolated from 96.8% of the broiler chicken diarrhea and/or abdominal cramps (14, 15, 18, 26, 27, 30). carcasses (n = 125) and from 85.7% of the fresh ground The clinical symptoms of A. butzleri suggest that it is a turkey samples (n = 7) (16). human pathogen (14, 15). A. butzleri organisms have also Proper identification of Areobaeter is needed in order to been isolated from water (6, 10, 13), poultry (2, 5, 10, 16, understand fully its role in causing human foodborne illness. 20), and pork (4, 5). The morphological similarities between Areobaeter spp. and Various enrichment methods have been used to recover Campylobaeter spp. may lead to the misidentification of the Areobaeter spp. from meats (2, 4, 5, 16). The Leptospira organisms when relying on the traditional plating methods semisolid medium, Ellinghausen-McCullough-Iohnson- and dark-field microscopy. The two organisms do show Rarris Polysorbate-80 (EMIR P-80), was first used to detect some physiological differences: Areobaeter spp. grow at Areobaeter in aborted livestock fetuses (8, 9). EMIR P-80 15°C in the presence of oxygen and in 1.5% NaC!, whereas has also been used to enrich for Areobaeter spp. in ground Campylobaeter spp. require growth at 37°C under microaero- pork (4). In that study, Areobaeter spp. was isolated from bic conditions (3 to 10% oxygen) (28). 89.9% of the pork samples (n = 149) tested during the first The use of oligonucleotide DNA probes (32) and polymerase chain reaction (PCR)-based methods (3, 12) provides an alternative method to identify Areobaeter spp. * Author for correspondence. Tel: 515-294-4733 (voice); Fax: 515-294- These methods are based on identifying sequences that are 6019; E-mail: jdickson@iastate,edu. t Journal paper no. J-17547 of the Iowa Agriculture and Home Economics specific for the 16S rRNA or 23S rRNA genes of Areobaeter Experiment Station, Ames, Iowa, Project No. 3250, and supported by the spp. andA. butzleri (3, 12,34). PCR-based DNA fingerprint- Hatch Act and State of Iowa funds. ing has elucidated the epidemiology of Areobaeter (29). :j: Names are necessary to report factually on available data; however, the This method relies on amplification of the enterobacterial U.S. Department of Agriculture (USDA) neither guarantees nor warrants repetitive intergenic consensus (ERIC) sequences found in the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be gram-negative organisms. The resultant DNA patterns allow suitable. for the differentiation of the isolates (31). In one study, DNA 1624 MANKE ET AL. J. Food Prot., Vol. 61, No. 12 fingerprints of outbreak-related strains of A. butzleri were Mechanically Separated Turkey Samples identical (29). In contrast, multiple patterns would indicate strain differences and thus suggest more than one source of contamination. Despite the application of PCR-based ERIC fingerprinting in epidemiological studies, the function of Enrichment in P-80 (7 Days, 30° C) these repetitive DNA sequences is unknown. The Nationwide Raw Ground Turkey Microbiological Survey conducted by the Food Safety and Inspection Service (FSIS) found that 25.4% of the raw ground turkey samples Subcultured in P-80 (3 Days, 30° C) (n = 295) were contaminated with Campylobaeter jejuni/ eoIi (11). Other surveys on turkey products indicate that C.jejuni contamination ranges from 0 to 90% (17, 35). Since Areobaeter is closely related phylogenetically to and is more PCR to Identify Arcobacter spp. Positive Samples aerotolerant than Campylobaeter, it is plausible that Areobae- ter may also be found at high levels in poultry. In two different studies, Areobaeter was isolated from 24.1 % of poultry samples (n = 220) (5) and six of seven fresh ground Southern Transfer of PCR Gel turkey samples (16). In a pilot study on turkey skin samples conducted in our laboratory, all samples (n = 12) were found to be positive for A. butzleri (4). The morphological similarities between Areobaeter Hybridization to Identify A. butzleri Positive Samples spp. and Campylobaeter spp. as well as their presence in FIGURE 1. General schematic flow diagramfor the identification turkey products led to surveying mechanically separated of Arcobacter spp. and A. butzleri for the winter mechanically turkey. Mechanically separated turkey is widely used in the separated turkey samples. production of both cooked and raw meat products. The presence of Areobaeter spp. in mechanically separated spp. and A. butzleri in the winter survey samples. Figure 2 shows a turkey could represent a potential foodborne hazard. general schematic flow diagram for the identification ofArcobacter The objective of this study was to determine the spp. and A. butzleri in the summer and fall survey samples. Upon prevalence of Areobaeter spp. and A. butzleri in mechani- delivery, 10 g of each mechanically separated turkey sample were cally separated turkey. In addition, the summer and fall enriched in 50-ml plastic centrifuge tubes (Blue Max, Becton A. butzleri isolates were analyzed for genetic variation in Dickinson, Lincoln Park, N.J.) containing 20 ml of EMJR P-80 ERIC sequences. semisolid media (7) supplemented with agar and 100 mg of 5-fluorouracil per liter (4,22). MATERIALS AND METHODS Sampling techniques. Three surveys were conducted. The Mechanically Separated Turkey Samples winter (initial) survey consisted of 100 mechanically separated turkey samples that were obtained from a poultry plant (A) on four separate dates in January and February 1996. Two additional Enrichment in P-80 (3 Days, 30° C) surveys, summer and fall 1996, were conducted after the initial survey data were analyzed. The summer and fall surveys were expanded to include the initial plant (A) along with two additional plants (B and C) in two different states. The summer survey Subcultured in P-80 (3 Days, 30° C) consisted of 145 mechanically separated turkey samples, 25 samples from each plant (except plant B, which furnished 45 samples), collected on two separate dates in July and August 1996. The fall survey consisted of 150 mechanically separated turkey PCR to Identify Areobaeter spp. Positive Samples samples, 25 samples from each plant collected on two separate dates in September 1996. The mechanically separated turkey used in the surveys was typical of that used in the meat industry. It consisted of a fresh homogeneous mixture composed of skeletal Extraction of DNA from the Areobaeter spp. Positive Samples tissue, skin, and nonmeat ingredients (salt and sodium nitrite). All samples were collected using the same method: 25 samples were sent per date with five samples (75 g each) from each of five randomly selected containers of product ("combos", ~900 kg DNA Dot Blot Hybridization with A. butzleri-specific Probe each). The samples were collected by plant personnel and were shipped overnight on ice to the National Animal Disease Center, Ames, Iowa. Flow diagrams for the isolation and identification of DNA Fingerprinting of A. butzleri Positive Samples Arcobacter spp. and A. butzleri for the winter (Fig. 1) and for the summer and fall surveys (Fig. 2) are given. FIGURE 2. General schematic flow diagramfor the identification Arcobacter spp. enrichment techniques. Figure I shows a of Arcobacter spp. and A. butzleri for the summer and fall general schematic flow diagram for the identification ofArcobacter mechanically separated turkey samples. J. Food Prot., Vol. 61, No. 12 ARCOBACTER IN MECHANICALLY SEPARATED TURKEY MEAT 1625

The winter samples were enriched (7 days, 30°C), subcultured PCR reaction mixture consisted of 25 pmol each of ERIC lR and (1 ml of enrichment into 9 ml of fresh EMJH P-80), and incubated ERIC 2,10 mmollitecl Tris-HCl, 50 mmollitecl KCl, 2.0 mmol for an additional 3 days at 30°C. The summer and fall samples were litecl MgCl2, and 200 mmolliter-I each of the four dNTPs and enriched (3 days, 30°C), subcultured (1 ml of enrichment into 9 ml 1.25 U of Taq polymerase (Boehringer Mannheim). PCR was of fresh EMJH P-80), and incubated (3 days at 30°C). performed in a thermal cycler (Perkin-Elmer Cetus, Norwalk, Pure cultures were obtained as follows; A 0.5-ml aliquot from Conn.) using previously described conditions (12). The PCR the EMJH-P-80 enrichment was filtered onto 0.45-llm membranes product was analyzed by gel electrophoresis as described above. placed onto the surface of brain-heart infusion agar (Difco) The photographs of the gels were scanned using the Gel Doc 1000 supplemented with 10% defibrinated bovine blood. After 30 min, (Bio-Rad, Hercules, Calif.) and profiles analyzed using the Molecu- the membranes were removed, the filtrate was streaked for colony lar Analyst Software (Bio-Rad). This allowed for molecular weight isolation, and plates were incubated aerobically (30°C for 48 to values to be assigned to each of the isolates for comparison of 72 h). banding patterns. Based on the molecular weights and visual inspection of the photographs, differences between isolates were Arcobacter spp. identification. For all surveys, a 250-I.tI obtained. aliquot (previously frozen) of each subculture was used to perform the PCR reaction for the detection of Arcobacter spp. The aliquots Statistical analysis. To determine if the incidence of Arcobac- were boiled (15 min, 1l0°C) and centrifuged (1 min, 1l,000 X g). ter and A. butzleri was significantly different in samples obtained A 5-1l1aliquot of the supernatant served as the PCR template. The from the three plants, a chi-square test of independence was reagents and conditions for the PCR reaction were as described conducted. P values of 0.05 or less were considered statistically (12). The amplified DNA product was analyzed by gel electropho- significant. To determine if the incidence of A. butzleri was resis (120 V, 1 h) on a 1.5% agarose gel (Seakem ME agarose, FMC disproportionately distributed between the three plants, a binomial Bioproducts, Rockland, Maine) using a 6.5 X lO-cm horizontal gel test for distribution was conducted. A binomial test P value of 0.05 bed (Minnie the Gel-Cicle, Hoefer Scientific Instruments, San was considered statistically significant (24). Francisco, Calif.) and TBE (0.09 M Tris, 0.09 M boric acid, 0.002 RESULTS M EDTA, pH 8.5) as the running buffer. The gel was stained with ethidium bromide, visualized with UV light, and photographed as The initial (winter) survey consisted of samples from described previously (12). only plant A. Arcobacter spp. were isolated from 92% (92 of Arcobacter butzleri identification. For the winter survey, the 100) of the samples with a total of 87% (80 of 92) positive agarose gel containing the Arcobacter spp. amplicons was dena- for A. butzleri. A representative dot-blot hybridization, tured in 0.5 M NaOH, 1.5 M NaCI (30 min, 4°C), neutralized in 1 which was used to identify A. butzleri, is shown in Figure 3. M Tris-base, 1.5 M NaC!, pH 5.5 (30 min, 4°C), and transferred The high contamination rate of Arcobacter in meat samples onto a nylon membrane (Nytron, Schleicher & Schuell, Keene, obtained from plant A in the winter survey led to summer N.H.) using the method of Southern (25) with the Turboblotter and fall surveys in which the same plant plus two additional Rapid Downward Transfer System (Schleicher & Schuell). After plants (B and C) were tested. Based on the three samplings, transfer, the membrane was placed on filters saturated with 0.4 N plant A had 96% (191 of 200) of the samples positive for NaOH (1 min) and then on filters saturated with 0.025 M Na2HP04 Arcobacter spp.; 80% (153 of 191) of these were identified (1 min). The immobilized DNA on the membrane was crosslinked as A. butzleri (Table 1). For plant B, 72% (68 of 95) of the on both sides using the UV Stratalinker 1800 (Stratagene, La Jolla, Calif.). samples were positive for Arcobacter spp. with 65% (44 of The membrane was prehybridized (3 h, 3rC) using the 68) identified as A. butzleri (Table 1). For plant C 44% (44 Genius System hybridization solution (Boehringer Mannheim, out of 100) of the samples were positive for Arcobacter spp. Indianapolis, Ind.) and then hybridized (18 h, 3rC) with the with 59% (26 of 44) positive for A. butzleri (Table 1). A total Genius System hybridization solution containing the digoxigenin of 77% (303 of 395) of the samples were positive for (DlG)-labeled A. butzleri species-specific probe (34). After hybrid- Arcobacter spp. with 74% (223 of 303) of these confirmed as ization, the membranes were washed and incubated with the DIG A. butzleri (Table 1). detection system according to the manufacturer's directions. The membrane was exposed (60 min, room temperature) to X-ray film (X-Omat, Kodak, Rochester, N.Y.) and developed using the X-Omat Film Processor (Kodak). Isolates of Arcobacter spp. from the summer and fall surveys were identified as A. butzleri by dot-blot hybridization using cesium-chloride-purified DNA and the species-specific probe (34). Purified DNA (2 Ilg) was immobilized on nylon membranes (Nytron, Schleicher & Schuell) as described (34). A. butzleri served as a positive control, whereas A. cryaerophilus lA and IB served as negative controls for the assay. The membrane was probed with the A. butzleri-specific oligonucleotide probe, washed, and exposed to X-ray film as described above. FIGURE 3. Dot-blot hybridization of Arcobacter butzleri DNA of DNA fingerprinting of isolates. Genetic variability of the field strains isolated from mechanically separated turkey and field strains was determined by PCR with primers targeting the hybridized with the A. butzleri species-specific probe. Each sample ERIC motifs. The DNA from 121 of the summer and fall A. butzleri was analyzed in duplicate. Wells AI-4 contain Arcobacter cry- isolates was amplified using primers, ERIC lR (5'-ATG-T AA- aerophilus (negative controls). Wells A5 to 6 contain A. butzleri GCT-CC T-GGG-GAT-TCA-C-3') and ERIC 2 (5'-AAG-TAA- (positive control). Wells BI to D12 contain field isolates. Wells A7 GTG-ACT-GGG-GTG-AGC-G-3') as described (29). The 50-Ill to 12 are empty. 1626 MANKE ET AL. J. Food Prot., Vol. 61, No. 12

TABLE 1. Recovery of Arcobacter spp. and A. butz1eri isolated P > 0.25), there was a disproportionately higher number from mechanically separated turkey samples contaminated with A. butzleri from plants A (64.7%;

No. positive Arcobacter/ No. positive A. butzleril P < 0.01) and B (80.1 %; P < 0.05). Plant no. samples tested (%) no. Arcobacter samples (%) A total of 121 summer and fall A. butzleri isolates were analyzed for distinct DNA amplification patterns by PCR- Aa 191/200 153/191 based DNA fingerprinting (29). Eighty-six different patterns (96) (80) were obtained from the 121 isolates. A representative set of Bb 68/95 44/68 DNA profiles is shown in Figure 4. Twenty of the 86 patterns (72) (65) were repeated at least twice in either the same plant or in two Cb 44/100 26/44 (44) (59) different plants. Overall, 71 % (86 different profiles for 121 TotalC 303/395 223/303 isolates) of the isolates displayed unique DNA amplification (77) (74) patterns. In plant A, 64% (38 different patterns for 59 isolates) of the isolates displayed unique patterns. In plant B, a Tested in winter, summer, and fall. 89% (32 different patterns for 36 isolates) of the isolates b Tested in the summer and fall. displayed unique patterns. In plant C, 81% (21 different C Combined data from all three plants. patterns for 26 isolates) of the isolates displayed unique patterns. The recovery of Arcobacter spp. using a 7-day (winter samples) versus 3-day (summer and fall) primary enrich- DISCUSSION ment was compared using the chi-square test for indepen- dence. For plant A, the only facility sampled in the winter, Arcobacter spp. (77%) and A. butzleri (56%) were 92% (92/100) of samples yielded Arcobacter; 80% (80/100) present in a total of 395 samples of mechanically separated yielded A. butzleri. In the summer and fall surveys, for plant turkey obtained from three processing sites. Species were A, Arcobacter spp. were detected in 99% (99/100) of the confirmed by dot-blot hybridization with the A. butzleri samples collected; A. butzleri was detected in 73% (73/100) species-specific probe (34). Differences in the recovery rate of the samples. Thus, the shorter primary enrichment used between plants were noted. Plant A had the highest recovery for the summer and fall samples yielded a statistically for Arcobacter spp. (96%) of which 80% of the positive significant increase in the recovery of Arcobacter spp. samples were confirmed as A. butzleri. Plant C yielded the (P < 0.04). The shorter primary enrichment, however, lowest percentage of Arcobacter (44%) with 59% (26 of 44) yielded no statistically significant differences in the recovery of these positive for A. butzleri. Differences in recovery of of A. butzleri (P > 0.20). Arcobacter were reported earlier for ground pork obtained The isolation rates of Arcobacter and A. butzleri for all from various sources (4). Collins et al. detected Arcobacter three plants were compared using the chi-square test for in 89% of ground pork samples (n = 149) obtained from a independence. Differences in recovery of Arcobacter from slaughter facility (plant 1). In a later survey involving plant 1 plants A, B, and C were statistically significant (P < 0.01). and four other premises (plants 2 through 5), 90% of Inspection of the data suggested that A. butzleri was samples from plant 1 were again positive, but only 5% of the recovered at higher levels in some of the plants. The total 120 samples from the four other facilities (plants 2 binomial distribution test for recovery of A. butzleri was through 5) yielded Arcobacter spp. (4). compared for all three facilities. Except for plant C (59.1 %; Significant differences were found in the recovery of Arcobacter between plants A, B, and C. In addition, a bp disproportionately high number of A. butzleri strains were recovered in plants A and B but not in C. Sources of turkeys and differences in plants (geographic location, age) could all account for the variations. In an earlier study, it was not

2176_ determined whether management practices at the source 1766- farms or the sanitary conditions during hog slaughter 1230_ 1033_ influenced the prevalence of Arcobacter spp. in ground pork (4). Likewise, in this study, although the causes of contami- 653- nation of the turkey product were not explored, variations 517- 453- between plants could be due to several factors, including the 394- 298- source of the birds, slaughter practices, and environmental 234/220- contamination during processing. Thus, the factors contribut- 154_ ing to these differences are unknown. Although few surveys have been conducted for the presence of aerotolerant -like organisms in FIGURE 4. Representative ERIC-based DNA fingerprints of Arco- meats, Arcobacter, like Campylobacter has been reported bacter butz1eri. Lane 1, molecular weight marker VI (Boehringer from poultry (2,5, 10, 16, 17, 20). In France, A. butzleri was Mannheim) was usedfor size comparison (bp). Lanes 2 to 7 and 9 recovered from 81 % of poultry carcasses examined to 12 contain A. butzlerifield strains. Lane 8 is blank. Lane 13 is a (n = 201). Nearly half of the poultry isolates in that study PCR negative control. were of serogroup 1 (20). In a survey of poultry products in J. Food Prot., Vol. 61, No. 12 ARCOBACTER IN MECHANICALLY SEPARATED TURKEY MEAT 1627

Canada, A. butzleri was recovered from 97% (121 of 125) of 2. Atabay, H. I., and J. E. L. Corryl. 1997. The prevalence of poultry carcasses obtained from five different processing campy10bacters and arcobacters in broiler chickens. J. Appl. Micro- bioI. 83:619-626. plants. In addition, A. butzleri was cultured from retail- 3. Bastyns, K., D. Cartuyve1s, S. Chappelle, P. Vandamme, H. Gossens, purchased whole and ground poultry, chicken, and turkey and R. De Wachter. 1995. A variable 23S rDNA regions a useful samples (16). As was the case in the French study, serotype 1 discriminating target for genus-specific and species-specific PCR was the predominant serotype isolated from Canadian amplification in Arcobacter species. Syst. Appl. Microbiol. 18:353- 356. poultry (16). In contrast, Arcobacter was detected in only 4. Collins, C. I., I. V. Wesley, and E. A. Murano. 1996. Detection of 24% (53 of 224) of retail-purchased poultry in the Nether- Areobaeter spp. in ground pork by modified plating methods. J. Food lands (5). Prot. 59:448-452. 5. DeBoer, E., J. J. H. C. Tilburg, D. L. Woodward, H. Lior, and W. M. Differences in published recovery rates could be attrib- Johnson. 1996. A selective medium for the isolation of Areobaeter uted to multiple factors, such as bias in plant selection, from meats. Lett. Appl. Microbiol. 23:64-66. hygienic conditions throughout production and processing, 6. Dhamabutra, N., P. Kamo1-Rathanakul, and K. Pienthaweechai. 1992. or differences in the sensitivity of isolation methods. In the Isolation of Campylobaeter from the canals of Bangkok metropolitan area. J. Med. Assoc. Thail. 75:3350-363. current study, to expedite the identification of Arcobacter 7. Ellinghausen, H. C., and W. G. McCullough. 1965. Nutrition of spp., the length of primary enrichment was reduced from 7 Leptospira pomona and growth of 13 other serotypes: fractionation of days (winter survey) to 3 days (summer and fall surveys). oleic albumin complex and a medium of bovine albumin and polysorbate 80. Am. J. Vet. Res. 26:45-51. The shorter primary enrichment used for the summer and 8. Ellis, W. A., S. D. Neill, J. J. O'Brien, H. W. Ferguson, and J. Hanna. fall samples yielded a statistically significant increase in the 1977. Isolation of SpirillumNibrio-like organisms from bovine recovery of Arcobacter spp. (P < 0.04). The shorter primary fetuses. Vet. Rec. 100:451-452. enrichment, however, yielded no statistically significant 9. Ellis, W. A., S. D. Neill, J. J. O'Brien, and J.Hanna. 1978. Isolation of spirillum-like organisms from pig fetuses. Vet. Rec. 102: 106. differences in the recovery of A. butzleri (P > 0.20). 10. Festy, B. E, E Squinazi, M. Marin, R. Derimay, and H. Lior. 1993. In earlier studies, PCR-mediated DNA fingerprinting Poultry meat and water as the possible sources of Arcobacter butzleri confirmed the genetic identity of A. butzleri isolates recov- associated with human disease in Paris, France. Acta Gastro-Enterol. ered from a nursery school outbreak and suggested a single Belg. 56(suppl. 34):35. 11. Food Safety and Inspection Service. 1996. Nationwide raw ground source of contamination (29). In this study, PCR-based turkey microbiological survey. Concept paper. U.S. Department of fingerprinting was used to distinguish the field strains of Agriculture. A. butzleri. By PCR amplification of repetitive ERIC 12. Harmon, K. M., and I. V. Wesley. 1996. Identification of Areobaeter sequences, 71 % of the DNA profiles of the summer and fall isolates by PCR. Lett. Appl. Microbiol. 23:241-244. 13. Jacob, J., H. Lior, and I. Feuerpfeil. 1993. Isolation of Areobaeter isolates of A. butzleri were unique. The multiple DNA butzleri from a drinking water reservoir in eastern Germany. Zent- fingerprints may indicate numerous sources of contamina- ra1bl. Bakteriol. Hyg. 193:557-562. tion. Interestingly, while plant A had the highest recovery for 14. Kieh1bauch, J. A., D. J. Brenner, M. A. Nicholson, C. N. Baker, C. M. Patton, A. G. Steigerwalt, and I. K. Wachsmuth. 1991. Campylobaeter both Arcobacter spp. (96%) and A. butzleri (76%), it butzleri sp. nov. isolated from humans and animals with diarrheal exhibited the lowest percentage (64%) of polymorphism in illness. J. Clin. Microbiol. 29:376-385. the ERIC sequences. Thus, plant A had more isolates with 15. Kiehlbauch, J. A., R. V. Tauxe, and I. K. Wachsmuth. 1991. Clinical similar DNA profiles than the other plants examined. features of Campylobaeter butzleri associate with diarrheal illness. Abstract (C14-2). Vlth International workshop on Campylobaeter, Further, plant C, with only 26 A. butzleri isolates, exhibited a Helieobaeter and related organisms. disproportionately high number (n = 21) of distinctive pro- 16. Lammerding, A. M. 1996. Isolation method for recovery of Areobae- files. ter butzleri from fresh poultry and poultry products, p. 329-333. In In conclusion, this study shows that Arcobacter spp. D. G. Newall and J. Ketley (ed.), Campylobaeter VIII. Plenum Publ. Corp., New York. especially A. butzleri is prevalent in mechanically separated 17. Lammerding, A. M., M. M. Garcia, E. D. Mann, Y. Robinson, W. J. turkey. The diversity of DNA patterns found among the Dorward, R. B. Truscott, and E Tittiger. 1988. Prevalence of A. butzleri isolates suggests multiple sources of contamina- Salmonella and thermophilic Campylobaeter in fresh pork, beef, veal and poultry in Canada. J. Food Prot. 52:47-52. tion. Thus, future studies should focus on the source of 18. Lerner, J., V. Brumberger, and V. Preac-Mursic. 1994. Severe diarrhea contamination, seasonal and geographical variations, and associated with Areobaeter butzleri. Eur. J. Clin. Microbiol. Infect. plant sanitation practices to reduce contamination. Dis. 13:660-662. 19. Logan, E. E, S. D. Neill, and D. P. Mackie. 1982. Mastitis in dairy cows associated with an aerotolerant campylobacter. Vet. Rec. ACKNOWLEDGMENTS 110:229-230. 20. Marinescu, M., A. Collilgnon, E Squinazi, D. Woodward, and H. Lior. We thank Sharon Franklin for her technical guidance, Dr. Joe Cordray 1996. Biotypes and serogroups of poultry strains of Areobaeter sp. for organizing sample delivery, Dr. Greg Phillips for use of his Gel Doc isolated in France, p. 519-520. In D. G. Newall and J. Ketley (ed.), 1000, and Mr. Bill Nimitz for scanning pictures. We acknowledge the Campylobaeter VIII. Plenum Publ. Corp., New York. statistical help of Mr. Harold Ridpath, USDA-APHIS-NVSL. This work 21. Neill, S. D., J. N. Campbell, J. J. 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