Isolation and Enumeration of Campylobacter jejuni from

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1983, p /83/ $02.00/0 Copyright 1983, American Society for Microbiology Vol. 46, No. 5 Isolation and Enumeration of Campylobacter jejuni from Poultry Products by a Selective Enrichment Methodt RONALD D. WESLEY, BALASUBRAMANIAN SWAMINATHAN,* AND WILLIAM J. STADELMAN Food Sciences Institute, Purdue University, West Lafayette, Indiana Received 6 June 1983/Accepted 29 August 1983 A direct selective enrichment procedure was developed for the isolation of Campylobacter jejuni from poultry products. The selective enrichment medium (ATB) consisted of (per liter) tryptose (20 g), yeast extract (2.5 g), sodium chloride (5 g), FBP supplement (ferrous sulfate [0.25 g], sodium metabisulfite [0.25 g], sodium pyruvate [0.25 g]), bicine (10 g), and agar (1 g). Hematin solution (6.25 ml; prepared by dissolving g of bovine hemin in 10 ml of 0.15 N sodium hydroxide solution and autoclaving at 0.35 kg/cm2 for 30 min), rifampin (25 mg), cefsulodin (6.25 mg), and polymyxin B sulfate (20,000 IU) were added after the medium was sterilized. The ph was adjusted to 8.0. Samples were enriched in the above medium at 42 C for 48 h under an atmosphere of 5% 02, 10% C02, and 85% N2. Enrichment cultures were streaked on a plating medium composed of Brucella agar, hematin solution, FBP supplement, and the above antibiotics. Plates were incubated under the same conditions as above. Suspect colonies from the plates were confirmed to be C. jejuni by morphological examination, growth characteristics, and biochemical tests. The above method yielded 25 isolates of C. jejuni from 50 samples of retail cut-up chicken and chicken parts, whereas a more complex method involving filtration, centrifugation, selective enrichment under a flowing atmosphere, and membrane filtration yielded only 6 positives from the same samples. The new isolation procedure was particularly effective in isolating C. jejuni in the presence of large numbers of Pseudomonas aeruginosa. This procedure yielded quantitative recoveries of C. jejuni from artificially inoculated (less than one cell per g) chicken parts, chicken liver, and mechanically deboned chicken meat, as determined by a three-tube, most-probable-number technique. Campylobacterjejuni is generally regarded as an important cause of disease in humans. It has been recognized as the most commonly isolated pathogen from stools of patients with diarrheal illness (3). There have also been several reports of C. jejuni infections associated with or causing other significant human disease syndromes (24). The organism has been isolated frequently from foods of animal origin, including chicken (22, 26), turkey (18, 26), pork (29), beef (29), lamb (28), and unpasteurized milk (11). Many suspected or proven outbreaks of C. jejuni gastroenteritis have been caused by contaminated foods (2). Since the infective dose of C. jejuni for human illness appears to be as low as 500 organisms (25), it is vital that methods of isolation from foods are sensitive enough to detect relatively low numbers of C. jejuni in the presence of large numbers of contaminants. Most of the currently t Journal paper no. 9496, Purdue University Agricultural Experiment Station, West Lafayette, IN accepted methods were developed to recover C. jejuni from stool specimens, where the organism is present in relatively large numbers (3, 7, 8, 27). Other methods designed for use in foods are either ineffective in recovering very small numbers of C. jejuni or excessively complex and labor intensive (1, 20, 22). One method (10) has been described which is more rapid and direct than other procedures for the isolation of C. jejuni from foods. The method was effective in recovering the organism from foods when it was present at levels ranging from 0.1 to 4 cells per g. However, difficulty in recovering C. jejuni when inoculated at these levels onto chicken skin may indicate its lack of sensitivity for use with poultry products. The objective of this study was to develop an enrichment procedure for the detection of very low numbers of C. jejuni in poultry products and to evaluate its effectiveness. MATERIALS AND METHODS Bacterial strains. C. jejuni CJ19 and CJ34 were isolated from cut-up chicken parts bought from a local 1097

2 1098 WESLEY, SWAMINATHAN, AND STADELMAN grocery store. C. jejuni ATCC (Campylobacter fetus subsp.jejuni; American Type Culture Collection, Rockville, Md.) was a human isolate. Three strains isolated from chicken, C (6a; 9/17/80), D (6a; 9/24/80), and E (9a; 9/30/80); two from turkey, F (12a; 11/25/80) and G (2a; 11/25/80); and one from pork, H (P20; originally from M. B. Skirrow, Royal Infirmary, Worcester, United Kingdom) were obtained from J. Hunt (Bacterial Physiology Branch, Division of Microbiology, Food and Drug Administration, Cincinnati, Ohio). All strains conformed to the biochemical and growth characteristics of C. jejuni (19). Pseudomonas aeruginosa PA2 and PA3 were isolated from mechanically deboned chicken meat (MDCM). They conformed to biochemical and growth characteristics of P. aeruginosa (21). Enrichment broth. The selective enrichment broth (ATB) was composed of tryptose (Difco Laboratories, Detroit, Mich.; 20 g/liter); yeast extract (Difco; 2.5 g/liter); sodium chloride (5 g/liter); FBP supplement (ferrous sulfate [0.25 g/liter], sodium metabisulfite [0.25 g/liter], sodium pyruvate [0.25 g/liter]); bicine (Sigma Chemical Co., St. Louis, Mo.; 10 g/liter); and agar (Difco; 1 g/liter). The ph of the medium was adjusted to 8.0 with a 5 M NaOH solution. After sterilization, rifampin (Sigma; 25,ug/ml); cefsulodin (Abbott Laboratories, North Chicago, Ill.; 6.25,ug/ml); and polymyxin B sulfate (Pfizer Laboratories Division, Pfizer Inc., New York, N.Y.; 20 IU/ml) were added. Also, alkaline hematin solution, prepared by dissolving 32 mg of hemin (Sigma) in 10 ml of 0.15 N NaOH and autoclaving for 30 min at 0.35 kg/cm2, was added to achieve 2 mg of hemin per liter (5). The medium was then either used immediately or stored overnight in the dark in a refrigerator before use. Plating medium. The selective plating medium (BA) was composed of Brucella agar (Difco) plus the same concentrations of antibiotics, FBP supplement, and alkaline hematin solution as in ATB. The antibiotics and alkaline hematin solution were added before pouring the plates. The plates were allowed to stand overnight in the dark on the laboratory bench before use. Microaerobic incubation. All plates and culture flasks (with loosened caps or one-hole stoppers fitted with bent-glass tubing to allow gas exchange) were placed in GasPak 150 vented jars (BBL Microbiology Systems, Cockeysville, Md.). The jars were evacuated to ca. 72 cm of Hg with an aspirator attached to a laboratory sink faucet and refilled to ca. 0.2 kg/cm2 from a gas cylinder containing 5% 02, 10% C02, and 85% N2. The evacuation and refilling procedure was done three times before the jars were placed in an incubator at 42 C. Microbial susceptibility testing. Strains of C. jejuni and P. aeruginosa were tested for microbial susceptibility to various antibiotics by a tube dilution method (23) that used ATB without antibiotics as the growth medium. Inocula consisted of 0.5-ml portions of 102 dilutions in ATB of each strain grown in fluid thioglycolate (Difco) for 24 h at 42 C. Incubation took place under microaerobic conditions at 42 C for 48 h. Growth was scored as positive (+) or negative (-) by visual observation of duplicate tube cultures. Recovery efficiency. A most-probable-number (MPN) method was used to determine the efficiency of recovery of low numbers of artificially inoculated C. APPL. ENVIRON. MICROBIOL. jejuni from chicken samples. Chicken fryer parts (livers, drumsticks, and wings) and MDCM were purchased locally and were determined not to contain viable C. jejuni. In the first experiment, three 25-g samples of liver were inoculated with 1.0 ml of 10-6, 10'-, and 108 dilutions, respectively, of a 24-h culture of strain CJ34 grown in fluid thioglycolate medium (Difco) that had been incubated at 42 C. The inoculum levels were estimated to yield approximate initial numbers of 100 to 500, 10 to 50, and 1 to 5 cells per 25 g of liver. One 25-g uninoculated sample was used as a control. ATB (25 ml) was added to each sample, and the mixtures were homogenized in a Stomacher 400 (Cooke Laboratories, Inc., Alexandria, Va.) for 30 s. Triplicate 10-, 1.0-, and 0.1-ml portions of the liver- ATB-CJ34 slurries were added to 100-, 100-, and 10-ml quantities, respectively, of ATB in standard milk dilution bottles (screw-capped test tubes [16 by 125 mm] for the 10-ml cultures). The milk dilution bottles were fitted with one-hole rubber stoppers with bentglass tubing which had one end extending above the fluid surface to allow atmospheric exchange when the bottles were placed horizontally in the jars. The screwcapped test tubes, with caps loosened, were placed as nearly horizontal as possible without permitting the contents to spill. The cultures were incubated microaerobically for 48 h at 42 C. One loopful of each culture was then streaked onto a BA plate. The plates were incubated microaerobically for 48 h at 42 C. Characteristic colonies of C. jejuni were colorless to grayish or tan, small (about 0.5 to 2 mm in diameter), and often extended along the streak lines in the agar surface. Suspect colonies from each plate were examined under wet-mount preparations and identified by biochemical and growth characteristics (19). C. jejuni inoculum levels were determined by viable plate counts of serial dilutions of the 24-h fluid thioglycolate cultures on BA plates. In addition, aerobic plate counts (14) were done on the food sample. The second experiment was a repetition of the first, except that three 25-g samples of C. jejuni-free MDCM were used instead of liver, and strain CJ19 replaced strain CJ34. In the third experiment, CJ19 was again used as the inoculum. The food samples were C. jejuni-free chicken drumsticks. To ensure that the samples did not contain C. jejuni, each sample was rubbed over its entire surface with a sterile, cotton-tipped swab before inoculation. The swabs were then placed in screwcapped test tubes containing 10 ml of ATB, incubated microaerobically for 48 h at 42 C, plated on BA, and examined for C. jejuni. The samples were inoculated with 0.1-ml portions of serial dilutions (10', 10', 10-6, and 10-') of a 24-h fluid thioglycolate culture which were spread on the skin surfaces with sterile, bent-glass rods. Inocula were allowed to adsorb into the skin surfaces by placing the inoculated chicken pieces in a refrigerator for 1 h. Each sample was then placed into a sterile plastic bag with 50 ml of ATB and massaged by hand for 1 min. Portions (10, 1, and 0.1 ml) of the rinse fluid from each sample were added to 100-ml quantities of ATB in triplicate, and the remainder of the procedure was the same as in the first experiment. A fifth chicken drumstick was sampled for an aerobic plate count (14). A fourth experiment was conducted in a manner identical to that of the third experiment, except that C.

3 VOL. 46, 1983 jejuni-free wings from a different source were used as the food sample and inoculated with strain CJ19. Comparative recovery. The enrichment procedure described above was directly compared with the method of Park et al. (22) to recover C. jejuni from a variety of chicken fryer giblets and cut-up parts, including wings, thighs, drumsticks, drumettes, livers, gizzards, and hearts, purchased from local grocery stores. A total of 50 food samples were evaluated by the two methods. Each sample was placed in a sterile plastic bag and massaged by hand (giblet samples were stomached for 30 s) in 50 ml of 0.1% peptone water. Next, 10 ml of this rinse fluid was placed in 100 ml of ATB in a standard milk dilution bottle fitted with a one-hole rubber stopper and bent-glass tube, as described above. After 48 h of incubation at 42 C under microaerobic conditions, one BA plate was streaked from each ATB culture and incubated microaerobically for 48 h at 42 C. Suspect colonies were examined by the methods of Luechtefeld et al. (19) for identification as C. jejuni. The remainder of the rinse fluid was subjected to the enrichment methods of Park et al. (22). It was filtered through a double layer of cheesecloth and centrifuged at 16,300 x g for 15 min. The sediment was suspended in 5 ml of Brucella broth (Difco). The suspension was then transferred to an enrichment broth consisting of 100 ml of Brucella broth with vancomycin (Sigma; 8,ug/ml), trimethoprim (Sigma; 4,ug/ml), and polymyxin B (Sigma; 8 IU/ml) in a 250-ml flask. During incubation for 3 days at 37 C, a constant flow of an atmosphere consisting of 5% 02, 10% CO2, and 85% N2 was bubbled through the culture in the flask at a rate of 5 to 7 mllmin. Then, 5 ml of the enrichment culture was filtered through a Lm membrane filter (Millipore Corp., Bedford, Mass.), and serial dilutions of the filtrate were plated in parallel on Skirrow's selective agar (27) and on Skirrow's selective agar containing a 10-fold increase in polymyxin B concentration. Isolates were identified as C. jejuni by the methods of Luechtefeld et al. (19). RESULTS Antibiotic susceptibility tests yielded the following results. (i) Cefsulodin inhibited both strains of P. aeruginosa (PA2 and PA3) at 25 g/ml but also inhibited one strain of C. jejuni (ATCC 29428) at 12.5,ug/ml. (ii) A concentration of 50 jig of rifampin per ml in the ATB medium was required to inhibit both strains of P. aeruginosa; however, C. jejuni ATCC was also inhibited at this level. (iii) Combinations of cefsulodin and polymyxin B were more prohibitive: at 6.25,ug of cefsulodin per ml and 2.5 to 20 jxg of polymyxin B per ml, the two strains of P. aeruginosa were completely inhibited, whereas there was no effect on the growth of any of the seven C. jejuni strains. Data from the recovery efficiency experiments are presented in Table 1. Of the three foods tested, the enrichment procedure was least effective in recovering C. jejuni from MDCM, which also had a very high aerobic plate count. Nevertheless, the procedure was CAMPYLOBACTER JEJUNI IN POULTRY PRODUCTS 1099 sensitive enough to recover C. jejuni from the MDCM at an inoculum level as low as 0.44 CFU/g in the presence of indigenous flora at a level of 1.4 x 108 CFU/g. The method was effective at recovering C. jejuni from the skin of chicken drumsticks at inoculum levels as low as 0.49 and 0.07 CFU/g and from chicken livers at an inoculum level of 0.44 CFU/g. In all samples of chicken liver and chicken drumsticks tested, the inoculum levels of C. jejuni per gram of sample fell between the upper and lower limits (95% confidence intervals) of the MPN, which were calculated from the results of the three-tube MPN procedure. For the MDCM samples, the initial numbers of C. jejuni were only slightly higher than the upper 95% confidence limits of the calculated MPN values. Table 2 shows a summary of the results of the comparison between the C. jejuni recovery rates of the ATB enrichment method described here and the vancomycin-trimethoprim-polymyxin (VTP) enrichment method of Park et al. (22). Overall, by the ATB method, 25 of 50 (50%) of the chicken fryer parts purchased at local grocery stores were found to contain C. jejuni. In contrast, only 6 of 50 (12%) of the same samples were C. jejuni-positive by the VTP method. Five of the samples yielded C. jejuni by both methods, 20 only by the ATB method, and 1 sample (a chicken heart) was found to contain C. jejuni only by the VTP method. DISCUSSION The ATB enrichment method described in this paper appears to be simple and effective in isolating C. jejuni in very low numbers from poultry products. Several other procedures for isolating C. jejuni from various substrates have been described previously (1, 3, 9, 12, 22, 29), but none has been shown to be as effective for use with chicken and poultry products. The method of Park et al. (22), which was modified subsequent to the comparison conducted in this study, was reported to have a sensitivity of as low as 0.2 cell per g of chicken in the presence of 104 to 106 cells of contaminants per g. However, because of the relative complexity of the Park et al. procedure, expenses in equipment, time, and labor, and its apparent inferiority in recovering C. jejuni from chicken parts, the ATB method seems to be the more efficient and reliable method. Most recently, Doyle and Roman (10) introduced a selective enrichment procedure for the recovery of C. jejuni and Campylobacter coli from food. The method involved incubation of food in an enrichment broth composed of Brucella broth with horse blood, sodium succinate, cysteine hydrochloride, and four antibiotics for 16 to 18 h with agitation under microaerobic

4 1100 WESLEY, SWAMINATHAN, AND STADELMAN TABLE 1. Recovery of C. jejuni from inoculated foods by selective enrichment with an MPN procedure Expt Fd s Aerobic plate C. jejuni inoculum Three-tube MPN MPN 95% confidence limits no. oo sampe count (CFU/g) level (CFU/g) values (CFU/g) Lower Upper 1 Chicken livers 2.2 x x x x x 10-2 <6.0 x x 10-3 <6.0 x MDCM 1.4 x x x x x 10-2 <1.0 x x x 10-2 <6.0 x Chicken drumsticks 1.5 x x 101 >1.6 x x x x x x x x 10-2 <2.0 x Chicken drumsticks 1.6 x x x x x x x x x x X 10-3 <2.4 x 10-2 conditions at 42 C and with subsequent plating on Campy-BAP agar (3). Effectiveness of recovery as good as 0.1 cell of Campylobacter spp. per g of food was reported. All isolates were recovered from hamburger and raw milk at an inoculum level of 1 to 4 cells per g, and 41 of 50 and 40 of 50 were recovered from hamburger and milk, respectively, at a level of 0.1 to 0.4 cells per g. However, it was much more difficult to recover isolates of both C. jejuni and C. coli from chicken skin. At an inoculum level of 1 to 4 cells per g, one porcine and two of six bovine isolates were not recovered; at a level of 0.1 to 0.4 cells per g, one porcine, two of three canine, and four of six bovine isolates were not recovered. Doyle and Roman (10) reported that the indigenous flora of chicken skin was a better competitor for C. jejuni, C. coli, and nalidixic acid-resistant thermophilic Campylobacter spp. than the flora of other foods tested in their study. The ATB enrichment may be more effective for the recovery of C. jejuni from chicken skin, since recovery was accomplished at inoculum levels of 0.49 and 0.07 CFU/g of chicken. The report of Tanner and Bullin (31) on the increase in effectiveness of recovery with small inocula (1 to 10 cells) when alkaline peptone water enrichment (ph 8.4; 43 C) was used provided the impetus for development of our procedure. Two other reports (26, 32) also stated that alkaline peptone enrichment enhanced the recovery of Campylobacter spp. in some cases. We found that an alkaline tryptose enrichment was more effective than alkaline peptone, and our later studies showed that a ph of 8.0 was better than ph values of 8.5 or 7.5 for the recovery of C. jejuni (data not shown). However, problems with overgrowth of indigenous poultry flora necessitated the addition of APPL. ENVIRON. MICROBIOL. constituents to enhance the growth of C. jejuni and to inhibit the growth of contaminants. We found, as did other researchers (5; M. H. H. Razi and R. W. A. Park, J. Appl. Bacteriol. 47:X, 1979), that the addition of an alkaline hematin solution to liquid and solid media effected the same growth stimulation of C. jejuni as did the incorporation of blood into the media. We found that it also eliminated any problem (due to the opacity of the medium) in counting blood agar plates. The addition of FBP supplement (13), which appears to act on culture media to quench toxic superoxide anions and enhance aerotolerance of C. jejuni (16), promoted the rate and extent of growth of pure cultures of C. jejuni in tryptose broth, as measured by an increase in the optical density of the medium. The effect of adding both alkaline hematin and FBP supplement was greatest at the higher ph levels tested (8.0 to 9.0) as opposed to the lower levels (7.0 and 7.5). The addition of 1% bicine to the medium slowed the drop in ph, which was caused by incubation in a high-co2 atmosphere, TABLE 2. Comparison of methods for isolation of C. jejuni from poultry products Enrichment methoda Chicken part ATB VTP Wing 5/9 0/9 Thigh 7/11 2/11 Drumstick 4/12 0/12 Gizzard 3/5 1/5 Heart 0/3 1/3 Liver 5/8 2/8 Drumette 1/2 0/2 a Number positive for C. jejuniltotal number of samples.

5 VOL. 46, 1983 CAMPYLOBACTER JEJUNI IN POULTRY PRODUCTS 1101 and did not adversely affect the growth of C. jejuni. Presumably, the longer the ph remained high (near 8.0), the greater was the inhibitory effect (if any) on competing flora. As the prototype ATB method was applied to a variety of poultry products to selectively isolate C. jejuni, it became evident that P. aeruginosa was the primary problem. In products containing high numbers of bacteria, especially in MDCM, obvious overgrowth of undesirable bacteria occurred in culture broths and on selective plates. We found that neither Butzler's (8) nor Campy-BAP (3) selective plating media was inhibitory enough to prevent excessive growth of isolates of P. aeruginosa from MDCM. At that point, our efforts toward improvement of the enrichment medium centered on resolution of the problem with P. aeruginosa overgrowth. Several inhibitory agents and antibiotics, including 5-fluorouracil, EDTA, gentamicin, claforan, and sulfacetamide, were tested at several concentrations by using a tube dilution method (23) modified for use with C. jejuni; all agents inhibited C. jejuni growth to a greater extent than they inhibited growth of P. aeruginosa and other contaminants. However, tests with cefsulodin showed that incorporation at a level of 25,ug/ml inhibited all strains of P. aeruginosa and only one of seven test strains of C. jejuni. When cefsulodin at 6.25,ug/ml and polymyxin B sulfate at concentrations of 2.5 to 20 IU/ml were incorporated into the ATB medium, there was no inhibition of the seven test strains of C. jejuni, whereas P. aeruginosa cultures showed no growth at high inoculum levels. Furthermore, a level of 25,ug of rifampin per ml caused no inhibition of C. jejuni strains but did cause a reduction in the growth of P. aeruginosa at 48 h. A selective medium similarly containing, among other antibiotics, both polymyxin B and rifampin, has been described for the recovery of C. jejuni and C. coli from animal and environmental specimens (4). Thus, cefsulodin (6.25,ug/ml), polymyxin B sulfate (20 IU/ml), and rifampin (25,ug/ml) were incorporated into the final formulation of the ATB enrichment and BA selective plating media. During the testing for the response of C. jejuni and P. aeruginosa to various levels of cefsulodin, a dramatic effect of manipulating the ratio of surface area to volume of the broth medium was noted. In 10-ml portions of enrichment medium containing cefsulodin as the only inhibitory agent, the cultures grown in 50-ml flasks showed severalfold-greater amounts of growth of all C. jejuni cultures than in vertically positioned test tubes. Cultures of P. aeruginosa produced heavy growth in test tubes, whereas identical inocula were nearly completely inhibited in the flasks. This effect was found repeatedly in subsequent experiments in which some culture flasks were positioned on their sides to increase surface-to-volume ratios and others were left upright. Incubation of cultures in closed flasks filled with a microaerobic atmosphere in a temperature-controlled water bath with agitation (10) may duplicate or enhance this effect. During the development of the BA plating medium and plating procedure, it became evident that the manner with which the plates were handled before and during incubation had an effect on the success of the method. If the plates were not allowed to stand for some time (presumably for reduction in moisture content), colonies of C. jejuni formed would be thin, watery, and effuse, and would often coalesce and mix with any contaminating flora on the plates. Counting of colonies on such plates was difficult or impossible. Allowing the plates to stand overnight after pouring and before incubation was enough to prevent this effect, which was also noted by Buck and Kelly (6). Furthermore, the conventional inverted position in stacking plates for incubation to prevent condensation from falling on the agar surface proved to be impractical with the BA plates. During or shortly after the evacuation procedure, the medium in the inverted plates would often fall into the lids of the plates and destroy the results of the experiments. Incubation of plates in the upright position was thereafter adopted, and few problems arose with moisture falling on the agar surfaces. Perhaps both of these difficulties could have been lessened or eliminated by increasing the agar concentration in the ATB, but the effect on C. jejuni growth would have to be determined. It should be stated that no attempt was made in this study to separate the species C. jejuni and C. coli. There appears to be a great deal of uncertainty concerning the taxonomic classification and distinguishing characteristics of strains of thermophilic Campylobacter spp. Hebert et al. (15) found that they could not separate C. coli and C. jejuni by published procedures, and they reported, as is probably the case in our study, that strains described as C. jejuni probably included some of both species. Leaper and Owen (17) found a great deal of variability among Campylobacter strains and proposed that classification according to cellular fatty acid composition may be useful to identify strains with no clear identity by biochemical means. They stated that C. coli appears to be as similar to C. jejuni as C. fetus subsp. fetus is to C. fetus subsp. venerealis and thus may better be considered as a subspecies of C. jejuni. Stern (30) also had difficulty in discriminating C. jejuni from C. coli by using growth ability at 30.5 C and 2,3,5- triphenyltetrazolium tolerance. Such a discrimination may not be of critical importance for food

6 1102 WESLEY, SWAMINATHAN, AND STADELMAN microbiologists, however, since both C. jejuni and C. coli have been shown to be pathogenic to humans (30). LITERATURE CITED 1. Acuff, G. R., C. Vanderzant, F. A. Gardner, and F. A. Golan Evaluation of an enrichment-plating procedure for the recovery of Campylobacterjejuni from turkey eggs and meat. J. Food Prot. 45: Blaser, M. J Campylobacterjejuni and food. Food Technol. (Chicago) 36: Blaser, M. J., I. D. Berkowitz, F. M. LaForce, J. Cravens, L. B. Reller, and W. L. Wang Campylobacter enteritis: clinical and epidemiologic features. Ann. Intern. Med. 91: Bolton, F. J., and L. Robertson A selective medium for isolating Campylobacter jejunilcoli. J. Clin. Pathol. 35: Border, M. M., B. D. Firehammer, and L. L. Myers Tube culture method for viable counts of Campylobacter fetus (Vibrio fetus). Appl. Microbiol. 28: Buck, G. E., and M. T. Kelly Effect of moisture content of the medium on colony morphology of Campylobacter fetus subsp. jejuni. J. Clin. Microbiol. 14: Butzler, J. P., P. Dekeyser, M. Detrain, and F. Dehaen Related vibrio in stools. J. Pediatr. 82: Butzler, J. P., and M. B. Skirrow Campylobacter enteritis. Clin. Gastroenterol. 8: Chan, F. T. H., and A. M. R. Mackenzie Enrichment medium and control system for isolation of Campylobacter fetus subsp. jejuni from stools. J. Clin. Microbiol. 15: Doyle, M. P., and D. J. Roman Recovery of Campylobacterjejuni and Campylobacter coli from inoculated foods by selective enrichment. Appl. Environ. Microbiol. 43: Doyle, M. P., and D. J. Roman Prevalence and survival of Campylobacter jejuni in unpasteurized milk. Appl. Environ. Microbiol. 44: Ehlers, J. G., M. Chapparo-Serrano, R. L. Richter, and C. Vanderzant Survival of Campylobacter fetus subsp. jejuni in cheddar and cottage cheese. J. Food Prot. 45: George, H. A., P. S. Hoffman, R. M. Smibert, and N. R. Krieg Improved media for growth and aerotolerance of Campylobacterfetus. J. Clin. Microbiol. 8: Gilliland, S. E., F. F. Busta, J. J. Brinda, and J. E. Campbell Aerobic plate count, p In M. L. Speck (ed.), Compendium of methods for the microbiological examination of foods. American Public Health APPL. ENVIRON. MICROBIOL. Association, Washington, D.C. 15. Hebert, G. A., D. G. Hollis, R. E. Weaver, M. A. Lambert, M. J. Blaser, and C. W. Moss years of campylobacters: biochemical characteristics and a biotyping proposal for Campylobacterjejuni. J. Clin. Microbiol. 15: Hoffman, P. S., H. A. George, N. R. Krieg, and R. M. Smibert Studies of the microaerophilic nature of Campylobacter fetus subsp. jejuni. II. Role of exogenous superoxide anions and hydrogen peroxide. Can. J. Microbiol. 25: Leaper, S., and R. J. Owen Identification of catalase-producing Campylobacter species based on biochemical characteristics and on cellular fatty acid composition. Curr. Microbiol. 6: Luechtefeld, N. W., and W. L. Wang Campylobacterfetus subsp. jejuni in a turkey processing plant. J. Clin. Microbiol. 13: Luechtefeld, N. W., W. L. Wang, M. J. Blaser, and L. B. ReDler Campylobacter fetus subsp. jejuni: background and laboratory diagnosis. Lab. Med. 12: Oosterom, J., M. J. G. M. Vereuken, and G. B. Engels Campylobacter isolation. Vet. Q. 3: Palleroni, N. J The Pseudomonas group, p Meadowfield Press Ltd., Bushey, England. 22. Park, C. E., Z. K. Stankiewicz, J. Lovett, and J. Hunt Incidence of Campylobacterjejuni in fresh eviscerated whole market chickens. Can. J. Microbiol. 27: Pfizer Inc Suggestions on testing microbial susceptibility by the tube dilution method. Pfizer Inc., New York. 24. Rettig, P. J Campylobacter infections in human beings. J. Pediatr. 94: Robinson, D. A Infective dose of Campylobacter jejuni in milk. Br. Med. J. 282: Simmons, N. A., and F. J. Gibbs Campylobacter spp. in oven-ready poultry. J. Infect. 1: Skirrow, M. B Campylobacter enteritis: a "new" disease. Br. Med. J. 2: Stern, N. J Campylobacterfetus ssp. jejuni: recovery methodology and isolation from lamb carcasses. J. Food Sci. 46: , Stern, N. J Recovery rate of Campylobacter fetus ssp. jejuni on eviscerated pork, lamb, and beef carcasses. J. Food Sci. 46:1291, Stern, N. J Methods of recovery of Campylobacter jejuni from foods. J. Food Prot. 45: , Tanner, E. I., and C. H. Bullin Campylobacter enteritis. Br. Med. J. 2: Wang, W. L., M. Blaser, and J. Cravens Isolation of campylobacter. Br. Med. J. 2:57.