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280 ISSN 0255-6162 GPN 2006900031 280 93 4 20 161-2 0226531318 http://www.nlfd.gov.tw 1

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280 3 2 FDA Dr. John Saldanha (WHO ) 3 8 Access 2002 18 3 16 ISO 14971 3 18 3 23 93 3 26 3 28 AOAC 3 29 Word Excel 6 3 30 3 31 3

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280 Fig. 1. The diagram of conventional method and rapid detection method for Campylobacter spp. in food Table 1. Application of genetic and immunological detection methods No.1-3* No.4-7* 8

280 *PCR polymerase chain reaction. Table 2, Commercial rapid detection kits for Campylobacter spp. NO. AOAC (NT$)/ 1 AccuProbe DNA probe 2 GENE-TRAK DNA probe 2 GEN-PROBE - 150 2 GENE-TRAK - 250 3 VIDAS CAM ELISA 2 biomerieux - 713 4 Campyslide LAT 4 Becton Dickinson - 140 5 INDX -Campy(jcl) LAT 4 Integrated Diagnostics 6 Microscreen Campylobacter LAT 4 Microgen Bioproducts - 140-150 7 API CAMPY 4 biomerieux - 558 1. 2. 2.1 100 15 15 CFU/ 2.2 2.2.1 2.2.2 2.2.3 5 3 2.2.4 1 ml 0.01 ml 5 10 ml 0.1 ml 2.2.5 (Pipette aid) 2.2.6 90-100 mm 15 mm 2.2.7 (Blender) (Stomacher) 2.2.8 400 ml 3.5 L 90 ml 450 ml () 2.2.9 0.2 2.2.10 1.0 2.2.11 Anaerobe container 3-3.4 L 5% O 2, 10% CO 2, 85% N 2 (gas-generating envelope) 2.5 L 9.5 L 2.5 L 5.5 L 125 ml 70% 9

280 2.2.12 ( 3 mm) 2.2.13 15 150 mm 2.2.14 0.22 µm 0.45 µm 25 mm47 mm 90 mm 2.2.15 2.2.14 2.2.16 2.2.17 6,000 g 0.1 g 200 g 0.1 mg 2.2.18 Hot plate 2.2.19 20,000 g 250 ml 50 ml 2.2.20 ph 2.2.21 ph ph 6-8 2.2.22 25 rpm 2.2.23 Vortex mixer 2.2.24 100 phase-contrast microscope 63 dark-field microscope 1,000 light microscope 2.2.25 2.2.26 2.2.27 2.2.28 0.25 2.2.29 McFarland nephelometer standard units 2.2.30 30 - 萘 - 腙 苄 苄 B B 吲 哚 N,N,N,N - (30 µg) (30 µg) 腖 腖 腖 腖 3 2.2.31 2.2.31.1 (Campylobacter enrichment broth) a. (Campylobacter enrichment broth base) 腖 (meat peptone) (lactalbumin hydrolysate) (yeast extract) (NaCl) (haemin) (sodium pyruvate) - (-ketoglutamic acid) (sodium metabisulphite) (sodium carbonate) b. (lysed horse blood) 10 g 5 g 5 g 5 g 0.01 g 0.5 g 1 g 0.5 g 0.6 g 1000 ml 50 ml 50 ml -20 c. 1) 腙 (sodium cefoperazone) 腙 0.5 g 100 ml 0.22 µm 4 5-20 14-70 5 20 mg/l () 2) i. 苄 (trimethoprim lactate) 苄 0.66 g 100 ml 0.22 µm 4 1 10

280 20 mg/l ii. 苄 (trimethoprim) 苄 0.5 g 0.05 N 30 ml 50 100 ml 20 mg/l i ii 3) (vancomycin) 0.5 g 100 ml 0.22 µm 4 2 20 mg/l 4) (cycloheximide) 1.25 g 20-30 ml 100 ml 0.22 µm 4 1 50 mg/l 2.2.31.3 B 5 ml 10 121 15 ph 7.4 0.2 50 ml 4 ml 2.2.31.2 (Preston broth for Campylobacter enrichment) a. 2 (nutrient broth No.2) (meat extract) 腖 (peptone) (NaCl) b. (rifampicin) 25 g 10 g 10 g 5 g 1000 ml 0.25 g 60-80 ml 100 ml 100 ml 0.22 µm -20 1 10 mg/l c. B (polymyxin B) 5 B 50 ml 0.22 µm -20 1 5000 IU/L ( ) 121 15 ph 7.4 0.2 2.2.31.1 50 ml 苄 2 ml 8 ml 4 B 5 ml 2.2.31.3 B 10 ml 2.2.31.3 - - (Abeyta-Hunt-Bark agar, A-H-B agar) a. (basal medium) heart infusion agar 500 g (beef heart, infusion from 500 g) (tryptose) (NaCl) (agar) (yeast extract) b. B (amphotericin B) 40 g 10 g 5 g 15 g 2 g 950 ml B 0.1 g 100 ml 0.22 µm -20 1 2 mg/l c. (ferrous sulphate, sodium metabisulphite, sodium pyruvate, FBP) 6.25 g 10-20 ml 6.25 g 11

280 6.25 g 100 ml 0.22 µm FBP -20 1-70 3 121 15 ph 7.4 0.2 50 2.2.31.1 50 ml 腙 6.4 ml2.2.31.2 B 2 ml FBP 2 ml 1520 ml 42 2.2.31.4 (Modified Campylobacter blood-free agar, mccda) CCDA 45.5 g 2 (nutrient broth No.2) 25 g (bacteriological charcoal) 4 g (casein hydrolysate) 3 g (sodium desoxycholate) 1 g (ferrous sulphate) 0.25 g (sodium pyruvate) 0.25 g (agar) 12 g (yeast extract) 2 g 1000 ml 121 15 ph 7.4 0.2 50 2.2.31.1 腙 6.4 ml2.2.31.3 4 ml B 4 ml 15-20 ml 42 2.2.31.5 - - Abeyta-Hunt-Bark agar without antibiotics - - 121 15 ph 7.4 0.2 502.2.31.1 FBP 4 ml 15-20 ml 2.2.31.6 (Modified semisolid medium for biochemical identification) 27.6 g (agar) 1.8 g 1000 ml 4 250 ml3 0.2% (neutral red) 2.5 ml (glycine)2.5 g 7.5 g (cysteine-hcl0.05 g 4 2.5 g 10 ml 121 15 ph 7.4 0.2 2.2.31.7 (Modified semisolid medium for culture storage) 27.6 g (sodium citrate) 0.1 g (agar) 1.8 g 1000 ml ph 7.4 0.210 ml 121 15 2.2.31.8 (Freezing medium)( ) 9.5 ml 0.22 µm 1 ml (fetal bovine serum) 10% (glycerol) 1 ml 2.2.31.9 (Triple sugar iron agar, TSI) (beef extract) (yeast extract) 腖 (peptone) 3 g 3 g 15 g 12

280 腖 (proteose peptone) 5 g (NaCl) 5 g (lactose) 10 g (sucrose) 10 g (glucose) 1 g (ferrous sulphate) 0.2 g (sodium thiosulfate) 0.3 g (phenol red) 0.024 g (agar) 12 g 1000 ml 1/3 121 15 ph 7.4 0.2 (polypeptone) 20 g (NaCl) 5 g (lactose) 10 g (sucrose) 10 g (glucose) 1 g (ferrous sulphate) 0.2 g (sodium thiosulfate) 0.2 g (phenol red) 0.025 g (agar) 13 g 1000 ml 1/3 118 15 ph 7.3 0.2 2.2.31.10 - (Oxidation-fermentation glucose semisolid medium, OF) 腖 (tryptone or trypticase) 2 g (NaCl) 5 g (dipotassium phosphate) 0.3 g (bromthymol blue) 0.03 g (agar) 2 g 1000 ml 10 g 5 ml ( ) 5 ml 121 15 ph 6.8 0.2 2.2.31.11 (MacConkey agar) 腖 (proteose peptone) 3 g (peptone) 17 g (lactose) 10 g 3 (bile salts No.3) 1.5 g (NaCl) 5 g (neutral red) 0.03 g (crystal violet 0.001 g (agar) 13.5 g 1000 ml 121 15 ph 7.1 0.2 15-20 ml 3 2.2.32 2.2.32.1 0.85% 8.5 g 1000 ml 121 15 2.2.32.2 (Butterfield s phosphate-buffered dilution water) 34 g 500 ml 1 ph 7.2 1000 ml 121 15 1.25 ml 1000 ml 121 15 2.2.32.3 0.1% 腖 (0.1% peptone diluent) 腖 1 g 1000 ml 121 15 ph 7.0 0.2 13

280 2.2.32.4 (Gram stain solution) (1) (Hucker's) ( ) A (crystal violet) 2 g 95% 20 ml B (ammonium oxalate) 0.8 g 80 ml A B 24 (2) ( ) 2 g 1 g 5-10 1 ml 5 ml 10 ml 300 ml (3) ( ) (basic fuchsin) 0.5 g 4.5 g (4115, v/v)100 ml 2.2.32.5 0.2% (Neutral red solution) 0.2 g 10 ml 100 2.2.32.6 (Nitrite detection reagents) A (sulfanilic acid) 1 g 5 N 125 ml B (-naphthol) 1 g 5 N 200 ml 2.2.32.7 3% (Hydrogen peroxide solution) 30% 5 ml 45 2.2.32.8 1% Hippurate solution (sodium hippurate)0.1 g 10 ml 0.22 µm 0.4 ml -20 2.2.32.9 (Ninhydrin solution) 3.5 g (11, v/v) 100 ml 2.2.32.10 酶 Oxidase reagent N,N,N,N - (N,N,N,N -tetramethyl-p-phenylenediamine.2hcl) 1 g 100 ml 2.2.32.11 1 N 2 N 40 g 80 g 1000 ml 2.2.32.12 0.1% (sodium hypochlorite solution) 5.25% 19 ml 1000 ml 2.2.32.13 1M 15.8 g 100 ml 0.22 µm 2.2.32.14 (Contrast stain) 11 (v/v) 2.2.32.15 20% 吲 哚 (Indoxyl acetate) 吲 哚 0.5 g 2.5 ml 2.2.33 吲 哚 20% 吲 哚 25 µl 4 (2) 2.3 (3) 2.3.1 2.3.2 2.3.8 25 g( 50 g) 100 ml 5 25 rpm 5 5 ( ) 2N ph 7.4 2.3.2 50 100 g 2.3.1 2.3.3 25 g ( ) 3.5 L 200 ml 10 0.1% 70% 14

280 250 ml 16,000 x g 15 10 ml 3 100 2.3.4 25 g 125 ml 16 16 25 ml 100 ml 130 16 130 2.3.5 100-200 g 60 25 g 400 ml 500 ml 225 ml 110 110 25 ml 225 ml 1100 110 1100 125 ml 2.3.6 2 4 L 1M 5 ml 0.45 µm (47 mm) 90 mm 100 1,000 ml( ) 125 ml 125 ml 2.3.7 10 ml 50 ml 2.3.8 2.3.8.1 ph ph 7.6 1 N 2 N ph 7.5 0.2 50 g 20,000 x g 40 10 ml 90 ml 2.3.8.2 2 5 18 45 15 2.3.8.1 2.3.8.3 50 g 50 ml 15-30 5 2.3.8.1 2.3.8.4 ( ) 50 g 100 ml 1. 0.1% 腖 0.85% 2. 3. (1Tween 80) 2-5 18 45 15 2.4 2.4.1 10 37 4 2.4.2 10 10 30 3 37 2 2.4.3 42( C. fetus 37 ) 48 28-29 ( C. fetus 52 ) 2.5 2.5.1 2.4 0.1 ml 9.9 ml 1 100 15

280 1100 A-H-B mccda 42 24-48 24 C. fetus 37 48-72 2.5.2 鈎 1 (63 ) (1,000 ) 鈎 1 0.1 ml 3-5 (1,000 ) 1.5-5 µm 10% 4 A-H-B 鈎 42 24-48 ( C. fetus 37 ) 鈎 2.5.3 A-H-B 鈎 (Gram stain) 酶 (oxidase test) 酶 (catalase test) 2.5.4 2.5.3.1 (1) 鈎 (2) 1 5 (3) 1 (4 ) 95 (5) 30 (6) (7) 2.5.3.2 酶 (1) 酶 24 10 3 (2) ( ) 10 (1)(2) 2.5.3.3 酶 鈎 13% 2.5.4 2.5.4.1 (Hippurate hydrolysis test) A-H-B 鈎 1% 0.4 ml 37 2 0.2 ml 10 ( ) C. jejunic. jejuni subsp. doylei 2.5.4.2 吲 哚 (Indoxyl acetate hydrolysis test) 12 吲 哚 A-H-B 鈎 吲 哚 20 2.5.4.3 (Glucose utilization test) A-H-B 鈎 OF ( ) 35-37 4 2.5.4.4 TSI (TSI test) 16

280 2.6 A-H-B 鈎 TSI 35-37 5 TSI H 2 S 2.5.4.5 (Antibiotic inhibition test) A-H-B 鈎 0.1% 5 ml 1 (McFarland No. 1) A-H-B (nalidixic acid) (cephalothin) 37 24-48 2.5.4.6 (Growth temperature tolerance) 2.5.4.5 3 A-H-B 4 4 3 25 35-37 42 3 2.5.4.7 (Growth on MacConkey agar) 2.5.4.5 4 (4 )35 3 2.5.4.8 1% (Growth in glycine) 2.5.4.5 0.1 ml 1% 35-37 3 2.5.4.9 3.5% (Growth in 3.5%NaCl) 2.5.4.5 0.1 ml 3.5% 35-37 3 2.5.4.10 (Production of H 2 S from cysteine) 2.5.4.5 0.1 ml 0.02% 35-37 3 2.5.4.11 (Nitrate reduction test) 2.5.4.5 0.1 ml 1% 35-37 5 AB 0.1-0.5 ml C. jejunic. colic. laric. fetus subsp. fetus C. jejuni subsp. doylei C. jejuni C. jejuni subsp. doylei C. coli C. lari C. fetus subsp. fetus C. hyointestinalis C. upsaliensis 25 D 35-37 42 D 3.5% 酶 酶 ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) (81%) 1% 吲 哚 17

280 C. jejuni C. jejuni subsp. doylei C. coli C. lari C. fetus subsp. fetus C. hyointestinalis C. upsaliensis TSI D (c) S b S S R R R S R R R R S c S S a 90%90% D 11-89% R S b. C. jejuni c. C. fetus subsp. fetus 2.7 1. Butzler, J. P., and Oosterom, J. 1991. Campylobacter pathogenicity and significance in foods. Int. J. Food Microbiol. 12:1-8. 2. Hodge, D. S., Borczyk, A., and Wat, L.-L. 1990. Evaluation of the indoxyl acetate hydrolysis test for the differentiation of campylobacters. J. Clin. Microbiol. 28:1482-1483. 3. Hunt, J. M., Abeyta, C., and Tran, T. 1998. Chapter 7. Campylobacter. In "Food and Drug Administration Bacteriological Analytical Manual." 8th ed., pp.7.01-7.27. AOAC International, Gaithersburg, MD, USA. 4. ISO 10272. 1995. Microbiology of food and animal feeding stuffshorizontal method for detection of thermotolerant Campylobacter. 5. Kapperud, G., Skjerve, E., Bean,N. H., Ostroff, S. M. and Lassen, J. 1992. Risk factors for sporadic Campylobacter infections: results of a case-control study in southeastern Norway. J. Clin. Microbiol. 30:3117-3121. 6. Morris, G. K., and Patton, C. M. 1985. Campylobacter. pp. 302-308. In "Manual of Clinical Microbiology," 4th ed., E. H. Lennette, A. Balows, W. J, Hausler (Jr.), and H. J. Shadomy. (ed.), American Public Health Assoc., Washington, D.C. 7. Nielsen, E. M., Engberg, J., and Madsen, M. 1997. Distribution of serotypes of Campylobacter jejuni and Campylobacter coli from Danish patients, poultry, cattle, and swine. FEMS Immunol. Med. Microbiol. 19:47-56. 8. Skirrow, M. B. 1977. Campylobacter enteritis: a "new" disease. Br. Med. J. 2:9-11. 9. Skirrow, M. B. 1990. Foodborne illness Campylobacter. The Lancet 336: 921-923. 10. Smibert, R. M. 1984. Genus II. Campylobacter, pp. 111-118. In "Bergey's Manual of Determinative Bacteriology," Vol. 1. N. R. Kreig and J. G. Holt (eds). Williams & Wilkins, Baltimore, MD, USA. 11. Stern, N. J., and Kazmi, S. U. 1989. Campylobacter jejuni. pp. 71-110. In "Foodborne Bacterial Pathogens", Ed. M. P. Doyle. Marcel Dekker Inc., New York, USA. 12. Stern, N. J., Patton, C. M., Doyle, M. P., Park, C. E., and McCardell, B. A. 1992. Chapter 29. Campylobacter. In "Compendium of Methods for the Microbiological, Examination of Foods." 3rd ed., pp. 475-495. Vanderzant, C. and Splittstoesser, D. F. (eds). American Public Health Association Inc., Washington, D. C., USA. 13. Tauxe, R. V. 1992. Epidemiology of Campylobacter jejuni infections in the United States and other industrialized nations. pp. 9-19. In ''Campylobacter jejuni Current Status and Future Trends,'' Nachamkin I., Blaser M. J., and Tompkins L. S. (ed.), American Society for Microbiology, Washington, D.C. 14. Yuki, N., and T. Miyatake. 1998. Guillain-Barre syndrome and Miller-Fisher s syndrome following Campylobacter jejuni infection. Ann. N. Y. Acad. Sci. 845:330 340. 18