J Vet Diagn Invest 3:368-373 (1991)
REVIEW ARTICLES
Cultivation of Mycobacterium paratuberculosis from bovine fecal specimens and a suggested standardized procedure Diana L. Whipple, Donald R. Callihan, Jerald L. Jarnagin
Paratuberculosis is a chronic granulomatous enteritis of ruminants caused by a small, fastidious, acidfast bacillus, Mycobacterium paratuberculosis. The disease now called paratuberculosis was described in 1826 as an enteritis found in some cattle with chronic diarrhea. 3 Johne and Frothingham further described the disease in 1895 and demonstrated the presence of acidfast bacilli in affected intestine.3 The first successful isolation of this organism was in 1910 by Twort, who named in Mycobacterium enteriditis chronicae pseu39,40 dotuberculosis bovis johne. The name of the organism has since been changed to Mycobacterium paratuberculosis, 1 and the clinical disease is called Johne’s disease. 3 Many tests have been developed to aid in diagnosis of paratuberculosis; however, isolation of M. paratuberculosis from tissue or fecal specimens is regarded 3,5 as the definitive diagnostic test in cattle. Cultivation of M. paratuberculosis from fecal specimens is the most frequently used diagnostic test and is the standard for comparison for nearly all other tests.8 Most paratuberculosis control programs use fecal culture results as the basis for management decisions;35 however, the procedures used have not been standardized. The purpose of this paper is to present a partial review of the literature describing procedures for cultivation of M.
paratuberculosis from fecal specimens and to suggest a standardized cultivation procedure. Review of the literature Isolation of the organism now known as M. paratuberculosis was first reported in 1910,39 and the complete description of the procedure was reported in 1912.40 An inspissated egg-base medium containing Mycobacterium tuberculosis was used. The authors included M. tuberculosis because they recognized that some type of “necessary foodstuff needed to support growth of M. paratuberculosis was missing from the medium. They theorized that this “foodstuff’ could be supplied by another acid-fast organism, such as M. tuberculosis. After successfully isolating M. paratuberculosis on egg medium containing M. tuberculosis, they substituted the timothy grass bacillus (M. phlei) for M. tuberculosis and observed growth that was superior. The “necessary foodstuff’ was extracted from M. phlei using hot ethanol and was used in medium instead of a suspension of M. phlei to support growth of M. paratuberculosis. Use of egg-based medium and supplementation with extracted “foodstuff’ (now called mycobactin) provided the foundation for subsequent procedures for isolation of M. paratuberculosis. Various types of media have been evaluated for their ability to support growth of mycobacteria. In 1931, egg yolk agar medium was reported to support growth of M. tuberculosis better than medium containing whole eggs because egg whites were inhibitory to the organism. 11 In 1942, 27 different media were evaluated, ineluding a modification of Herrold’s egg yolk medium (HEYM), for their ability to support growth of M. para28 tuberculosis. Media containing whole eggs were superior to media containing egg yolks alone. In addition, samples could be decontaminated with antiformin (1020%, final concentration), and either brilliant green or malachite green could be included in the medium to help control contamination and enhance visibility of colonies.
From the Leptospirosis/Mycobacteriosis Research Unit, National Animal Disease Center, USDA, Agricultural Research Service, Ames, IA 50010 (Whipple), the Diagnostic Laboratory, New York State College of Veterinary Medicine, Cornell University, Ithaca, NY 14853 (Callihan), and the Mycobacteria, Brucella Reagents Section, Diagnostic Bacteriology Laboratory, National Veterinary Services Laboratories, USDA, Animal and Plant Health Inspection Services, Ames, IA 50010 (Jamagin). Current address (Callihan): the Department of Pathology, Pitt County Memorial Hospital, and the Department of Clinical Pathology and Laboratory Medicine, East Carolina University School of Medicine, Greenville, NC 27835. Received for publication February 11, 1991. 368
Downloaded from vdi.sagepub.com at Univ of Connecticut / Health Center / Library on May 16, 2015
Review article: Cultivation of M. paratuberculosis
Microscopic and cultural examination of fecal specimens for diagnosis of Johne’s disease was described in 1948.15 In that study, Dunkins medium containing whole eggs, liver, and a glycerated extract of M. phlei was used. Samples were decontaminated in antiformin. The author concluded that microscopic examination of fecal specimens was more practical for diagnosis than was cultivation of the organism from fecal specimens and that culture was most useful for confirming the presence of M. paratuberculosis in specific cases of Johne’s disease. In 1950, 8 different media for primary isolation of M. paratuberculosis were compared.34 Media containing at least 50% egg yolk were more reliable than complex mixtures containing whole eggs or liver. Finlayson’s medium,9 which contains 60% egg yolk, was identified as the preferred medium. Decontaminating agents also were evaluated. Although oxalic acid was less inhibitory to M. paratuberculosis, use of antiformin was recommended because of more effective decontamination of fecal specimens. The author stated, “there is at present no completely reliable method of obtaining M. johnei in culture from infected bovine faeces, particularly from samples in which they may be present in very small numbers.” Various decontaminating agents in conjunction with two media were compared in 1953 .33 Modified Dubo’s medium was judged superior to Taylor’s modification of Finlayson’s medium 34 for cultivation of M. paratuberculosis from infected material. Decontaminants compared were 4% NaOH, 10% trisodium phosphate, 5% oxalic acid, 5% sulphuric acid, and 10-20% antiformin. The efficacy of including penicillin and chloramphenicol in media to control contamination without inhibition of M. paratuberculosis also was tested. Contamination was best controlled by pretreating fecal specimens with 20% antiformin and incorporating penicillin into the medium. A review of procedures for diagnosis of paratuberculosis was published in 1959. 12 The authors stated, “Cultural examination of the faeces is not only complicated by contamination problems, but is of little practical use for diagnosis because of the slow growth of the organisms, and this procedure is reserved almost solely for experimental work. If faeces examinations are practical only in the scouring animal, as has been suggested, this test has only limited diagnostic value and has no direct application to eradication or control measures.” In 1963, specimens were decontaminated using 10% sulfuric acid and inoculated onto Lowenstein-Jensen medium containing 5% killed bovine tubercle bacilli.30 Inoculation of more tubes of medium increased the chances of obtaining a positive diagnosis by culture. Culture was positive in some cases that were negative
369
by microscopic evaluation; however, some cases were microscopically positive and culture negative. The prolonged time required for culture was thought to diminish its value for diagnosis. The purpose of a study reported in 196421 was to develop procedures for primary isolation of M. paratuberculosis from specimens that contained too few organisms to be detected microscopically. A modification of HEYM was compared with lymph node egg yolk medium,22 each containing M phlei suspended in glycerin as a source of mycobactin. The following decontaminants were also evaluated: 4% NaOH, 0.2% NaOCI, 0.5% phenol, and 0.1-1.0% benzalkonium chloride. The combination of benzalkonium chloride as a decontaminant and modified HEYM was superior to microscopic examination for detecting M. paratuberculosis in a specimen. Use of 0.3% benzalkonium chloride for l-3 days for decontamination of fecal specimens was recommended. In 1968, Merkal et a1.23 recommended 1) the cultural examination of fecal specimens for diagnosis of subclinical paratuberculosis and 2) the examination by twice yearly fecal culture of herds suspected of having paratuberculosis and the slaughter of culture-positive animals. Although mycobactin from M. phlei was isolated and characterized in 1953,10 most investigators continued to use a suspension of M. phlei in media as a mycobactin source. The HEYM described in 1968 contained an extract of M. phlei as a crude mycobactin source .23 Mycobactin from M. phlei was later designated mycobactin P. 18 In 1970 and 1973, 50-100 organisms per gram of feces were reported as required for detection of M . paratuberculosis using the recommended cultivation procedure. 18,19 In 1972, decontamination of fecal specimens in benzalkonium chloride followed by either mixing the inoculum in a suspension containing amphotericin B or by inoculation onto media containing amphotericin B was recommended to better control fungal contaminants. 26 Amphotericin B, an antifungal agent, had no detrimental effect on M. paratuberculosis. Growth and metabolic characteristics of M. paratuberculosis were described in 1974.20 Dependence of M. paratuberculosis on exogenous mycobactin could be circumvented by adding 1% ferric ammonium citrate to the medium, and growth of most strains tested was enhanced by adding 4.1% sodium pyruvate to the medium. In 1980, fecal culture results were compared with antibody response as determined by the complement fixation test.8 The decontaminant used was 0.2% alkylbenzyl-dimethyl-ammonium chloride, and the medium was HEYM containing mycobactin P. The au-
Downloaded from vdi.sagepub.com at Univ of Connecticut / Health Center / Library on May 16, 2015
370
Whipple, Callihan, Jamagin
thors stated, “fecal culture therefore appears to be a good basis on which to evaluate various immunological reactions of infected cattle.” The disadvantages of using fecal culture were prolonged incubation and contamination problems. In 1982, an alternative medium for cultivation of M. paratuberculosis from bovine fecal specimens was described. 13 Modified Lowenstein-Jensen medium, containing mycobactin, sodium pyruvate, cycloheximide, penicillin, and chloramphenicol (Jorgensen’s medium), was used and was superior to standard Lowenstein-Jensen medium containing mycobactin for cultivation of M. paratuberculosis. Later studies were done to compare Jorgensen’s medium with HEYM. In 1985, Jorgensen’s medium was reported as able to support growth of M. paratuberculosis; however, this medium was more adversely affected by contamination than was HEYM.43 A 1989 report indicated that colonies were more numerous and appeared earlier on Jorgensen’s medium than on HEYM (Hennager SG: 1989, Abstr Conf Res Workers Anim Dis. #113, p. 21). The author recommended that both types of medium be used for cultivation of M. paratuberculosis. In 1982, mycobactin J, a new mycobactin isolated from M. paratuberculosis strain 18, was described.25 When growth of M. paratuberculosis on HEYM containing mycobactin J was compared with that on HEYM containing mycobactin P, colonies appeared earlier and more colonies were present on medium containing mycobactin J. 7,25,42 In addition, some isolates from goats were recovered only on medium containing mycobactin J.7,25 Oxalic acid, NaOH, benzalkonium chloride, and hexadecylpyridinium chloride (HPC) were evaluated as decontaminants in 1982,24 1983, 42 and 1988 (Kenefick KB, Himrich CD, Collins MT: 1988, Abstr Conf Res Workers Anim Dis #137, p. 24). HPC had the least detrimental effect on growth of M. paratuberculosis. In addition, contamination was controlled slightly better by HPC than by benzalkonium chloride.24,42 In contrast, results of other comparisons showed that HPC was not superior to benzalkonium chloride (unpublished data); therefore, benzalkonium chloride has been used as the decontaminant in some laboratories.41 In 1986, the most common types of bacterial and fungal contaminants that interfere with cultivation of M. paratuberculosis from fecal specimens were identified.36 Various antimicrobial agents were then tested to determine if any could be used to inhibit growth of contaminants without inhibition of Mycobacterium paratuberculosis. 36,37 Ketaconazole and cycloheximide could be added to HEYM without inhibiting M. para2,38 tuberculosis. In addition, incorporation of miconazole into HEYM significantly reduced contamination. 14
Studies comparing results from samples centrifuged prior to inoculation with those from samples that were not centrifuged have been recently described (Whitlock RH, Bruce JB, Spencer PA, Hutchinson L: 1988, Abstr Conf Res Workers Anim Dis #134, p. 24).14,37,44 A n increase in sensitivity of almost 300% was observed for centrifuged versus noncentrifuged samples.44 In a separate study, a lo-fold increase in the number of organisms isolated was observed when samples were centrifuged. 37 Ten to 50 colony-forming units per gram of feces may be detected when centrifugation is used. 31,32,46 In contrast, a separate study showed no significant increase in the number of positive samples when centrifuged samples were compared with noncentrifuged samples. 14 Centrifuged samples were contaminated more often than noncentrifuged samples. The procedures used in this study were different from those used in previous studies. Therefore, direct comparison of results was not possible. Incubation times reported in the literature vary considerably. In 1989, Whitlock reported that >98% of positive cultures were detectable by 12 weeks incubation and that 90% of cultures with > 6 colonies were detected by 8 weeks. Therefore, it was possible to report culture results after 12 weeks incubation.45 In addition to cultivation of M. paratuberculosis using solid media, methods using liquid media containing 14C-labeled fatty acids have been described? When compared with conventional cultivation procedures using HEYM, increased sensitivity and decreased incubation time were achieved using filter concentration of fecal specimens and radiometric cultivation procedures 16 The radiometric cultivation method described was more rapid, sensitive, and economical than conventional cultivation methods for diagnosis of paratuberculosis. 16 Many procedures for cultivation of M. paratuberculosis from fecal specimens have been developed and are in use in various laboratories. However, no specific method is recognized as the standard procedure for fecal culture. A standardized procedure would provide uniformity from laboratory to laboratory, facilitate comparison of results among laboratories, and provide a reference point for evaluation of other diagnostic tests, including improved cultivation procedures. Suggested standardized procedure for cultivation of M. paratuberculosis from bovine fecal specimens Safety
Although M. paratuberculosis generally is not considered to be infectious to humans, an M. paratuberculosis-like organism has been isolated from some patients with Crohn’s disease.4,17 Fecal specimens and
Downloaded from vdi.sagepub.com at Univ of Connecticut / Health Center / Library on May 16, 2015
Review article: Cultivation of M. paratuberculosis
suspensions of organisms should be handled in a certified level 2 biological safety cabinet.29 Phenolic-based compounds, such as 2% Amphyl,a are effective disinfectants. 27 Specimens
Collect feces rectally from each animal using a clean, dry examination glove. Place about 10 g of feces into a clean, sealable sample container that has been labeled to permit unequivocal identification of the animal from which the sample was collected. Samples should be packed in an insulated container with refrigerated (not frozen) cool packs to protect from temperature extremes during transport then sent to the laboratory by overnight delivery. Samples should be processed within 96 hours of collection; however, if this is not possible, samples should be placed in a -70 C freezer. Frozen samples should be thawed rapidly by placing them in a 35 C waterbath or incubator. Procedures
Place 1 g of feces in a 50-ml centrifuge tube with 35-40 ml sterile deionized or distilled water. Shake or vortex samples to make a uniform suspension. Samples may be shaken 30 minutes on a horizontal shaker. After the suspension has been set upright and allowed to settle for 30 minutes, transfer 5 ml from the upper half of the supernatant to another 50-ml centrifuge tube containing 25 ml of 0.9% HPC (final concentration, 0.75% HPC). Invert tubes to mix the sample suspension with decontaminant then set tubes upright at room temperature. After overnight (16-24 hours) decontamination, inoculate 4 tubes of HEYM with 0.1 ml of the sediment that forms at the bottom of the sample tube. Samples submitted from cattle suspected of having paratuberculosis should be inoculated onto 3 tubes of HEYM containing mycobactin J and 1 tube of HEYM with no mycobactin. Samples submitted for whole herd testing and certification programs should be inoculated onto 4 tubes of HEYM containing mycobactin J. Place inoculated tubes in a horizontal position with loosened caps for at least 1 week at 37 C. When no visible moisture remains on the surface of the medium, tighten caps and continue incubation in a vertical position. Examine tubes at least once every 4 weeks for at least 12 weeks. Some laboratories report results after 12 weeks incubation, whereas others allow cultures to incubate for 16 weeks. Approximately 97% of positive cultures will be visible after 12 weeks incubation, and an additional 2-3% will be positive after 16 weeks incubation. Colonies that require >3 weeks incubation to be visible, that have a morphology consistent with M . paratuberculosis. and that stain acid-fast can be iden-
371
tified as M. paratuberculosis with a high level of confidence. Cultures may be tested for mycobactin dependence to confirm identification. Any sample from which M. paratuberculosis is isolated should be considered positive, even in the presence of contamination. Negative samples must have at least 2 of 4 tubes free of contamination for the test result to be considered valid. Samples with 3 or more tubes contaminated should be retested if M. paratuberculosis has not been isolated. Mycobactin dependency test
Suspend suspect colony in 0.5 ml of sterile distilled water so that the turbidity is about 0.01 optical density units at 540 nm. Inoculate 0.1 ml of the suspension onto a single tube of HEYM containing mycobactin J and 0.1 ml onto a single tube that does not contain mycobactin. Incubate tubes for 1 week with loosened caps at 37 C. Examine tubes weekly for the presence of growth. Acid-fast cultures that grow only in the presence of mycobactin are identified as M. paratuberculosis. Modified Herrold’s egg yolk medium
Combine the following: 9.0 g peptone, 4.5 g sodium chloride, 15.3 g Noble agar, 2.7 g beef extract, and 4.1 g sodium pyruvate. Add 27 ml glycerine and 870 ml distilled water. Mix ingredients on a stir plate to form a suspension and add sufficient 1 N NaOH so that the pH is 8.1-8.4. The pH of the final product should be 7.0-7.5 when the suspension prior to autoclaving is pH 8.1-8.4. Autoclave at 121 C for 25 minutes then cool to 56 C. Prepare eggs as follows, using 6-7 eggs/liter of medium: 1) scrub eggs with a brush, using water containing a mild household detergent; 2) rinse eggs in water then soak in 70% isopropanol for 30 minutes; 3) allow eggs to air dry by placing them on a sterile towel and covering them with part of the towel; 4) using sterile forceps, crack and remove the top part of the eggshell; 5) remove egg white and yolk membrane using sterile forceps and gravity; 6) carefully remove egg yolk and place in a sterile beaker. Use at least 120 ml of egg yolk/liter of medium. If fresh eggs from a disease-free antibiotic-free source are not available, 100 ml of Bacto egg yolk suspensionb may be used. After the medium has cooled to 56 C, add the prepared egg yolks, 5 ml of sterile malachite green oxalate (2% w/v in distilled water; mix well before removing the 5 ml aliquot), 4 ml of ferric mycobactin J (2 mg mycobactin dissolved in 4 ml 95% ethanol), and 5 ml of 10 mg amphotericin B/ml sterile distilled water. Allow medium to mix thoroughly then dispense 9 ml aliquots into sterile 20 mm x 125 mm screw-cap tubes. Allow medium to solidifv by placing tubes in a slanted
Downloaded from vdi.sagepub.com at Univ of Connecticut / Health Center / Library on May 16, 2015
Whipple, Callihan, Jamagin
372
nosition so that the surface area of the medium is maximized. Quality control procedures for HEYM
Allow medium to solidify overnight and test the pH. Final DH should be 7.0-7.5. Sterility of the medium should be evaluated by randomly selecting at least 10% of the tubes and incubating at 37 C. Tubes should be examined after 3-7 days for evidence of bacterial or fungal contamination. Test the ability of the medium to support growth of M. paratuberculosis by inoculating at least 2 tubes with a light suspension of a known field isolate of M. paratuberculosis. In addition, inoculate 2 tubes of medium with a light suspension of a strain or species of mycobacteria that does not require mycobactin (suitable organisms would be M. paratuberculosis strain 18 or M. intracellulare ATCC 13950). Tubes should be read at 2-week intervals, making note of the number of colonies and speed of growth. Typical growth of the M. paratuberculosis field isolate should be evident 46 weeks after inoculation on medium containing mycobactin. The medium should be stored at 4-8 C in the dark. Be certain that all caps have been tightened to prevent desiccation during long-term storage. Shelf life of HEYM has not been determined, but it is at least 6 months (personal observation). Although cultivation of M. paratuberculosis from fecal specimens is not an ideal diagnostic test, it is considered the definitive test for diagnosis of paratuberculosis. 5 Research is continuing at several laboratories to improve current procedures. Based on currently available information, the suggested standardized procedure is a reliable method for routine cultivation of M. paratuberculosis from bovine fecal specimens. Sources and manufacturers a. National Laboratories, Lehn and Fink Industrial Products Division of Sterling Drugs, Montavale, NJ. b. Difco Laboratories, Detroit, MI.
References 1. Bergey DH, Harrison FC, Breed RS, et al.: 1923, Mycobacteria. In: Bergey’s manual of determinative bacteriology, pp. 374375. Williams & Wilkins Co., Baltimore, MD. 2. Callihan DR: 1986, Growth and effects of antifungal agents on Mycobacterium paratuberculosis using the BACTEC system. Proc 3rd Northeast Reg Meet Paratuberculosis, p. 36. 3. Chiodini RJ, van Kruiningen HJ, Merkal RS: 1984, Ruminant paratuberculosis (Johne’s disease): the current status and future prospects. Cornell Vet 74:218-262. 4. Chiodini RJ, van Kruiningen HJ, Thayer WR, et al.: 1984, Characteristics of an unclassified Mycobacterium species isolated from patients with Crohn’s disease. J Clin Microbiol 20: 966-971. 5. Colgrove GS, Thoen CO, Blackbum BO, Murphy CD: 1989,
Paratuberculosis in cattle: a comparison of three serologic tests with results of fecal cultures. Vet Microbiol 19: 183-187. 6. Collins MT, Kenefick KB, Sockett DC, et al.: 1990, Enhanced radiometric detection of Mycobacterium paratuberculosis by using filter-concentrated bovine fecal specimens. J Clin Microbiol 28:2514-2519. 7. Coloe PJ, Lightfoot D, Fromm W: 1983, Comparison of the use of mycobactin J and mycobactin P for the detection of Johne’s disease in Australian dairy herds. Proc Int Colloq Res Paratuberculosis, Ames, IA, pp. 71-76. 8. de Lisle GW, Samagh BS, Duncan JR: 1980, Bovine paratuberculosis. II. A comparison of fecal culture and the antibody response. Can J Comp Med 44: 183-191. 9. Finlayson MK: 1946, Stimulation of the growth of mycobacteria by egg yolk. J Pathol Bacterial 58:88-93. 10. Francis J, Macturk HM, Madinaveitia J, Snow GA: 1953, Mycobactin, a growth factor for Mycobacterium johnei. 1. Isolation from Mycobacterium phlei. Biochem J 55:596-607. 11. Herrold RD: 1931, Egg yolk agar medium for the growth of tubercle bacilli. J Infect Dis 48:236-241. 12. Hole NH, Maclay MH: 1959, I. The diagnosis of Johne’s disease in cattle and the identification of M. johnei infection. Vet Rec 71:1145-1149. 13. Jorgensen JB: 1982, An improved medium for culture of Mycobacterium paratuberculosis from bovine faeces. Acta Vet Stand 23:325-335. 14. Kim YG, Beth-Nielsen S, Gordon JC, et al.: 1989, Comparison of two methods for isolation of Mycobacterium paratuberculosis from bovine fecal samples. Am J Vet Res 50: 1110-1113. 15. Levi ML: 1948, The diagnosis of Johne’s disease of cattle by microscopical and cultural examination of faeces. Vet Rec 60: 336-337. 16. McDonald JM, Collins MT, Lambrecht RS: 1989, Radiometric detection of Mycobacterium paratuberculosis from clinical specimens. In: Johne’s disease: current trends in research, diagnosis, and management, ed. Milner A, Wood P, pp. 146-152. CSIRO, Melbourne, Australia. 17. McFadden JJ, Butcher PD, Chiodini R, Hermon-Taylor J: 1987, Crohn’s disease-isolated mycobacteria are identical to Mycobacterium paratuberculosis, as determined by DNA probes that distinguish between mycobacterial species. J Clin Microbiol 25: 796-801. 18. Merkal RS: 1970, Diagnostic methods for detection of paratuberculosis (Johne’s disease). Proc Annu Meet US Anim Health Assoc 74:620-623. 19. Merkal RS: 1973, Laboratory diagnosis of bovine paratuberculosis. J Am Vet Med Assoc 163: 1100-1102. 20. Merkal RS, Curran BJ: 1974, Growth and metabolic characteristics of Mycobacterium paratuberculosis. Appl Microbiol 28: 276-279. 21. Merkal RS, Kopecky KE, Larsen AB, Thurston JR: 1964, Improvements in the techniques for primary cultivation of Mycobacterium paratuberculosis. Am J Vet Res 25: 1290-1293. 22. Merkal RS, Larsen AB: 1962, Improved methods for primary cultivation of Mycobacterium paratuberculosis. Am J Vet Res 23: 1307-1309. 23. Merkal RS, Larsen AB, Kopecky ISE, Ness RD: 1968, Comparison of examination and test methods for early detection of paratuberculous cattle. Am J Vet Res 29: 1533-1538. 24. Merkal RS, Lyle PAS, Whipple DL: 1982, Decontamination, media, and culture methods for Mycobacterium paratuberculosis. Proc Annu Meet US Anim Health Assoc 86:5 19-522. 25. Merkal RS, McCullough WG: 1982, A new mycobactin, mycobactin J, from Mycobacterium paratuberculosis. Curr Microbiol 7:333-335. 26. Merkal RS, Richards WD: 1972, Inhibition of fungal growth
Downloaded from vdi.sagepub.com at Univ of Connecticut / Health Center / Library on May 16, 2015
Review article: Cultivation of M. paratuberculosis in cultural isolation of mycobacteria. Appl Microbiol 24:205207. 27. Merkal RS, Whipple DL: 1982, Effectiveness of disinfectants on Mycobacterium paratuberculosis. Proc Annu Meet US Anim Health Assoc 86:514-518. 28. Minett FC: 1942, The diagnosis of Johne’s disease of cattle by cultural methods. J Pathol Bacteriol 54:209-219. 29. Richardson JH, Barkley WE, eds.: 1988, Biosafety in microbiological and biomedical laboratories, 2nd ed. US Department of Health and Human Services, Public Health Service, Centers for Disease Control and National Institutes of Health, Washington, D.C. 30. Ringdal G: 1963, Culture of Mycobacterium johnei. Acta Vet Stand 4: 85-91. 31. Ris DR, Hamel KL, Ayling JM: 1988, The detection of Mycobacterium paratuberculosis in bovine faeces by isolation and the comparison of isolation with the examination of stained smears by light microscopy. NZ Vet J 36: 112-114. 32. Sanftleben P: 1990, Quest continues for fast, reliable test for bovine paratuberculosis. J Am Vet Med Assoc 197:299-305. 33. Smith HW: 1953, Modifications of Dubo’s media for the cultivation of Mycobacterium johnei. J Pathol Bacteriol 66:375381. 34. Taylor AW: 1950, Observation on the isolation of Mycobacterium johnei in primary culture. J Pathol Bacteriol 62:647650. 35. Thoen CO, Baum KH: 1988, Current knowledge of paratuberculosis. J Am Vet Med Assoc 192: 1609-1611. 36. Turcotte C, Brooks BW, Dion WM, Marenger R: 1986, Bacterial and fungal contaminants interfering with the isolation of Mycobacterium paratuberculosis. Proc Annu Meet Am Assoc Vet Lab Diagn 29:49-58. 37. Turcotte C, Brooks BW, Duncan JR: 1986, Influence of sample processing on the sensitivity of culture for the isolation of Mycobacterium paratuberculosis. Proc 3rd Northeast Reg Meet Paratuberculosis, p. 27.
373
38. Turcotte C, Dion WM, Brooks BW: 1986, Identification and control of fungal contaminants in fecal cultures for the isolation of Mycobacterium paratuberculosis. Proc 3rd Northeast Reg Meet Paratuberculosis, p. 26. 39. Twort FW: 1910, A method for isolating and growing the lepra bacillus of man (preliminary note). Proc R Soc Lond Ser B 83: 156-158. 40. Twort FW, Ingram GLY: 1912, A method for isolating and cultivating Mycobacterium enteritidis chronicae pseudotuberculosis bovis, Johne, and some experiments on the preparation of a diagnostic vaccine for pseudotuberculosis enteritis of bovines. Proc R Soc Lond Ser B 84:517-542. 41. US Department of Agriculture: 1985, Laboratory methods in veterinary mycobacteriology, pp. 59-69. National Veterinary Services Laboratories, Animal and Plant Health Inspection Service, Ames, IA. 42. Whipple DL, Merkal RS: 1983, Modifications in the techniques for cultivation of Mycobacterium paratuberculosis. Proc Int Colloq Res Paratuberculosis, Ames, IA, pp. 82-92. 43. Whipple DL, Merkal RS: 1985, Procedures for the field and laboratory processing of fecal specimens for the isolation of Mycobacterium paratuberculosis. Proc Annu Meet US Anim Health Assoc 89:475-479. 44. Whitlock RH, Bruce JL: 1986, Mycobacterium paratuberculosis: culture using centrifugation a more sensitive technique? Proc 3rd Northeast Reg Meet Paratuberculosis, p. 28. 45. Whitlock RH, Rosenberger AE, Spencer PA: 1989, Laboratory culture techniques for Johne’s disease: a critical evaluation of contamination and incubation times. Proc Annu Meet US Anim Health Assoc 93:382-386. 46. Whitlock RH, Sweeney RW: 1990, Johne’s disease: current aspects of transmission and the art of fecal culture testing. Proc Annu Meet Livest Conserv Inst, pp. 24-30.
J Vet Diagn Invest 3:373-383 (1991)
Ruminant paratuberculosis—a century of progress and frustration John M. Kreeger Since 1895 when paratuberculosis was first described genesis in paratuberculosis been more closely examas a clinical entity by Johne and Frothingham, study ined. Yet with all of these combined efforts directed of the disease has assembled an impressive number of towards the understanding of the disease, paratuberdevoted followers who have committed themselves to culosis remains a significant problem to the researcher, a life-long pursuit of unraveling various aspects of the diagnostician, and animal producer. disease. Disease chronicity and resistance to therapeuHistory tic measures have concentrated efforts on mechanisms of disease transmission, factors influencing susceptiAlong with describing the clinical aspects of the disbility, serologic identification of nonclinical carrier an- ease, Johne and Frothingham also identified the presimals, and other means of early diagnosis. Only in the ence of acid-fast organisms in the granulomatous lelast 10 years or so have mechanisms of disease patho- sions and speculated that this disease was probably an atypical form of ruminant tuberculosis. It was not until 102,103 From the University of Missouri, Veterinary Medical Diagnostic 1910 that the organism was first isolated by Twort and given the name Mycobacterium enteriditis chronLaboratory, Columbia, MO 65211. Received for publication May 13, 1991. icae txeudotuberculosae bovis johne. Some time later.
Downloaded from vdi.sagepub.com at Univ of Connecticut / Health Center / Library on May 16, 2015
REVIEW ARTICLES
Cultivation of Mycobacterium paratuberculosis from bovine fecal specimens and a suggested standardized procedure Diana L. Whipple, Donald R. Callihan, Jerald L. Jarnagin
Paratuberculosis is a chronic granulomatous enteritis of ruminants caused by a small, fastidious, acidfast bacillus, Mycobacterium paratuberculosis. The disease now called paratuberculosis was described in 1826 as an enteritis found in some cattle with chronic diarrhea. 3 Johne and Frothingham further described the disease in 1895 and demonstrated the presence of acidfast bacilli in affected intestine.3 The first successful isolation of this organism was in 1910 by Twort, who named in Mycobacterium enteriditis chronicae pseu39,40 dotuberculosis bovis johne. The name of the organism has since been changed to Mycobacterium paratuberculosis, 1 and the clinical disease is called Johne’s disease. 3 Many tests have been developed to aid in diagnosis of paratuberculosis; however, isolation of M. paratuberculosis from tissue or fecal specimens is regarded 3,5 as the definitive diagnostic test in cattle. Cultivation of M. paratuberculosis from fecal specimens is the most frequently used diagnostic test and is the standard for comparison for nearly all other tests.8 Most paratuberculosis control programs use fecal culture results as the basis for management decisions;35 however, the procedures used have not been standardized. The purpose of this paper is to present a partial review of the literature describing procedures for cultivation of M.
paratuberculosis from fecal specimens and to suggest a standardized cultivation procedure. Review of the literature Isolation of the organism now known as M. paratuberculosis was first reported in 1910,39 and the complete description of the procedure was reported in 1912.40 An inspissated egg-base medium containing Mycobacterium tuberculosis was used. The authors included M. tuberculosis because they recognized that some type of “necessary foodstuff needed to support growth of M. paratuberculosis was missing from the medium. They theorized that this “foodstuff’ could be supplied by another acid-fast organism, such as M. tuberculosis. After successfully isolating M. paratuberculosis on egg medium containing M. tuberculosis, they substituted the timothy grass bacillus (M. phlei) for M. tuberculosis and observed growth that was superior. The “necessary foodstuff’ was extracted from M. phlei using hot ethanol and was used in medium instead of a suspension of M. phlei to support growth of M. paratuberculosis. Use of egg-based medium and supplementation with extracted “foodstuff’ (now called mycobactin) provided the foundation for subsequent procedures for isolation of M. paratuberculosis. Various types of media have been evaluated for their ability to support growth of mycobacteria. In 1931, egg yolk agar medium was reported to support growth of M. tuberculosis better than medium containing whole eggs because egg whites were inhibitory to the organism. 11 In 1942, 27 different media were evaluated, ineluding a modification of Herrold’s egg yolk medium (HEYM), for their ability to support growth of M. para28 tuberculosis. Media containing whole eggs were superior to media containing egg yolks alone. In addition, samples could be decontaminated with antiformin (1020%, final concentration), and either brilliant green or malachite green could be included in the medium to help control contamination and enhance visibility of colonies.
From the Leptospirosis/Mycobacteriosis Research Unit, National Animal Disease Center, USDA, Agricultural Research Service, Ames, IA 50010 (Whipple), the Diagnostic Laboratory, New York State College of Veterinary Medicine, Cornell University, Ithaca, NY 14853 (Callihan), and the Mycobacteria, Brucella Reagents Section, Diagnostic Bacteriology Laboratory, National Veterinary Services Laboratories, USDA, Animal and Plant Health Inspection Services, Ames, IA 50010 (Jamagin). Current address (Callihan): the Department of Pathology, Pitt County Memorial Hospital, and the Department of Clinical Pathology and Laboratory Medicine, East Carolina University School of Medicine, Greenville, NC 27835. Received for publication February 11, 1991. 368
Downloaded from vdi.sagepub.com at Univ of Connecticut / Health Center / Library on May 16, 2015
Review article: Cultivation of M. paratuberculosis
Microscopic and cultural examination of fecal specimens for diagnosis of Johne’s disease was described in 1948.15 In that study, Dunkins medium containing whole eggs, liver, and a glycerated extract of M. phlei was used. Samples were decontaminated in antiformin. The author concluded that microscopic examination of fecal specimens was more practical for diagnosis than was cultivation of the organism from fecal specimens and that culture was most useful for confirming the presence of M. paratuberculosis in specific cases of Johne’s disease. In 1950, 8 different media for primary isolation of M. paratuberculosis were compared.34 Media containing at least 50% egg yolk were more reliable than complex mixtures containing whole eggs or liver. Finlayson’s medium,9 which contains 60% egg yolk, was identified as the preferred medium. Decontaminating agents also were evaluated. Although oxalic acid was less inhibitory to M. paratuberculosis, use of antiformin was recommended because of more effective decontamination of fecal specimens. The author stated, “there is at present no completely reliable method of obtaining M. johnei in culture from infected bovine faeces, particularly from samples in which they may be present in very small numbers.” Various decontaminating agents in conjunction with two media were compared in 1953 .33 Modified Dubo’s medium was judged superior to Taylor’s modification of Finlayson’s medium 34 for cultivation of M. paratuberculosis from infected material. Decontaminants compared were 4% NaOH, 10% trisodium phosphate, 5% oxalic acid, 5% sulphuric acid, and 10-20% antiformin. The efficacy of including penicillin and chloramphenicol in media to control contamination without inhibition of M. paratuberculosis also was tested. Contamination was best controlled by pretreating fecal specimens with 20% antiformin and incorporating penicillin into the medium. A review of procedures for diagnosis of paratuberculosis was published in 1959. 12 The authors stated, “Cultural examination of the faeces is not only complicated by contamination problems, but is of little practical use for diagnosis because of the slow growth of the organisms, and this procedure is reserved almost solely for experimental work. If faeces examinations are practical only in the scouring animal, as has been suggested, this test has only limited diagnostic value and has no direct application to eradication or control measures.” In 1963, specimens were decontaminated using 10% sulfuric acid and inoculated onto Lowenstein-Jensen medium containing 5% killed bovine tubercle bacilli.30 Inoculation of more tubes of medium increased the chances of obtaining a positive diagnosis by culture. Culture was positive in some cases that were negative
369
by microscopic evaluation; however, some cases were microscopically positive and culture negative. The prolonged time required for culture was thought to diminish its value for diagnosis. The purpose of a study reported in 196421 was to develop procedures for primary isolation of M. paratuberculosis from specimens that contained too few organisms to be detected microscopically. A modification of HEYM was compared with lymph node egg yolk medium,22 each containing M phlei suspended in glycerin as a source of mycobactin. The following decontaminants were also evaluated: 4% NaOH, 0.2% NaOCI, 0.5% phenol, and 0.1-1.0% benzalkonium chloride. The combination of benzalkonium chloride as a decontaminant and modified HEYM was superior to microscopic examination for detecting M. paratuberculosis in a specimen. Use of 0.3% benzalkonium chloride for l-3 days for decontamination of fecal specimens was recommended. In 1968, Merkal et a1.23 recommended 1) the cultural examination of fecal specimens for diagnosis of subclinical paratuberculosis and 2) the examination by twice yearly fecal culture of herds suspected of having paratuberculosis and the slaughter of culture-positive animals. Although mycobactin from M. phlei was isolated and characterized in 1953,10 most investigators continued to use a suspension of M. phlei in media as a mycobactin source. The HEYM described in 1968 contained an extract of M. phlei as a crude mycobactin source .23 Mycobactin from M. phlei was later designated mycobactin P. 18 In 1970 and 1973, 50-100 organisms per gram of feces were reported as required for detection of M . paratuberculosis using the recommended cultivation procedure. 18,19 In 1972, decontamination of fecal specimens in benzalkonium chloride followed by either mixing the inoculum in a suspension containing amphotericin B or by inoculation onto media containing amphotericin B was recommended to better control fungal contaminants. 26 Amphotericin B, an antifungal agent, had no detrimental effect on M. paratuberculosis. Growth and metabolic characteristics of M. paratuberculosis were described in 1974.20 Dependence of M. paratuberculosis on exogenous mycobactin could be circumvented by adding 1% ferric ammonium citrate to the medium, and growth of most strains tested was enhanced by adding 4.1% sodium pyruvate to the medium. In 1980, fecal culture results were compared with antibody response as determined by the complement fixation test.8 The decontaminant used was 0.2% alkylbenzyl-dimethyl-ammonium chloride, and the medium was HEYM containing mycobactin P. The au-
Downloaded from vdi.sagepub.com at Univ of Connecticut / Health Center / Library on May 16, 2015
370
Whipple, Callihan, Jamagin
thors stated, “fecal culture therefore appears to be a good basis on which to evaluate various immunological reactions of infected cattle.” The disadvantages of using fecal culture were prolonged incubation and contamination problems. In 1982, an alternative medium for cultivation of M. paratuberculosis from bovine fecal specimens was described. 13 Modified Lowenstein-Jensen medium, containing mycobactin, sodium pyruvate, cycloheximide, penicillin, and chloramphenicol (Jorgensen’s medium), was used and was superior to standard Lowenstein-Jensen medium containing mycobactin for cultivation of M. paratuberculosis. Later studies were done to compare Jorgensen’s medium with HEYM. In 1985, Jorgensen’s medium was reported as able to support growth of M. paratuberculosis; however, this medium was more adversely affected by contamination than was HEYM.43 A 1989 report indicated that colonies were more numerous and appeared earlier on Jorgensen’s medium than on HEYM (Hennager SG: 1989, Abstr Conf Res Workers Anim Dis. #113, p. 21). The author recommended that both types of medium be used for cultivation of M. paratuberculosis. In 1982, mycobactin J, a new mycobactin isolated from M. paratuberculosis strain 18, was described.25 When growth of M. paratuberculosis on HEYM containing mycobactin J was compared with that on HEYM containing mycobactin P, colonies appeared earlier and more colonies were present on medium containing mycobactin J. 7,25,42 In addition, some isolates from goats were recovered only on medium containing mycobactin J.7,25 Oxalic acid, NaOH, benzalkonium chloride, and hexadecylpyridinium chloride (HPC) were evaluated as decontaminants in 1982,24 1983, 42 and 1988 (Kenefick KB, Himrich CD, Collins MT: 1988, Abstr Conf Res Workers Anim Dis #137, p. 24). HPC had the least detrimental effect on growth of M. paratuberculosis. In addition, contamination was controlled slightly better by HPC than by benzalkonium chloride.24,42 In contrast, results of other comparisons showed that HPC was not superior to benzalkonium chloride (unpublished data); therefore, benzalkonium chloride has been used as the decontaminant in some laboratories.41 In 1986, the most common types of bacterial and fungal contaminants that interfere with cultivation of M. paratuberculosis from fecal specimens were identified.36 Various antimicrobial agents were then tested to determine if any could be used to inhibit growth of contaminants without inhibition of Mycobacterium paratuberculosis. 36,37 Ketaconazole and cycloheximide could be added to HEYM without inhibiting M. para2,38 tuberculosis. In addition, incorporation of miconazole into HEYM significantly reduced contamination. 14
Studies comparing results from samples centrifuged prior to inoculation with those from samples that were not centrifuged have been recently described (Whitlock RH, Bruce JB, Spencer PA, Hutchinson L: 1988, Abstr Conf Res Workers Anim Dis #134, p. 24).14,37,44 A n increase in sensitivity of almost 300% was observed for centrifuged versus noncentrifuged samples.44 In a separate study, a lo-fold increase in the number of organisms isolated was observed when samples were centrifuged. 37 Ten to 50 colony-forming units per gram of feces may be detected when centrifugation is used. 31,32,46 In contrast, a separate study showed no significant increase in the number of positive samples when centrifuged samples were compared with noncentrifuged samples. 14 Centrifuged samples were contaminated more often than noncentrifuged samples. The procedures used in this study were different from those used in previous studies. Therefore, direct comparison of results was not possible. Incubation times reported in the literature vary considerably. In 1989, Whitlock reported that >98% of positive cultures were detectable by 12 weeks incubation and that 90% of cultures with > 6 colonies were detected by 8 weeks. Therefore, it was possible to report culture results after 12 weeks incubation.45 In addition to cultivation of M. paratuberculosis using solid media, methods using liquid media containing 14C-labeled fatty acids have been described? When compared with conventional cultivation procedures using HEYM, increased sensitivity and decreased incubation time were achieved using filter concentration of fecal specimens and radiometric cultivation procedures 16 The radiometric cultivation method described was more rapid, sensitive, and economical than conventional cultivation methods for diagnosis of paratuberculosis. 16 Many procedures for cultivation of M. paratuberculosis from fecal specimens have been developed and are in use in various laboratories. However, no specific method is recognized as the standard procedure for fecal culture. A standardized procedure would provide uniformity from laboratory to laboratory, facilitate comparison of results among laboratories, and provide a reference point for evaluation of other diagnostic tests, including improved cultivation procedures. Suggested standardized procedure for cultivation of M. paratuberculosis from bovine fecal specimens Safety
Although M. paratuberculosis generally is not considered to be infectious to humans, an M. paratuberculosis-like organism has been isolated from some patients with Crohn’s disease.4,17 Fecal specimens and
Downloaded from vdi.sagepub.com at Univ of Connecticut / Health Center / Library on May 16, 2015
Review article: Cultivation of M. paratuberculosis
suspensions of organisms should be handled in a certified level 2 biological safety cabinet.29 Phenolic-based compounds, such as 2% Amphyl,a are effective disinfectants. 27 Specimens
Collect feces rectally from each animal using a clean, dry examination glove. Place about 10 g of feces into a clean, sealable sample container that has been labeled to permit unequivocal identification of the animal from which the sample was collected. Samples should be packed in an insulated container with refrigerated (not frozen) cool packs to protect from temperature extremes during transport then sent to the laboratory by overnight delivery. Samples should be processed within 96 hours of collection; however, if this is not possible, samples should be placed in a -70 C freezer. Frozen samples should be thawed rapidly by placing them in a 35 C waterbath or incubator. Procedures
Place 1 g of feces in a 50-ml centrifuge tube with 35-40 ml sterile deionized or distilled water. Shake or vortex samples to make a uniform suspension. Samples may be shaken 30 minutes on a horizontal shaker. After the suspension has been set upright and allowed to settle for 30 minutes, transfer 5 ml from the upper half of the supernatant to another 50-ml centrifuge tube containing 25 ml of 0.9% HPC (final concentration, 0.75% HPC). Invert tubes to mix the sample suspension with decontaminant then set tubes upright at room temperature. After overnight (16-24 hours) decontamination, inoculate 4 tubes of HEYM with 0.1 ml of the sediment that forms at the bottom of the sample tube. Samples submitted from cattle suspected of having paratuberculosis should be inoculated onto 3 tubes of HEYM containing mycobactin J and 1 tube of HEYM with no mycobactin. Samples submitted for whole herd testing and certification programs should be inoculated onto 4 tubes of HEYM containing mycobactin J. Place inoculated tubes in a horizontal position with loosened caps for at least 1 week at 37 C. When no visible moisture remains on the surface of the medium, tighten caps and continue incubation in a vertical position. Examine tubes at least once every 4 weeks for at least 12 weeks. Some laboratories report results after 12 weeks incubation, whereas others allow cultures to incubate for 16 weeks. Approximately 97% of positive cultures will be visible after 12 weeks incubation, and an additional 2-3% will be positive after 16 weeks incubation. Colonies that require >3 weeks incubation to be visible, that have a morphology consistent with M . paratuberculosis. and that stain acid-fast can be iden-
371
tified as M. paratuberculosis with a high level of confidence. Cultures may be tested for mycobactin dependence to confirm identification. Any sample from which M. paratuberculosis is isolated should be considered positive, even in the presence of contamination. Negative samples must have at least 2 of 4 tubes free of contamination for the test result to be considered valid. Samples with 3 or more tubes contaminated should be retested if M. paratuberculosis has not been isolated. Mycobactin dependency test
Suspend suspect colony in 0.5 ml of sterile distilled water so that the turbidity is about 0.01 optical density units at 540 nm. Inoculate 0.1 ml of the suspension onto a single tube of HEYM containing mycobactin J and 0.1 ml onto a single tube that does not contain mycobactin. Incubate tubes for 1 week with loosened caps at 37 C. Examine tubes weekly for the presence of growth. Acid-fast cultures that grow only in the presence of mycobactin are identified as M. paratuberculosis. Modified Herrold’s egg yolk medium
Combine the following: 9.0 g peptone, 4.5 g sodium chloride, 15.3 g Noble agar, 2.7 g beef extract, and 4.1 g sodium pyruvate. Add 27 ml glycerine and 870 ml distilled water. Mix ingredients on a stir plate to form a suspension and add sufficient 1 N NaOH so that the pH is 8.1-8.4. The pH of the final product should be 7.0-7.5 when the suspension prior to autoclaving is pH 8.1-8.4. Autoclave at 121 C for 25 minutes then cool to 56 C. Prepare eggs as follows, using 6-7 eggs/liter of medium: 1) scrub eggs with a brush, using water containing a mild household detergent; 2) rinse eggs in water then soak in 70% isopropanol for 30 minutes; 3) allow eggs to air dry by placing them on a sterile towel and covering them with part of the towel; 4) using sterile forceps, crack and remove the top part of the eggshell; 5) remove egg white and yolk membrane using sterile forceps and gravity; 6) carefully remove egg yolk and place in a sterile beaker. Use at least 120 ml of egg yolk/liter of medium. If fresh eggs from a disease-free antibiotic-free source are not available, 100 ml of Bacto egg yolk suspensionb may be used. After the medium has cooled to 56 C, add the prepared egg yolks, 5 ml of sterile malachite green oxalate (2% w/v in distilled water; mix well before removing the 5 ml aliquot), 4 ml of ferric mycobactin J (2 mg mycobactin dissolved in 4 ml 95% ethanol), and 5 ml of 10 mg amphotericin B/ml sterile distilled water. Allow medium to mix thoroughly then dispense 9 ml aliquots into sterile 20 mm x 125 mm screw-cap tubes. Allow medium to solidifv by placing tubes in a slanted
Downloaded from vdi.sagepub.com at Univ of Connecticut / Health Center / Library on May 16, 2015
Whipple, Callihan, Jamagin
372
nosition so that the surface area of the medium is maximized. Quality control procedures for HEYM
Allow medium to solidify overnight and test the pH. Final DH should be 7.0-7.5. Sterility of the medium should be evaluated by randomly selecting at least 10% of the tubes and incubating at 37 C. Tubes should be examined after 3-7 days for evidence of bacterial or fungal contamination. Test the ability of the medium to support growth of M. paratuberculosis by inoculating at least 2 tubes with a light suspension of a known field isolate of M. paratuberculosis. In addition, inoculate 2 tubes of medium with a light suspension of a strain or species of mycobacteria that does not require mycobactin (suitable organisms would be M. paratuberculosis strain 18 or M. intracellulare ATCC 13950). Tubes should be read at 2-week intervals, making note of the number of colonies and speed of growth. Typical growth of the M. paratuberculosis field isolate should be evident 46 weeks after inoculation on medium containing mycobactin. The medium should be stored at 4-8 C in the dark. Be certain that all caps have been tightened to prevent desiccation during long-term storage. Shelf life of HEYM has not been determined, but it is at least 6 months (personal observation). Although cultivation of M. paratuberculosis from fecal specimens is not an ideal diagnostic test, it is considered the definitive test for diagnosis of paratuberculosis. 5 Research is continuing at several laboratories to improve current procedures. Based on currently available information, the suggested standardized procedure is a reliable method for routine cultivation of M. paratuberculosis from bovine fecal specimens. Sources and manufacturers a. National Laboratories, Lehn and Fink Industrial Products Division of Sterling Drugs, Montavale, NJ. b. Difco Laboratories, Detroit, MI.
References 1. Bergey DH, Harrison FC, Breed RS, et al.: 1923, Mycobacteria. In: Bergey’s manual of determinative bacteriology, pp. 374375. Williams & Wilkins Co., Baltimore, MD. 2. Callihan DR: 1986, Growth and effects of antifungal agents on Mycobacterium paratuberculosis using the BACTEC system. Proc 3rd Northeast Reg Meet Paratuberculosis, p. 36. 3. Chiodini RJ, van Kruiningen HJ, Merkal RS: 1984, Ruminant paratuberculosis (Johne’s disease): the current status and future prospects. Cornell Vet 74:218-262. 4. Chiodini RJ, van Kruiningen HJ, Thayer WR, et al.: 1984, Characteristics of an unclassified Mycobacterium species isolated from patients with Crohn’s disease. J Clin Microbiol 20: 966-971. 5. Colgrove GS, Thoen CO, Blackbum BO, Murphy CD: 1989,
Paratuberculosis in cattle: a comparison of three serologic tests with results of fecal cultures. Vet Microbiol 19: 183-187. 6. Collins MT, Kenefick KB, Sockett DC, et al.: 1990, Enhanced radiometric detection of Mycobacterium paratuberculosis by using filter-concentrated bovine fecal specimens. J Clin Microbiol 28:2514-2519. 7. Coloe PJ, Lightfoot D, Fromm W: 1983, Comparison of the use of mycobactin J and mycobactin P for the detection of Johne’s disease in Australian dairy herds. Proc Int Colloq Res Paratuberculosis, Ames, IA, pp. 71-76. 8. de Lisle GW, Samagh BS, Duncan JR: 1980, Bovine paratuberculosis. II. A comparison of fecal culture and the antibody response. Can J Comp Med 44: 183-191. 9. Finlayson MK: 1946, Stimulation of the growth of mycobacteria by egg yolk. J Pathol Bacterial 58:88-93. 10. Francis J, Macturk HM, Madinaveitia J, Snow GA: 1953, Mycobactin, a growth factor for Mycobacterium johnei. 1. Isolation from Mycobacterium phlei. Biochem J 55:596-607. 11. Herrold RD: 1931, Egg yolk agar medium for the growth of tubercle bacilli. J Infect Dis 48:236-241. 12. Hole NH, Maclay MH: 1959, I. The diagnosis of Johne’s disease in cattle and the identification of M. johnei infection. Vet Rec 71:1145-1149. 13. Jorgensen JB: 1982, An improved medium for culture of Mycobacterium paratuberculosis from bovine faeces. Acta Vet Stand 23:325-335. 14. Kim YG, Beth-Nielsen S, Gordon JC, et al.: 1989, Comparison of two methods for isolation of Mycobacterium paratuberculosis from bovine fecal samples. Am J Vet Res 50: 1110-1113. 15. Levi ML: 1948, The diagnosis of Johne’s disease of cattle by microscopical and cultural examination of faeces. Vet Rec 60: 336-337. 16. McDonald JM, Collins MT, Lambrecht RS: 1989, Radiometric detection of Mycobacterium paratuberculosis from clinical specimens. In: Johne’s disease: current trends in research, diagnosis, and management, ed. Milner A, Wood P, pp. 146-152. CSIRO, Melbourne, Australia. 17. McFadden JJ, Butcher PD, Chiodini R, Hermon-Taylor J: 1987, Crohn’s disease-isolated mycobacteria are identical to Mycobacterium paratuberculosis, as determined by DNA probes that distinguish between mycobacterial species. J Clin Microbiol 25: 796-801. 18. Merkal RS: 1970, Diagnostic methods for detection of paratuberculosis (Johne’s disease). Proc Annu Meet US Anim Health Assoc 74:620-623. 19. Merkal RS: 1973, Laboratory diagnosis of bovine paratuberculosis. J Am Vet Med Assoc 163: 1100-1102. 20. Merkal RS, Curran BJ: 1974, Growth and metabolic characteristics of Mycobacterium paratuberculosis. Appl Microbiol 28: 276-279. 21. Merkal RS, Kopecky KE, Larsen AB, Thurston JR: 1964, Improvements in the techniques for primary cultivation of Mycobacterium paratuberculosis. Am J Vet Res 25: 1290-1293. 22. Merkal RS, Larsen AB: 1962, Improved methods for primary cultivation of Mycobacterium paratuberculosis. Am J Vet Res 23: 1307-1309. 23. Merkal RS, Larsen AB, Kopecky ISE, Ness RD: 1968, Comparison of examination and test methods for early detection of paratuberculous cattle. Am J Vet Res 29: 1533-1538. 24. Merkal RS, Lyle PAS, Whipple DL: 1982, Decontamination, media, and culture methods for Mycobacterium paratuberculosis. Proc Annu Meet US Anim Health Assoc 86:5 19-522. 25. Merkal RS, McCullough WG: 1982, A new mycobactin, mycobactin J, from Mycobacterium paratuberculosis. Curr Microbiol 7:333-335. 26. Merkal RS, Richards WD: 1972, Inhibition of fungal growth
Downloaded from vdi.sagepub.com at Univ of Connecticut / Health Center / Library on May 16, 2015
Review article: Cultivation of M. paratuberculosis in cultural isolation of mycobacteria. Appl Microbiol 24:205207. 27. Merkal RS, Whipple DL: 1982, Effectiveness of disinfectants on Mycobacterium paratuberculosis. Proc Annu Meet US Anim Health Assoc 86:514-518. 28. Minett FC: 1942, The diagnosis of Johne’s disease of cattle by cultural methods. J Pathol Bacteriol 54:209-219. 29. Richardson JH, Barkley WE, eds.: 1988, Biosafety in microbiological and biomedical laboratories, 2nd ed. US Department of Health and Human Services, Public Health Service, Centers for Disease Control and National Institutes of Health, Washington, D.C. 30. Ringdal G: 1963, Culture of Mycobacterium johnei. Acta Vet Stand 4: 85-91. 31. Ris DR, Hamel KL, Ayling JM: 1988, The detection of Mycobacterium paratuberculosis in bovine faeces by isolation and the comparison of isolation with the examination of stained smears by light microscopy. NZ Vet J 36: 112-114. 32. Sanftleben P: 1990, Quest continues for fast, reliable test for bovine paratuberculosis. J Am Vet Med Assoc 197:299-305. 33. Smith HW: 1953, Modifications of Dubo’s media for the cultivation of Mycobacterium johnei. J Pathol Bacteriol 66:375381. 34. Taylor AW: 1950, Observation on the isolation of Mycobacterium johnei in primary culture. J Pathol Bacteriol 62:647650. 35. Thoen CO, Baum KH: 1988, Current knowledge of paratuberculosis. J Am Vet Med Assoc 192: 1609-1611. 36. Turcotte C, Brooks BW, Dion WM, Marenger R: 1986, Bacterial and fungal contaminants interfering with the isolation of Mycobacterium paratuberculosis. Proc Annu Meet Am Assoc Vet Lab Diagn 29:49-58. 37. Turcotte C, Brooks BW, Duncan JR: 1986, Influence of sample processing on the sensitivity of culture for the isolation of Mycobacterium paratuberculosis. Proc 3rd Northeast Reg Meet Paratuberculosis, p. 27.
373
38. Turcotte C, Dion WM, Brooks BW: 1986, Identification and control of fungal contaminants in fecal cultures for the isolation of Mycobacterium paratuberculosis. Proc 3rd Northeast Reg Meet Paratuberculosis, p. 26. 39. Twort FW: 1910, A method for isolating and growing the lepra bacillus of man (preliminary note). Proc R Soc Lond Ser B 83: 156-158. 40. Twort FW, Ingram GLY: 1912, A method for isolating and cultivating Mycobacterium enteritidis chronicae pseudotuberculosis bovis, Johne, and some experiments on the preparation of a diagnostic vaccine for pseudotuberculosis enteritis of bovines. Proc R Soc Lond Ser B 84:517-542. 41. US Department of Agriculture: 1985, Laboratory methods in veterinary mycobacteriology, pp. 59-69. National Veterinary Services Laboratories, Animal and Plant Health Inspection Service, Ames, IA. 42. Whipple DL, Merkal RS: 1983, Modifications in the techniques for cultivation of Mycobacterium paratuberculosis. Proc Int Colloq Res Paratuberculosis, Ames, IA, pp. 82-92. 43. Whipple DL, Merkal RS: 1985, Procedures for the field and laboratory processing of fecal specimens for the isolation of Mycobacterium paratuberculosis. Proc Annu Meet US Anim Health Assoc 89:475-479. 44. Whitlock RH, Bruce JL: 1986, Mycobacterium paratuberculosis: culture using centrifugation a more sensitive technique? Proc 3rd Northeast Reg Meet Paratuberculosis, p. 28. 45. Whitlock RH, Rosenberger AE, Spencer PA: 1989, Laboratory culture techniques for Johne’s disease: a critical evaluation of contamination and incubation times. Proc Annu Meet US Anim Health Assoc 93:382-386. 46. Whitlock RH, Sweeney RW: 1990, Johne’s disease: current aspects of transmission and the art of fecal culture testing. Proc Annu Meet Livest Conserv Inst, pp. 24-30.
J Vet Diagn Invest 3:373-383 (1991)
Ruminant paratuberculosis—a century of progress and frustration John M. Kreeger Since 1895 when paratuberculosis was first described genesis in paratuberculosis been more closely examas a clinical entity by Johne and Frothingham, study ined. Yet with all of these combined efforts directed of the disease has assembled an impressive number of towards the understanding of the disease, paratuberdevoted followers who have committed themselves to culosis remains a significant problem to the researcher, a life-long pursuit of unraveling various aspects of the diagnostician, and animal producer. disease. Disease chronicity and resistance to therapeuHistory tic measures have concentrated efforts on mechanisms of disease transmission, factors influencing susceptiAlong with describing the clinical aspects of the disbility, serologic identification of nonclinical carrier an- ease, Johne and Frothingham also identified the presimals, and other means of early diagnosis. Only in the ence of acid-fast organisms in the granulomatous lelast 10 years or so have mechanisms of disease patho- sions and speculated that this disease was probably an atypical form of ruminant tuberculosis. It was not until 102,103 From the University of Missouri, Veterinary Medical Diagnostic 1910 that the organism was first isolated by Twort and given the name Mycobacterium enteriditis chronLaboratory, Columbia, MO 65211. Received for publication May 13, 1991. icae txeudotuberculosae bovis johne. Some time later.
Downloaded from vdi.sagepub.com at Univ of Connecticut / Health Center / Library on May 16, 2015
