ESTIVE DISEASES
`ND SCIENC
`
`Formerly Published as The American Journal of Digestive Diseases
`'itill111111M111111&
`
`•
`
`W Series Volume 29, Number 12
`1st Year of Continuous Publication
`
`DECEMBER 1984
`
`ISSN 01Q-2116
`DDSCDJ 29(12) 1073-1184 (1984)
`This issue completes Volunae 29
`
`Possible Role of Mycobacteria in Inflammatory Bowel Disease. I. An Unclassified Mycobacterium Species Isolated from Patients with Croll, s
`Disease. Rodrick J. Chiodini, Herbert J. Van Kruiningen, Walter R. Thayer, Richard S. Merkal, and Jessica A. Coutu
`
`Possible Role of Mycobacteria in Inflammatory Bowel Disease. II. Mycobacterial Antibodies in Crohn's Disease. Walter R. Thayer Jr., Je, 'ca A.
`Coutu, Rodrick J. Chiodini, Herbert J. Van Kruiningen, and Richard S. Merkal
`
`Original Articles
`
`h1,01111•111
`
`Is Crohn's Disease a Mycobacterial Disease After All? Gary Gitnick
`
`Editorial
`
` Original Articles
`
`
`
`Immunological Findings in Whole and Parotid Saliva of Patients with Crohn's Disease and Healthy Controls. Graciela Crama-Bohbouth, Petra
`Lems-van Kan, Irene T. Weterman, I. Biemond, and A.S. Pella
`
`9 Relationship Between Clinical and Laboratory Parameters and Length of Lesion in Crohn's Disease of Small Bowel. Cosimo Prantera, Carlo
`Luzi, Piero Olivotto, Susan Levenstein, Paola Cerro, and Angelo Fanucci
`
`II Hydrogen Breath Test Quantification and Clinical Correlation of Lactose Malabsorption in Adult Irritable Bowel Syndrome and Ulcerative
`Colitis. G. Sciarretta, G. Giacobazzi, A. Verri, P. Zanirato, G. Garuti, and P. Malaguti
`
`Is
`
`Achalasia as a Risk Factorfor Esophageal Carcinoma: A Reappraisal. Jackie J.H. Chuong, Suzanne DuBovik, and Richard W. McCallum
`
`II Plasma Cholecystokinin Concentrations in Patients with Pancreatic Insufficiency Measured by Sequence-Specific Radioimmunoassays. Jan B.M.J.
`Jansen, Wim P.M. Hopman, and Cornelis B.H.W. Lamers
`I8 Lack of Correlation Between Serum Lipoproteins and Biliary Cholesterol Saturation in Patients with Gallstones. Ja' W. Marks, Patricia A.
`Cleary, and John J. Albers
`
`Effect of Chenodeoxycholic and Ursodeoxycholic Acids on Isolated Adult Human Hepatocytes. Kohji Miyazaki, Fumio Nakayama, and Akitoshi
`Koga
`
`Antisecretory Activity of Fenoctimine in Rat and Dog. H.I. Jacoby, A.C. Bonfilio, T. Corcoran, I. Lopez, and M. Scott
`
`Transcatheter Arterial Embolization of Ruptured Hepatocellular Carcinoma Associated with Liver Cirrhosis. Toshihiko Nouchi, Masanobu
`Nishimura, Manabu Maeda, Testutaro Funatsu, Yasushi Hasumura, and Jugoro Takeuchi
`
`Possible Role of Endogenous Prostaglandins in Alkaline Response in Rat Gastric Mucosa Damaged by Ilypertonic NaCI. Youichi Nobuhara and
`Koji Takeuchi
`
`I
`In Vitro Bile Acid Adsorption by Bismuth Subsalicylate and Montmorillonite. Samuel A. Kocoshis, Cameron N. Ghent, and Joyce D. Gryboski
`
`13
`
`Comparison of Tritiated Thymidine and Metaphase Arrest Techniques of Measuring Cell Production in Rat Intestine. J.G. Sharp and N.A.
`Wright
`
`Acute Gastric Hemorrhage Secondary to Wandering Spleen. Ulf Angeras, Bengt Almskog, Pavel Lukes, Sven Lundstam, and Lilian Weiss
`
`Exercise-Induced Hypoglycemia Following Propranolol in a Patient After Gastric Fundoplication Surgery. Gary P. Zaloga and Robert F. Dons
` News and Notices (cid:9)
`
`
`
`11
`
`Case Reports
`
`Thank You to Reviewers
`
`Author Index
`
`Subject Index
`
`Gratitude
`
`Annual Indexes
`
`rr ,S)*
`
`4
`
`•
`
`Ex. 1037 - Page 1
`
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`While acid reduction speeds ulcer h (cid:9)
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`
`You can increase acid suppression with more potent therapy.
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`with `Tagamet':
`
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`
`Tablets: 200 mg.,
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`
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`
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`
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`
`*The exact degree and duration of acid suppression necessary
`to heal ulcers are not known.
`1. Graham, D.Y, et al.: Am. I. Gastmenterol. 76(6): 500-505, Dec. 1981.
`Before prescribing, please see brief summary of prescribing information on adjacent page.
`
`Ex. 1037 - Page 3
`
`(cid:9)
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`While acid reduction speeds ulcer h (cid:9)
`gastric acid has
`a number of important physiologic functions.'
`
`You can increase acid suppression with more potent therapy.
`But you can't improve upon the benefits your patients get
`with `Tagamet':
`
`• Rapid Healing
`• Early Pain Relief
`• Proven Long-term Safety Profile
`• Less Disruption of Normal Gastric Physiology
`
`,
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`
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`
`*The exact degree and duration of acid suppression necessary
`to heal ulcers are not known.
`1. Graham, D.Y, et al.: Am. I Gastroenterol. 76(6): 500-505, Dec. 1981.
`Before prescribing, please see brief summary of prescribing information on adjacent page.
`
`•
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`
`•
`
`Ex. 1037 - Page 4
`
`

`

`DIGESTIVE DISEASES
`AND SCIENCES
`
`EDITOR
`FRANK P. BROOKS, MD, ScD (MED)
`Gastrointestinal Section and
`Department of Physiology
`School of Medicine, Hospital of
`the University of Pennsylvania
`Philadelphia, Pennsylvania
`
`ASSOCIATE EDITORS
`
`SIDNEY COHEN, MD
`Chief, Gastrointestinal Section
`Hospital of the University
`of Pennsylvania
`Philadelphia, Pennsylvania
`
`BOOK REVIEWS
`
`S. P. BRALOW, MD
`Graduate Hospital
`Philadelphia, Pennsylvania
`
`EDITORIAL BOARD
`
`ELLIOT ALPERT, MD
`Houston, Texas
`AGOSTINHO BETTARELLO, MD
`Sao Paulo, Brasil
`SERGE J. BONFILS, MD
`Paris, France
`THOMAS F. BURKS, PHD
`Tucson, Arizona
`DONALD CASTELL, MD
`Bethesda, Maryland
`EDWARD COPELAND, MD
`Houston, Texas
`SVERRE EMAS, MD
`Stockholm, Sweden
`JOHN F. FIELDING, MD
`Dublin, Ireland
`GIORGIO GABELLA, MD
`London, England
`
`EDITOR FOR LIVER
`
`BRUCE W. TROTMAN, MD
`Chairman and Professor
`Department of Medicine
`Meharry Medical College
`Nashville, Tennessee
`
`RICHARD WECHSLER, MD
`Clinical Associate Professor
`of Medicine
`Pittsburgh, Pennsylvania
`
`EDITORS EMERITI
`
`HENRY JANOWITZ, MD
`E. CLINTON TEXTER, JR., MD
`JOHN T. FARRAR, MD
`SYDNEY F. PHILLIPS, MD
`
`YI-FU SHIAU, MD, PHD
`Gastrointestinal Section
`Veterans Administration Hospital
`Philadelphia, Pennsylvania
`
`EDITORIAL ASSISTANT
`
`BARBARA NELSON
`Gastrointestinal Section
`School of Medicine, Hospital of
`the University of Pennsylvania
`Philadelphia, Pennsylvania
`
`RALPH A. GIANNELLA, MD
`Cincinnati, Ohio
`ALLEN L. GINSBERG, MD
`Washington, D.C.
`GARY L. GITNICK, MD
`Los Angeles, California
`VAY LIANG W. Go, MD
`Rochester, Minnesota
`HARALD GOEBELL, MD
`Essen, Germany
`HENRY I. GOLDBERG, MD
`San Francisco, California
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`Philadelphia, Pennsylvania
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`San Diego, California
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`Milwaukee, Wisconsin
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`Maebashi, Japan
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`Cincinnati, Ohio
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`San Francisco, California
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`Charlottesville, Virginia
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`Melbourne, Australia
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`Urbana, Illinois
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`Little Rock, Arkansas
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`Torrance, California
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`Rochester, Minnesota
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`FRANCIS J. TEDESCO, MD
`Augusta, Georgia
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`Salford, England
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`Pittsburgh, Pennsylvania
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`Leuven, Belgium
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`FRANCISCO VILARDELL
`Barcelona, Spain
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`Boston, Massachusetts
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`SIDNEY J. WINAWER, MD
`New York, New York
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`London, England
`
`DIGESTIVE DISEASES AND SCIENCES is abstracted or indexed in Beck Medical Information, Biological Abstracts, Chemical Abstracts, Current Awareness in
`Biological Sciences, Current Contents, Current Surgery, Index Medicus, International Abstracts in Gastroenterology, Referativnyi Zhurnal, and Science
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`VES1'171"zo-f--tawmts.:$ (cid:9)
`
`ii4);;1"-N-4:"
`
`Ex. 1037 - Page 5
`
`

`

`Digestive Diseases and Sciences, Vol. 29, No. 12 (December 1984), pp. 1073-1079
`
`Possible Role of Mycobacteria in
`Inflammatory Bowel Disease
`I. An Unclassified Mycobacterium Species Isolated from
`Patients with Crohn's Disease
`
`RODRICK J. CHIODINI, PhD, HERBERT J. VAN KRUININGEN, DVM, PhD, WALTER R.
`THAYER, MD, RICHARD S. MERKAL, PhD, and JESSICA A. COUTU, BA
`
`A previously unrecognized Mycobacterium species was isolated from two patients with
`Crohn's disease. The organism is an acid-fast, mycobactin-dependent Mycobacterium
`that has characteristics which do not conform to any of the presently recognized species.
`It belongs to the Runyon group III mycobacteria and is most closely related to
`Mycobacterium paratuberculosis. Animal inoculation revealed pathogenicity for mice
`when injected intravenously or intraperitoneally, but not for rats, guinea pigs, rabbits, or
`chickens. The mice developed hepatic and splenic granulomas which contained numerous
`acid-fast mycobacteria. A 7-day-old goat which was inoculated orally with 50 mg of the
`organism developed both humoral and cell-mediated immunologic responses in two to
`three weeks and granulomatous disease of the distal small intestine, with noncaseating
`tuberculoid granulomas in five months. Acid-fast bacilli were not demonstrable in
`sections of the intestine, but a single organism was seen in each of two microgranulomas
`of the mesenteric lymph node. The Mycobacterium species was reisolated from the lymph
`node but not from intestine. Our findings raise the possibility that a Mycobacterium plays
`an etiologic role in at least some cases of Crohn's disease.
`
`Crohn's disease is a granulomatous form of enteritis
`which, historically was noted for its resemblance to
`tuberculosis. Scientific progress was thought to
`have been made when these two processes were
`convincingly distinguished. Nevertheless, over the
`years, recurrences of the notion that Crohn's dis-
`
`Manuscript received January 20, 1984; revised manuscript
`received August 7, 1984; accepted August 15, 1984.
`From the Department of Pathobiology, University of Connect-
`icut, Storrs, Connecticut; the Department of Gastroenterology,
`Rhode Island Hospital and Brown University, Providence,
`Rhode Island; and the Mycobacteriosis Research Unit, National
`Animal Disease Center, Ames, Iowa.
`Supported by a grant from the National Foundation for Ileitis
`and Colitis Inc.
`Address for reprint requests: Dr. R.J. Chiodini, Department of
`Pathobiology, U-89, University of Connecticut, Storrs, Connect-
`icut 06268.
`
`Digestive Diseases and Sciences, Vol. 29, No. 12 (December 1984)
`0163-2116/84/1200-1073$03.50/0 (cid:9)
`
`ease might, in fact, be mycobacterial in origin have
`persisted.
`The first accepted description of Crohn's disease
`appeared in the literature in 1932 (1). Prior to that
`time, Crohn's disease was often confused with
`intestinal tuberculosis. Early investigators (2, 3)
`searched through their pathologic material in an
`attempt to find acid-fast organisms, and finding
`none, concluded that cases of intestinal tuberculo-
`sis occurred in which it was "difficult or impossi-
`ble" to demonstrate the bacilli (4, 5). Even the
`earliest recognized description of ulcerative colitis
`showed confusion with both tuberculosis and
`Crohn's disease (6). Following recognition of the
`clinical distinctions among these entities, investiga-
`tors continued to look for mycobacteria in resected
`specimens (7, 8). In a more recent era, Golde
`1073 (cid:9)
`
`1984 Plenum Publishing Corporation
`
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`Ex. 1037 - Page 6
`
`(cid:9)
`

`

`suggested that the resemblance of Crohn's disease
`to other mycobacterial diseases, such as tuberculo-
`sis or Johne's disease of ruminants, and its response
`to sulfasalazine, a drug related to the antituberculo-
`sis drug, para-aminosalicylic acid, should make us
`reconsider the possible causative role of mycobac-
`teria (9).
`In October 1982, at a workshop sponsored by the
`National Foundation for Ileitis and Colitis,* we first
`reported the isolation of an unclassified Mycobacte-
`rium species from a 15-year-old female with
`Crohn's disease and production of a chronic ileitis
`in a goat following oral inoculation. The following
`describes those findings and the isolation of a
`similar Mycobacterium species from an additional
`patient.
`
`MATERIALS AND METHODS
`
`Patient 1. The patient was a 15-year-old black female
`with a chief complaint of intermittent abdominal pain,
`diarrhea, and weight loss of 1-year's duration. She had a
`2-year history of juvenile rheumatoid arthritis of the right
`wrist and left elbow. She was easily fatigued, had some
`decrease in appetite, and experienced occasional fevers
`up to 38.9° C associated with episodes of diarrhea. There
`was some tenderness in both lower quadrants of the
`abdomen. Stools contained streaks of blood.
`Three months prior to admission to the Rhode Island
`Hospital, a barium enema revealed a filling defect of the
`cecum. A tentative diagnosis of Crohn's disease was
`made. The patient was treated with prednisolone (30
`mg/day), but the pain persisted. On admission she was
`thin and had abdominal tenderness. An ill-defined cecal
`mass was noted in the lower right quadrant. Sigmoidosco-
`py showed a slightly friable, pale mucosa. A cecal mass, a
`small bowel fistula, and ulcerations of the terminal ileum
`were revealed by a gastrointestinal series. Chest radio-
`graphs were normal and the patient had a negative
`response to tuberculin-purified protein derivative (PPD).
`At surgery 8.0 cm of cecum and 13 cm of terminal ileum
`were resected, and the ileum was anastomosed to the
`ascending colon. The patient underwent an uneventful
`postoperative course and has done well since that time.
`Histologic examination of resected tissues, conducted by
`the Department of Pathology, Rhode Island Hospital,
`confirmed the diagnosis of Crohn's disease. Villi of
`several sections of the ileum were reduced in size; villous
`cores and submucosa appeared edematous and contained
`round aggregates of lymphocytes on a background of
`plasma cells and a few eosinophils. In one section, the
`basal lamina propria contained a solitary granuloma.
`Submucosa, intermuscular septum, and subserosa con-
`tained variable-sized lymphoid aggregates, some of which
`were associated with lymphatics. Lymphatics were often
`
`*Inflammatory Bowel Disease: Future Research Approaches,
`Princeton, New Jersey, October 28-31, 1982.
`
`1074
`
`CHIODINI ET AL
`
`distended, had prominent endothelium, or were sur-
`rounded by chronic inflammatory cells. Auerbach's plex-
`uses were prominent and neurons appeared degenerate.
`Muscle layers were thickened. Acid-fast bacilli were not
`demonstrable in tissue sections and cultures for Myco-
`bacterium tuberculosis were negative.
`Patient 2. The patient was a 12-year-old white male
`with a chief complaint of recurrent fevers (38.9°-40° C)
`over a period of two weeks; diarrhea, black stools, and
`cramping abdominal pain of two month's duration; and
`weight loss and decreased energy for the past 1.5 years.
`The child's father had had ulcerative colitis since 18 years
`of age, and there was a family history of rheumatoid
`arthritis. On physical examination there was a tender
`right submandibular lymph node, tenderness of the right
`upper and lower quadrants, hemorrhoids, and a focus of
`erythema nodosum on the left tibia. Abnormal laboratory
`findings included an elevated erythrocyte sedimentation
`rate and hypochromic, microcytic anemia. Chest radio-
`graphs were normal and the patient did not respond to
`tuberculin PPD. A clinical diagnosis of Crohn's disease
`was made, the rectal abscess was incised and drained, the
`patient was discharged.
`The patient was readmitted nine months later with
`fever and abdominal pain. There were hyperactive bowel
`sounds consistent with an incomplete small bowel
`obstruction, and abdominal radiographs showed multiple
`gas—fluid levels in small intestine. Because of the inade-
`quacy of medical management and growth retardation,
`elective surgery was performed. Multiple fibrous adhe-
`sions of the terminal ileum, appendix, and proximal colon
`prompted resection of 19 cm of ileum and 22 cm of
`proximal colon. Ileum was anastomosed to transverse
`colon. Histologic examination conducted at the Depart-
`ment of Pathology, Rhode Island Hospital, confirmed a
`diagnosis of Crohn's disease. Ileal and colonic mucosa
`contained frequent microulcers, with lymphocytic, plas-
`macytic bases, some of which were coated with fibrino-
`purulent exudate. Each section contained abscesses.
`Submucosa and muscularis contained moderate numbers
`of lymphoid nodules, some located perilymphatically.
`The normal lymphoid nodules of the superficial submuco-
`sa were enlarged, and one contained an aggregate of
`histiocytic cells. Subserosa contained granulomas, with
`swirling, but without giant cells. There was chronic
`lymphangitis, vasculitis, and perivasculitis of the subser-
`osa. Acid-fast bacilli were not demonstrable in tissue
`sections. The patient was discharged and treated with
`sulfasalazine.
`Culture Techniques. Freshly resected gastrointestinal
`tissues were obtained from Rhode Island Hospital. Imme-
`diately following surgery, resected tissues were trans-
`ported, wrapped in sterile gauze, to the Department of
`Surgical Pathology, Rhode Island Hospital, where sec-
`tions were removed for histologic examination under
`aseptic conditions using sterile gloves and sterile instru-
`ments. Remaining tissues were rewrapped in sterile
`gauze, placed into sterile plastic containers, and trans-
`ported, packed in ice, to the Department of Pathobiology,
`University of Connecticut for further processing. Tissues
`were washed in sterile water, aseptically trimmed of fat,
`and placed into sterile Butterfield's buffer (pH 7.2) over-
`
`Digestive Diseases and Sciences, Vol. 29, No. 12 (December 1984)
`
`Ex. 1037 - Page 7
`
`

`

`MYCOBACTERIA IN CROHN'S DISEASE
`
`night at 3-5° C (10). The following day, approximately 20
`g of mucosa was aseptically scraped from the surface and
`placed into 50 ml of sterile 2.5% trypsin (1:250, pH 7.5)
`with continuous stirring for 30-45 min. Tissues were
`filtered through sterile cheesecloth and centrifuged at
`4,340g for 30 min in sterile test tubes. The resulting
`sediment was aseptically suspended in 40 ml of sterile
`0.1% benzalkonium chloride. After standing at room
`temperature for 18-24 hr, 0.1-0.2 ml of the resulting
`sediment was aseptically distributed onto each of eight
`slants of Herrold's egg yolk medium (HEYM). Cultures
`were supplemented with mycobactin (2 lig/m1), iron-
`chelating growth factors, prepared from M. phlei or M.
`paratuberculosis by ethanol extraction, iron chelation,
`chloroform extraction, and column chromatography (11).
`Tubes were incubated at 37° C for 4-6 days in a slanted
`position, with caps slightly loose, to allow for evaporation
`of the inoculum. Tubes were then sealed with paraffin and
`incubated at 37° C until growth was evident.
`When growth developed, it was transferred to a fresh
`tube of HEYM with mycobactin and incubated at 37° C
`until luxurious growth was obtained. Colonies were sub-
`cultured on additional tubes of HEYM with and without
`mycobactin; Middlebrook's 7H10 medium with OADC
`(oleic acid, albumin, dextrose, and catalase) and myco-
`bactin; and Middlebrook's 7H9 broth with OADC, Tween
`80, and mycobactin. Biochemical tests were performed
`
`on medium supplemented with mycobactin by standard
`methods (12).
`Animal Inoculations. In laboratory animals, pathogenic-
`ity of the first isolate was determined in New Zealand
`rabbits, Hartley guinea pigs, chickens, rats, and Balb/c
`mice. Fifty milligrams (wet weight) of viable bacilli were
`suspended in 10 ml sterile physiologic saline, and 1.0 ml
`was injected into three animals of each species by either
`the intravenous, intraperitoneal, or subcutaneous routes.
`A single mouse was inoculated in the hind footpads with
`1.5 mg of bacilli in physiologic saline. Control animals
`were inoculated with sterile physiologic saline by all
`routes. Animals were autopsied at 28 days postinocula-
`tion.
`In ruminants, experimental animals originated from a
`herd known to be free of tuberculosis and paratuberculo-
`sis. Disease-free status was determined by the failure of
`all adult animals in the herd to respond to skin testing
`with tuberculin PPD and paratuberculin (johnin). These
`methods are reliable for determining herd infectivity (13).
`In addition, randomly selected animals, including the dam
`and young to be used in the experiments, were examined
`for humoral antibodies to M. tuberculosis and M. paratu-
`berculosis by the enzyme-linked immunosorbent assay
`(ELISA) method (14). A 7-day-old Nubian goat was
`inoculated orally with 50 mg (wet weight) of viable bacilli
`suspended in a 50:50 mixture of cream and fresh bovine
`
`TABLE 1. COMPARATIVE FEATURES OF SEVERAL PATHOGENIC MYCOBACTERIA
`
`Mycobacterium species
`
`Linda*
`
`4-6
`
`Tubercu-
`losis
`
`2-3
`
`Avium
`
`Kansasii
`
`Bovis
`
`Para-
`tuberculosis
`
`2-3
`4-
`
`1-3
`
`2-3
`
`8-12
`
`R
`<45
`
`S
`<45
`
`S
`<45
`
`R
`>45
`
`S
`<45
`
`V
`<45
`
`NAt
`NA
`NA
`
`Characteristic
`
`Growth rate (weeks)
`Mycobactin dependency
`Pigment production
`Niacin production
`Urease
`Sodium tolerance
`Catalase (RT)
`Catalase, quantitative (mm)
`Catalase, 68°C
`Tween 80 hydrolysis
`Arylsulfatase, 3 days
`2 weeks
`Tellurite reduction
`Nitrate reduction
`Pyrazinamidase, 4 days
`7 days
`Susceptibility to:
`Thiophene-2-carboxylic acid (TCH)
`Isoniazid (INH)
`Neotetrazolium chloride (NT)
`Streptomycin (ST)
`Rifampicin (RIF)
`Pathogenicity in:
`Guinea pigs
`Mice
`Rabbits
`Chickens
`
`* Mycobacterium species isolated from patients with Crohn's disease.
`tResults not available.
`
`Digestive Diseases and Sciences, Vol. 29, No. 12 (December 1984)
`
`1075
`
`Ex. 1037 - Page 8
`
`

`

`milk (a method widely used in the transmission of paratu-
`berculosis). A control littermate, reared separately, re-
`ceived only cream and milk. Sera from both control and
`inoculated goats were obtained weekly, and humoral
`immune responses were monitored by the ELISA method
`(14). Delayed hypersensitivity was monitored weekly by
`skin testing.
`Purified antigens from our isolates were not available;
`separate studies by Thayer et al (14) demonstrated cross-
`reactivity between our first isolate and M. paratuberculo-
`sis, therefore M. paratuberculosis antigen was used to
`monitor our responses in our goats. One milliliter of
`tuberculin (from M. tuberculosis) and johnin (from M.
`paratuberculosis) were injected intradermally on the lat-
`eral abdomen. Indurations were measured with a skin
`caliper and responses recorded as changes in skin thick-
`ness. Both animals were autopsied at 5 months of age and
`the liver, terminal ileum, ileocecal valve, colon, and
`ileocecal lymph node were cultured for mycobacteria.
`Sections of all major organs, mesenteric lymph nodes,
`and various segments of the gastrointestinal tract were
`removed from both animals and fixed in buffered 10%
`formalin. Tissues were processed for routine paraffin
`embedding, sectioned at 6 p.m, and stained with Harris's
`hematoxylin and eosin and by the Ziehl-Neelsen method
`for acid-fast bacilli.
`
`RESULTS
`
`Bacteriology. After 31/2 and 51/2 months of incuba-
`tion, a small, brilliant white, mucoid, irregular
`colony was observed on a slant inoculated with
`tissues from a 15-year-old (strain Linda) and a 12-
`year-old (strain Dominic) patient with Crohn's dis-
`ease, resepectively. The isolates were identified as
`acid-fast mycobactin-dependent Mycobacterium
`species which did not have characteristics conform-
`ing to those of any presently recognized species.
`The organism belongs to the Runyon group III
`mycobacteria (Mycobacterium avium-intracellu-
`lare, M. paratuberculosis, etc). It most closely
`resembles M. paratuberculosis, but has distinguish-
`ing features which are summarized in Table 1. A
`detailed description of the characteristics of this
`organism will be published elsewhere (15).
`Experimental Infections. Lesions did not occur in
`rabbits, chickens, rats, or guinea pigs following
`intraperitoneal, intravenous, or subcutaneous injec-
`tion of 5 mg of viable bacilli. No effect was ob-
`served in mice inoculated subcutaneously or in
`footpads. Mice inoculated intravenously developed
`noncaseating granulomas of the liver, spleen, and
`mesenteric lymph nodes (Figure 1). Mice inoculated
`intraperitoneally developed hepatic granulomas.
`Giant cells were not present. Large numbers of
`acid-fast bacilli were demonstrable within granulo-
`mas. Lesions were not observed in control animals.
`
`1076
`
`CHIODINI ET AL
`
`Fig 1. Hepatic granuloma in the liver of a mouse inoculated
`intraperitoneally with Mycobacterium species, strain Linda.
`
`The goat inoculated with strain Linda had a
`demonstrable delayed hypersensitivity to johnin at
`three weeks after inoculation, which progressively
`increased until the time of autopsy (Figure 2). A
`response to tuberculin did not develop. Humoral
`immunoglobulins of the IgM class were detected
`only during the first and second weeks postinocula-
`tion (Figure 3). An IgG response did not occur.
`
`3-
`
`E
`
`2.
`
`Johnin PPD--
`Tuberunn PPD--
`
`0 (cid:9)
`
`1 (cid:9)
`
`3 (cid:9)
`
`2 (cid:9)
`Months Post Inoculation
`
`fa'45
`4
`
`Fig 2. Skin-test reactivity to intradermal administration of johnin
`and tuberculinin PPD in a goat infected with Mycobacterium
`derived from resected Crohn's disease tissue.
`
`Digestive Diseases and Sciences, Vol. 29, No. 12 (December 1984)
`
`Ex. 1037 - Page 9
`
`

`

`MYCOBACTERIA IN CROHN'S DISEASE
`
`1 5-
`
`8
`
`10
`
`0
`
`5
`
`a
`
`-
`-
`
`Paratuberculosis
`Tuberculosis
`
`1
`..-- (cid:9)
`
`, 11°N. (cid:9)
`.A ,.P.-. (cid:9)
`„ (cid:9)
`.1 „-.\ /-fl (cid:9)
`..
`.,
`,.
`t Nix* • ------‘
`
`.
`•
`
`3
`2 (cid:9)
`MonthS Post Inoculation
`
`4 (cid:9)
`
`
`
`5
`
`Fig 3. IgM response in a goat infected with Mycobacterium
`species, strain Linda.
`
`Clinical disease did not develop during the course of
`this study. At autopsy, approximately 20 cm of the
`terminal ileum was thickened, contained numerous
`transverse corrugations, and focal hyperemia (Fig-
`ure 4). The regional mesenteric lymph nodes were
`enlarged. Histologically, the Peyer's patches of the
`terminal ileum contained multiple noncaseating tu-
`berculoid granulomas with giant cells (Figure 5).
`The adjacent mucosa was thickened with lympho-
`cytes, macrophages, and occasional giant cells (Fig-
`ure 6). Regional lymph nodes had multiple tubercu-
`loid granulomas within the cortical regions (Figure
`7). Acid-fast bacilli were not demonstrable in any
`gastrointestinal section; however, a single acid-fast
`
`bacillus was present in each of two granulomas of
`the lymph nodes. A Mycobacterium species with
`characteristics identical to those of the inoculum
`was reisolated from the mesenteric lymph node.
`Organisms were not recovered from intestine. The
`control littermate remained free of disease and
`immunologic responses, and mycobacteria were not
`isolated from any tissue. Infectious agents known to
`cause ileitis in ruminants were not isolated from
`either experimental animal.
`
`DISCUSSION
`
`The organism we have isolated is exceptionally
`fastidious; long incubation periods on complex me-
`dia with mycobactin are required for primary isola-
`tion. Luxurious growth was never obtained on
`primary culture. Equal difficulty was encountered
`in recovering this organism from experimentally
`infected animals. Our inability to see the organism
`in gastrointestinal sections of patients and experi-
`mental animals suggests their sparsity in tissues.
`The special concentration methods we employed
`may account for our success in isolating this orga-
`nism.
`To date we have cultured specimens from 11
`patients with Crohn's disease and three with ulcer-
`ative colitis. Our inability to recover similar myco-
`bacteria from other patients tested thus far is disap-
`pointing. Experience suggests that our current bac-
`teriologic methods may not be optimum for primary
`
`Fig 4. Corrugated intestinal mucosa (arrows) of the terminal ileum from a goat inoculated orally
`with Mycobacterium species, strain Linda.
`
`Digestive Diseases and Sciences, Vol. 29, No. 12 (December 1984)
`
`1077
`
`•
`
`•
`
`
`
`•
`
`c
`
`IA •
`•
`ra •
`
`Ex. 1037 - Page 10
`
`(cid:9)
`

`

`
`••• Rt
`
`CHIODINI ET AL
`
`are occasional reports of the isolation of mycobac-
`teria from the gastrointestinal tract of patients with-
`out intestinal disease. Some isolates are sapro-
`phytes and will not cause disease when introduced
`experimentally into animals. Others are pathogens
`shed from lymph nodes or other sites and represent
`transients in th