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John Hermon-Taylor 1, Nick Barnes 2, Chris Clarke 3, and Caroline Finlayson 4.
1 Professor of Surgery.
Department of Surgery,
St. George's Hospital Medical School,
London SW17 ORE. U.K.
2 Consultant Paediatrician.
Cambridge CB2 2QQ.
3 Senior Lecturer in Veterinary Pathology.
Dept of Veterinary Pathology,
University of Edinburgh,
Veterinary Field Station,
Easter Bush, Roslin,
Midlothian EH25 9RG.
4 Consultant Senior Lecturer in Histopathology.
Department of Histopathology
St. George's Hospital Medical School.
London SW17 ORE. U.K.
Proceedings of a Grand Round held on March 27th 1997 at St. George's Hospital and Medical School introduced by Professor George Griffin, Professor of Medicine.
In September 1988 a seven year old boy living in a village outside Cambridge, developed non-tuberculous mycobacterial cervical lymphadenitis. The nodes were removed and later shown to contain abundant Mycobacterium paratuberculosis. This pathogen causes chronic inflammation of the intestine ranging from a pluribacillary 'lepromatous' type to a paucimicrobial 'tuberculoid' type, in many species of animals including primates. Five years later he presented with chronic inflammation of the intestine affecting the terminal ileum and ascending colon, similar to Crohn's disease. This resolved on prolonged treatment with a combination of rifabutin and clarithromycin, leaving a healed ileal scar which required excision. As with M. bovis earlier this century, infection with M. paratuberculosis was probably acquired by the consumption of milk contaminated with these robust versatile organisms.
Mycobacterium paratuberculosis was first described from Dresden by Johne (Fig 1a) and Frothingham in 18951, as the cause of a chronic inflammatory disease of the intestine in a German cow. The organism was called Johne's bacillus and the illness, in which millions of acid fast mycobacteria were visible in the diseased tissues, Johne's disease. In 1901 Thomas K. Dalziel (Fig 1b), a surgeon at the Western Infirmary in Glasgow, operated on a colleague with chronic inflammation of the intestine. He was aware of the recent description of Johne's disease and of the subsequent bacteriological research in the field. He collected other cases and published his observations on "Chronic Interstitial Enteritis" in the British Medical Journal in 1913 2. He wrote that the "histological characters" of the disease he had described in humans were so similar to Johne's disease "as to justify a proposition that the diseases may be the same". 'Dalziel's dilemma'3, was that he could not see acid fast mycobacteria in the diseased intestine in humans.
M. paratuberculosis belongs to the M. avium- intracellulare group4. The term 'paratuberculosis' suggests close similarity to M. tuberculosis, but in truth it is very different. Unlike M. tuberculosis, M. paratuberculosis can survive in the environment, and is highly resistant in vivo to most standard anti-tuberculous drugs. M. paratuberculosis cannot be reliably detected by culture in the laboratory, particularly when present in low abundance or in spheroplast form without a bacillary cell wall; different sub-types of the organism with different preferred hosts 5,6, range from very slow growing to unculturable. M. paratuberculosis can cause chronic inflammation of the intestine in many species of animals including primates 7-9. The disease in animals demonstrates a broad range of histopathological types, from a pluribacillary (lepromatous) form with millions of typical acid fast bacilli visible in the tissues, to a paucimicrobial (tuberculoid) form in which M. paratuberculosis cannot be seen in the tissues and cannot be detected by culture, but in which there is a florid chronic granulomatous inflammatory response 10. Intermediate paucibacillary forms of the disease, in which only a very few acid fast mycobacteria may be visible, also occur.
Mycobacterium paratuberculosis has been cultured in the laboratory from humans with chronic inflammation of the intestine of the Crohn's disease type, but only very rarely 11-15. When it has been grown, the organism has first appeared in the culture in an unrecognisable spheroplast form, taking months or years of incubation to alter its phenotype to that of a mature bacillus with an established lipid rich cell wall able to be identified by conventional means. Detection and characterisation of M. paratuberculosis has been advanced by the advent of DNA-based methods using as their target a 1451bp DNA insertion element IS900, of which there are about 18 copies stably integrated into the M. paratuberculosis genome 16,17. These new techniques are central to our presentation today of a young boy who developed cervical lymphadenitis involving M. paratuberculosis followed five years later by a chronic terminal ileitis similar to Crohn's disease which healed on treatment with a combination of rifabutin and clarithromycin. Since an understanding of the nature of M. paratuberculosis disease in animals is desirable, a brief description is presented by Chris Clarke from the Department of Veterinary Pathology at the University of Edinburgh.
In September 1988, JL, a previously healthy boy aged 7 years 10 months living in a small village outside Cambridge U.K., developed enlarged lymph nodes on the right side of his neck. He was asymptomatic, Mantoux negative (PPD 1:10,000 0.1ml id) and had a normal chest x-ray. He had not had BCG. He was referred to the surgical service at Addenbrooke's Hospital and the enlarged lymph nodes were removed. The histology, which will be described by Caroline Finlayson, suggested a mycobacterial infection. Samples of the diseased lymph nodes were incubated on Lowenstein-Jensen slopes with duplicates including pyruvate, but not mycobactin, for 12 weeks at room temperature, 30oC and 37oC. He was treated with rifampicin 450mg, isoniazid 150mg once daily, and pyrazinamide 250mg 3 times daily for two months but when the cultures were negative drug treatment was stopped. The remaining lymph nodes on the right side of the neck progressively enlarged and were removed by block dissection in November 1989. Mycobacterial culture again proved negative. He remained well until March 1993 when he developed an arthritis affecting both knees, and an anaemia refractory to iron. By the end of 1993, he was an ill child with daily abdominal pain, anorexia, 2-3 loose bowel motions a day, weight loss and lethargy.
His weight was 37kg (on the 30th centile) and his height 153cms. He looked pale and ill and had angular stomatitis. Firm lymph nodes were still palpable on the right side of the neck. There was no clubbing. In the abdomen, an enlarged, tender loop of bowel was easily felt in the right iliac fossa. Radiologically the small gut showed narrowing and distortion of a 10cm segment of terminal ileum and prominent cobblestoning of the mucosa (Fig.2). White cell scan at one hour showed intense uptake of the label over the affected intestine (Fig.3a). These findings were considered typical of Crohn's disease.
He was treated with rifabutin 300 mg and clarithromycin 500 mg daily and was advised to have UHT (long-life) milk only. Eight days after the start of treatment, lymph nodes on the left side of the neck and residual nodes on the right, became enlarged and firm with surrounding oedema. At the same time he had a transient episode of subacute intestinal obstruction which subsided on conservative management. With continued rifabutin and clarithromycin treatment the nodes gradually subsided. By mid 1994 after six months treatment he was virtually asymptomatic and he was completely so by February 1995, when his weight had increased to 49kg and his height to 160 cM. White cell scanning then showed a complete resolution of the previously inflamed gut (Fig.3b). However, he was beginning to get obstructive symptoms. Barium enema showed no active disease but there was a tight stricture of the terminal ileum with upstream dilatation. A limited resection was therefore carried out with removal of the terminal ileum and adjacent ascending colon for a pale fibrotic stricture followed by end-to-end anastomosis. The histology of the resected stricture will be described. There was no other visible evidence of inflammatory disease throughout the gut. Rifabutin and clarithromycin treatment and restriction to UHT milk was continued. The drugs were stopped at the end of October 1996 after a total of 32 months treatment. By January 1997 his weight was 67kg (80th centile) and his height was 170cM. He was asymptomatic and a well developed adolescent on physical examination.
Sections of the cervical lymph nodes initially removed showed areas of typical caseous necrosis with a very few acid fast bacilli visible on ZN staining. Elsewhere there were marked chronic inflammatory changes with typical Langerhans giant cells, non-caseating granulomata and an epithelioid cell infiltrate with focal microcalcifications (Fig 4a & b). The macroscopical appearances at operation were of a white fibrous stricture 5mm in diameter and about 2cm long in the terminal ileum (Fig 4c), with encroachment of fat over the serosal surface. There was no evidence of chronic inflammatory disease elsewhere in the gut. Microscopically the mucosa over the ileal stricture was intact with little gland distortion and villous atrophy, but the submucosa and gut wall showed extensive transmural fibrosis. There was no evidence of active inflammatory disease or granulomata (Fig 4d). The mesenteric lymph nodes showed reactive hyperplasia, but were otherwise normal.
Two of the paraffin embedded cervical lymph nodes removed in 1988 (Fig 5a), were subjected to DNA extraction by Dr Douglas Millar using standard protocols, together with simultaneous process controls. IS900 PCR for Mycobacterium paratuberculosis was then performed on the DNA extract in triplicate using the primers p90 (5'-GAAGGGTGTTCGGGGCCGTCGCTTAGG-3') and p91 (5'-GGCGTTGAGGTCGATCG CCCACGTGAC-3')18. Amplification products were run on agarose gel electrophoresis and identified by hybridisation at high stringency using a 32p-labelled 229-bp internal probe followed by autoradiography, as previously described18. The DNA extract of the cervical lymph nodes was strongly positive in triplicate for Mycobacterium paratuberculosis in the presence of negative process controls and correctly reporting positive and negative internal PCR controls (Fig5b). The strongly positive IS900 PCR signal obtained on the crude DNA extract from the cervical lymph nodes without using hybridisation-capture of target DNA18, probably reflects a relatively high microbial abundance of M. paratuberculosis in the tissues at an early stage of the infection.
Johne's disease, or M. paratuberculosis infection in animals occurs in domestic ruminants especially cattle,sheep and goats. However, studies at the San Diego Zoo and elsewhere have shown that a broad range of animals including sub-human primates can be affected 8. Recently, M. paratuberculosis has been identified in the wild rabbit population, in Tayside, Scotland19. In sub-clinically infected cows M. paratuberculosis is secreted abundantly in the milk so that infection is acquired early in the life of new-born animals when they are particularly susceptible20. The gut is the main target organ, and M. paratuberculosis is shed in the faeces, but it can traffic widely throughout the animal in macrophages. A long latent interval usually occurs before clinical disease emerges. The signs of this in cattle are weight loss and diarrhoea, but diarrhoea is not a common feature of the clinical presentation in sheep. The pathology of the pluribacillary 'lepromatous' type of disease, is a chronic enteritis with diffuse thickening of the gut wall especially the terminal ileum, but extension to involve the colon is common. Mesenteric lymph nodes are enlarged. On opening the gut the mucosa is reddened with crevicing and marked oedema but not usually with ulceration. Microscopically there are millions of small acid-fast M. paratuberculosis living in macrophages (Fig 6a).
Our own work over the last few years particularly in sheep10, has shown that in about one quarter of cases there is a different form of Johne's disease. Although the clinical signs and gross pathology are similar, microscopically there is a pronounced chronic granulomatous enteritis with heavy lymphocytic infiltration, typical Langerhans-type giant cells and scattered granulomata. With prolonged searching a few nests of acid-fast bodies can be found in about half of these cases of 'tuberculoid' Johne's disease (Fig 6b). In the others, no bacillary-form M. paratuberculosis are visible in the tissues at all (Fig 6c and d). Only about 50% of sheep with this extreme paucimicrobial form of Johne's disease are IS900 PCR positive when the test is run directly on crude DNA extracts of the involved intestines.
In the early part of this century when milk supplies were at times heavily contaminated with M. tuberculosis and M. bovis 21, tuberculous cervical lymphadenitis was common. The organism entered via the oropharyngeal lymphoid tissues, but also through the gut, causing tuberculous ileitis and disease in other parts of the body. The problem was overcome by tuberculin testing of dairy herds and the mandatory introduction of milk pasteurisation, using conditions which ensured the destruction of these well recognised pathogens.
Work carried out between 1990-9522 suggests that the risk that something similar is now happening with M. paratuberculosis, is substantial although the organism is much less virulent for humans than M. tuberculosis. Subclinical infection of dairy cows with M. paratuberculosis is widespread in Britain and elsewhere in Europe 23 and such animals shed the organism in their milk 20,22. Conditions in the laboratory simulating commercial pasteurisation, which demonstrably kill M. bovis, do not guarantee the destruction of the more environmentally robust M. paratuberculosis 24,25. Outcomes analysis of the treatment of humans with chronic enteritis of the Crohn's disease type with a combination of drugs predicted to be active against spheroplast forms of M. paratuberculosis in vivo, suggests that their use can cause substantial remission in many patients with active Crohn's disease 26.
In the present case, JL was probably infected by consuming milk contaminated with non-tuberculous mycobacteria including M. paratuberculosis. The organisms would have entered his cervical lymphatics as well as his terminal ileum, as also occurs in animals 27. After a latent interval of about five years, first an arthritis and then a chronic enteritis emerged. This was similar clinically, radiologically and on white cell scanning to Crohn's disease. Shortly after starting rifabutin and clarithromycin he demonstrated a transient flare of the disease with widespread cervical lymph node enlargement and an episode of subacute intestinal obstruction, similar to disease exacerbations reported in some cases of leprosy and tuberculosis after starting treatment 28,29. In JL's case, the gut healed on prolonged treatment leaving a scar which required excision.
|Professor Angus Dalgleish||As M. tuberculosis is associated with caseation and M. paratuberculosis is not, could both have been involved there ?|
|Caroline Finlayson||I think that the involvement of M. tuberculosis is unlikely as it was not cultured from the lymph nodes, and JL was Mantoux negative. Other organisms such as M. Scrofulaceum can cause caseation in cervical lymph nodes. The information we have on JL suggests that this was a mixed non-tuberculous mycobacterial infection which on this occasion, also included M. paratuberculosis in considerable abundance. We do not of course see caseation in the intestinal lesions of Crohn's disease.|
|Professor George Griffin||Why don't we all get Crohn's disease ?|
|John Hermon-Taylor||Probably for the same reasons that not everybody got tuberculosis earlier this century, before pasteurisation was introduced and when milk supplies were at times heavily contaminated with M. tuberculosis 21. In addition, M. paratuberculosis has a much lower pathogenicity for humans than M. tuberculosis. One or more susceptibility factors are required for chronic enteritis and clinical disease due to M. paratuberculosis to develop. These include an inherited susceptibility 30 well known in Crohn's disease clinically especially amongst Jewish people 31, but possibly also in Celtic races. An understanding of the molecular genetics of susceptability is just beginning to emerge 32,33. Secondly, intercurrent microbial infection particularly gastroenteritis, is well known to be associated with the emergence of Crohn's disease. Lastly, psychological stress factors 34 which are known to be able to trigger the emergence of Johne's disease in animals, may be associated with both the development of Crohn's disease and its exacerbation.|
|Mr Devinder Kumar||If M. paratuberculosis is a ubiquitous environmental organism how do you explain the geographical distribution of Crohn's disease?|
|John Hermon-Taylor||The environmental distribution of M. paratuberculosis is not known. However, efficient farming methods in industrialised societies may have created localised conditions favouring the amplification of M. paratuberculosis in our domestic livestock and their controlled habitats over the course of the century. Persistence of the pathogen would be favoured in temperate regions with a high water table in the soil 35. Such conditions may also have increased the mutational frequency of M. paratuberculosis and resulted in the emergence of a strain with increased pathogenicity for humans.|
|Professor Robert Boyd||There is clearly a need for a randomised controlled trial of rifabutin and clarithromycin treatment in Crohn's disease sufferers.|
|Nick Barnes||This certainly seems to be the case. A trial in children would seem to be particularly appropriate because they present relatively early on during the course of the disease before they have developed the irreversible tissue damage and the range of complications that often occurs in adults.|
|Dr Dillip Banerjee||In paucibacillary or paucimicrobial M. paratuberculosis infections in animals do you find an immunological picture similar to that seen in tuberculoid leprosy ?|
|Chris Clarke||Yes. We have looked at antigen-specific lymphocyte responses in lamina propria, mesenteric lymph nodes, and circulating lymphocyte populations and have found significant reactivity. Lymphocytes isolated from the gut of animals with the lepromatous type of the disease show only weak reactivity. The cytokine profiles in paucibacillary M. paratuberculosis infection are also similar to tuberculoid leprosy.|
|Caroline Finlayson||The slide of the lepromatous type of M. paratuberculosis infection in animals reminded me strongly of the appearances of the small intestine in HIV positive patients with mycobacterial infections. Crohn's disease in humans would fit much better with the tuberculoid type of response.|
M. paratuberculosis research in the Department of Surgery, St. George's Hospital Medical School is supported by Action Research, The Dinwoodie Trust, The Ileostomy Association and The Wellcome Trust. The study in milk was also supported by The Ministry of Agriculture, Fisheries and Food. Dr Chris Clarke is supported by the BBSRC. We are grateful to Mr Jon Ford for assistance and to Astra Pharmaceuticals for their sponsorship of the meeting.
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Legend to Figure 1
(a)Dr Johne from Dresden who, together with Dr Frothingham visiting his laboratory from Boston Mass., described chronic enteritis affecting a German cow in 1895 1, caused by what is now known as M. paratuberculosis. (b) Thomas Kennedy Dalziel from the Western Infirmary in Glasgow, who operated on his first case of 'Chronic Interstitial Enteritis' in 1901 and reported his findings on this and other cases in the BMJ 1913 2, linking them to Johne's disease in animals.
Legend to Figure 2
Barium meal and follow through study performed on JL by Dr Alan Freeman at Addenbrooke's Hospital in December 1993, a little more than 5 years after the removal of cervical lymph nodes containing abundant M. paratuberculosis. The examination showed narrowing and distortion of a 10 cm segment of terminal ileum with classical 'cobbelstoning' of the mucosa (arrowed). The appearances are the same as those seen in Crohn's disease.
Legend to Figure 3
The appearances of white cell scans (at one hour) performed at St. George's Hospital in February 1994 before treatment, and in February 1995 after a year of chemotherapy using a combination of rifabutin and clarithromycin. Although there are technical differences between the two scans, uptake of labelled cells by the inflamed ileum characteristic of Crohn's disease, and its apparent complete resolution a year later, are clearly seen.
Legend to Figure 4
(a) x 400 and (b) x 250 microscopic appearances of the enlarged cervical lymph nodes from JL showing typical Langerhans giant cells with epithelioid cells, together with a non-caseating granuloma. Elsewhere there were foci of typical caseous necrosis with a very few acid-fast bacilli visible on ZN staining. (c) and (d) naked eye and microscopical appearances x 100 of the ileal stricture which developed after a year of rifabutin and clarithromycin treatment. The white scarred ileum with extensive intramural fibrosis microscopically and intact overlying mucosa, are clearly seen.
Legend to Figure 5.
(a) The enlarged paraffin-embedded cervical lymph nodes removed in September 1988 from which the DNA extract was prepared. (b) The results of IS900 PCR with amplification products analysed by agarose gel electrophoresis and autoradiography as described. Lanes 1, 2 and 3 are simultaneous process controls correctly reporting negative; lanes 4, 5 and 6, the DNA extract from the enlarged nodes showing strongly positive triplicate reactions for Mycobacterium paratuberculosis, then negative and positive (10fg Mptb target DNA) internal PCR controls.
Legend to Figure 6
Photomicrographs of sections of intestinal wall, illustrating the range of histological appearances and abundance of visible M. paratuberculosis organisms in infected sheep with different forms of Johne's disease. (a) ZN stain (x1600) showing classical pluribacillary Johne's disease in which large numbers of small acid-fast M. paratuberculosis organisms are seen within macrophages, but with little chronic granulomatous response. (b) ZN stain (x1600) showing the paucibacillary form of Johne's disease with scant M. paratuberculosis visible in the tissues and prominent lymphocytic infiltration. (c) ZN stain (x800) showing the paucimicrobial form of Johne's disease diagnosed by IS900 PCR, in which no bacillary-form M. paratuberculosis can be seen. (d) Haematoxylin and eosin stain (x800) of paucimicrobial Johne's disease in a sheep showing typical Langerhans giant cells and pronounced chronic granulomatous inflammation.