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The type of tubercle bacillus found in tuberculosis of the cat
GENERAL ARTICLES.
THE TYPE OF TUBERCLE BACILLUS FOUND IN TUBERCULOSIS OF THE CAT.
By LOUIS COBBETT, M.D., F.R.C.S., Lecturer in Pathology, The University, Cambridge. IN his recent paper on Tuberculosis of the Cat, in the last number of this Journal, A. S. Griffith refers to two tuberculous cats which are mentioned in my book on "The Causes of Tuberculosis" (p. 505) as having yielded on investigation cultures of tubercle bacilli of bovine type; but he did not include them in his summary of collected cases, because the details of the investigation had not been published. Therefore, as all the notes of this investigation are still preserved, as well as the organs of the cats, it seems desirable to put them on record. It wasin 1912 that the investigation was made. The results were not published at the time because it was hoped to obtain further cases which would enable me to write a more complete account of tuberculosis in this species than was then possible. But no more material came to hand, and, the war coming on, the investigation was abandoned. The two cats in question came from the Cambridge Pharmacological Laboratory, where they were about to be used for some experiment; but when, on their bodies being opened under ana::sthesia, disease of the lungs was revealed, the experiment was abandoned and the animals killed and handed over to me. Cat No. I.-The lungs were affected more severely than the other organs. They were thickly beset with little grey tubercles, hard to the touch and projecting from the surface of the collapsed organ, and with larger patches of consolidation which appeared to be partly caseous. In the spleen was a tuberculous nodule as large as a little pea, and in the liver a few small tubercles were just visible. In the portal fissure a small caseous lymph gland was seen. The bronchial glands, as well as one situated just over the manubrium sterni, were enlarged , and on section showed irregular star-shaped caseous centres. The mesenteric glands were tuberculous, and the disease appeared to be more advanced in them than in the bronchials. One group of glands was fused into a mass an inch long, containing three caseous centres, each roughly a quarter of an inch in diameter. The mucous membrane of the large intestine showed several raised spots, each with a central depression. These intestinal lesions were subsequently proved, as we shall see, to be tuberculous in nature. They, together with the lesions in the mesenteric glands, were taken to be the primary lesions, and the pulmonary disease was believed to be secondary and caused by tubercle bacilli carried to the lungs by the blood stream. The kidneys were normal, and so were the mouth, pharynx, and submaxillary lymph glands. Tubercle bacilli were seen in smears made from all the lesions, including lungs, bronchial glands, mesenteric glands, spleen, and
GENERAL ARTICLES.
143
liver; but they were present in s mall numbers only; none were found in a preparation from the discrete grey tubercles in the lung, and very few e ven in the caseous portions of that organ. They were m ore numerou s, however, in th e caseous matter of a mesenteric gland. I t is recorded that they were short. After hardening and preserv a tion in glycerine solution for several years the organs of this cat were re-examin ed and sections were cut for histological investigation. Inspection with the naked eye again showed that the lun gs were closely beset with tubercles and patches of conso lidation. Under the stereoscopic bin oc ular microscope (X6) many of the consolidated areas could be seen to be composed of a number of small er tubercles which had coalesced. In spite therefore of the patchy appearance
FIG.
I.
Miliary tuberculosis of lung of cat, showi ng caseation commencing at the centres of the larger tubercles. x 30.
of the lesions at the time of death their distribution does not seem inconsistent with a hrematogenous origin. Microscopic examination of a portion of the hardened lung containing only the smaller and more discrete tubercles showed that , in spite of their grey and transluscent appearance when freshly seen with the naked eye, caseation was actually occurring in the centres of many of them ; while in some the presence of two or more such centres supported the view, already put forward. that the larger tubercles were the result of the fusion of several smaller ones. Under the high power objective the central areas of some of these little tubercles were seen to be structureless, and were stained a pale pinkish colour after treating with hrematoxylin and eosin; while around each caseous area was a darker zone stained with hrema-
GENERAL ARTICLES.
toxylin, and containing fragments and droplets of nuclear matter. This may be called the caseating zone in contradistinction to the more caseous central area. It is in such a zor.e that tubercle bacilli may be expected to be found in greatest abundance, and it is here one should more diligently search when they are scanty. In some animals, such as a duck of whose tuberculous organs I have sections, and, occasionally, the monkey, they may be so numerous here as to form a ring, visible, after appropriate staining, to the naked eye. In the tubercles of other animals they may be hard to find, as in the very acute pulmonary tuberculosis of a calf artificially infected with tubercle bacilli, or generally, I think, in the pig. In this cat only an ,occasional bacillus could be seen.! When found they were of moderate length and some of them were granular. The peripheral zones of these tubercles presented a marked .alveolar structure, showing that they had grown by setting up a ,pneumonic process in the infundibula and air cells immediately ,around them. These alveoli were filled with epithelial cells and polymorph and other leucocytes. 2 In this region, too, were occasionally to be seen characteristic erythrocytes contained in blood vessels which seemed to be alveolar capillaries incorporated into the tubercle, and, contrary to what one reads about the blood
GENERAL ARTICLES.
145
lymphocytes. There were no giant cells or signs of caseation. A few rather short tubercle bacilli were seen. The most notable thing about these tubercles was that they were surrounded by, or rather, I think I may say, their peripheral zones were composed of, tissue arranged circumferentially-like a capsule in course of formationand in which were so large a number of red corpuscles as to make these zones appear in sections stained feebly with hrematoxylin and strongly with eosin
FIG.
2.
Tuberculous lung of guinea· pig, showing a blood-vessel cut across in the middle of a tubercle.
A culture raised in 1912 from one of the caseous mesenteric glands was dysgonic in mode of growth. The separate little colonies which appeared on the original egg cultures were, indeed, definitely raised and nodular, but subcultures on this medium appeared as a dull, barely visible layer of growth, with a surface like that of ground glass. It was noted, however, that the bacilli were rather long. Four large rabbits injected with emulsions of tuberculous tissues (mesenteric glands two rabbits, lung and spleen one rabbit each) died of general tuberculosis in from fifty-two to eighty-six days. In addition two rabbits were injected with a culture, but these died prematurely of some intercurrent disorder with only early signs of tuberculosis. Cat No.2, like its fellow, was more severely affected in the lungs
GENERAL ARTICLES.
than anywhere else; and there were un mistakable signs that the oldest tuberculous lesions were connected with the intestine, for in a mesenteric gland was a hard calcareous nodule of the size of a small pea which was dry and crumbly when cut, and which shelled out cleanly from the surrounding glandular tissue. Another mesenteric gland had a smaller nodule as hard as a stone. The lungs appeared to be the seat of more recent disease. They were, as we have said, affected severely; tubercles were scattered throughout their substance, and in places these had become confluent, forming consolidated areas reaching, in the base of the right lung, to as much as half an inch in length. In the centre of one of those nodules was a small cavity. Sections of the less affected portions of the organ showed little tubercles tending to become confluent. Their structure was like that of the tubercles of Cat I. The peripheral zones were alveolar, the centres sometimes caseous. Sometimes several caseous foci were seen in one nodule, indicating coalescence of as many tubercles. The lung was congested and showed petechial hao:morrhages. Red blood cells were seen in some of the air cells outside the tubercles. In the peripheral zones of the tubercles there was often blood, not distributed uniformly but in patches. Sometimes it appeared to be in alveolar capillaries which had become incorporated into the tubercle, but often the red blood corpuscles were mixed indiscriminately with other cells, obviously the result of hao:morrhage. Even in the caseous areas there were a few collections of red cells. , The bronchial glands appeared normal, except for one gland close to the trachea which contained a small tubercle. Liver, spleen, kidneys, and cervical glands were free from obvious disease. Tubercle bacilli were found in the caseous material and in the contents of the cavity in the lung. It was noted that in the latter situation they were unusually long. Tubercle bacilli were found also in a mesenteric gland. A young rabbit was injected with an emulsion of the caseous nodule from the lung of the cat. It was killed four months later and found to have severe tuberculosis of the lungs and some tubercles in the kidneys. Cultures were raised from this animal and proved to be dysgonic. I njections were made from them into three rabbits. One received '01 milligram me intravenously; it died of general tuberculosis in twenty-five days. The other two, which were injected subcutaneously with '1 and 1 milligram me respectively, died of general tuberculosis, the former in sixty-eight and the latter in forty-seven days. I n these two cats, then, the tubercle bacilli were proved to be of bovine type, and it can hardly be doubted, in view of the pathological evidence, that the infection had started in both of them in the intestine or mesenteric glands. The probability that they were infected by cow's milk is, of course, almost overwhelming. Bovine tubercle bacilli are usually held to be relatively short, and it is perhaps worth recalling that in the notes of these observations the bacilli, though sometimes described as short, are on three occasions stated to have been long, and on two occasions beaded, and
GENERAL ARTICLES.
147
this not alone in the tissues of the cats, but in artificial cultures on egg and serum made from them, and on one occasion in the organs of a rabbit infected from the cat. I do not know whether a tendencv to grow to greater length than usual has been observed in tubercl~ bacilli obtained from other cats, and on this account these observations may be worth a brief notice. But t do not attach much importance to them because of the great variation in length and other morphological characters which may be seen in any given strain of tubercle bacilli under different circumstances of growth. I have recently seen them very long in a rabbit. Cat No. l-A third cat was the subject of a partial investigation. Its kidneys alone came into my hands. I believe there was very
FIG.
3.
A tubercle in the liver of a cat, surrounded by a highly vascular zone, in which the red cor· puscles stand out as conspicuous dark objects; stained with hrematoxy lin and eosin.
x
160.
little obvious disease elsewhere, for had there been I do not think the veterinary surgeon who kindly sent me the kidneys would have omitted to include the other organs also. The kidneys differed much in size, one being six times as large as the other; the former was as large as a bantam's egg, and weighed 27~ grammes, and the latter no bigger than that of a thrush, and weighed 4! grammes. The larger kidney was, apparently. normal. Of the smaller, the pelvis was full of curdy pus containing some granular tubercle bacilli. This material was sown on egg medium and injected intra peritoneally into a rabbit and subcutaneously into a guinea-pig. Both animals died of general tuberculosis with the K
14 8
GENERAL ARTICLES.
usual lesions, the former in I I2 days and the latter in sixty-six. It is recorded of the tubercle bacilli in the tuberculous omentum of the rabbit that they were short. Unfortunately the record of the cultures has been lost; but from the animal experiments alone there can be no doubt that the strain of tubercle bacilli with which the cat was injt cted was, like those of Cats I and 2, of the bovine type. If these three cases be added to those collected by Griffith, it brings the number of tuberculous cats in which the type of tubercle bacillus found in the lesions has been determined to sixteen. In all these cases the bacilli were typical examples of the bovine type, and of full virulence for the rabbit.
LAMB
DYSENTERY.
AN ACCOUNT OF SOME EXPERIMENTAL FIELD WORK IN I925 AND I926. By T. DALLING, M.R.C.V.S., Langley Court, Beckenham, Kent. SECTION 1.
Artificial Reproduction of Lamb Dysentery. FURTHER experimental field work in lamb dysentery infected districts in Northumberland and the south of Scotland has been cond llcted during 1925 and 1926 to investigate the methods of the artificial production of the disease and the possibility of protecting lambs against its natural occurrence. A considerable amollnt of time was spent each spring in the field, and a temporary laboratory was set up in Northumberland. The first records of the successful artificial reproduction in lambs of a disease clinically identical with the naturally occurring condition, were in I923, (I) when the feeding of young healthy lambs with (a) intestinal contents of naturally infected lambs, and (b) a mixture of cultures of B. welchii and B. coli, apparently gave rise to the disease. During I925 and 1926 we have carried out experimental work on similar lines, and in addition have investigated the results of the introduction of similar substances into young lambs by routes other than the mouth. In I925 twenty-five young healthy lambs were used. They were procured from clean farms on which lamb dysentery has never been known, and on removal from their mothers they were kept at the field laboratory, cow's milk forming their diet during experimentation. In I926 lambs born from thirty Cheviot ewes were used; these ewes carne from a farm and flock in which lamb dysentery is unknown; they were removed a few days prior to the beginning of
THE TYPE OF TUBERCLE BACILLUS FOUND IN TUBERCULOSIS OF THE CAT.
By LOUIS COBBETT, M.D., F.R.C.S., Lecturer in Pathology, The University, Cambridge. IN his recent paper on Tuberculosis of the Cat, in the last number of this Journal, A. S. Griffith refers to two tuberculous cats which are mentioned in my book on "The Causes of Tuberculosis" (p. 505) as having yielded on investigation cultures of tubercle bacilli of bovine type; but he did not include them in his summary of collected cases, because the details of the investigation had not been published. Therefore, as all the notes of this investigation are still preserved, as well as the organs of the cats, it seems desirable to put them on record. It wasin 1912 that the investigation was made. The results were not published at the time because it was hoped to obtain further cases which would enable me to write a more complete account of tuberculosis in this species than was then possible. But no more material came to hand, and, the war coming on, the investigation was abandoned. The two cats in question came from the Cambridge Pharmacological Laboratory, where they were about to be used for some experiment; but when, on their bodies being opened under ana::sthesia, disease of the lungs was revealed, the experiment was abandoned and the animals killed and handed over to me. Cat No. I.-The lungs were affected more severely than the other organs. They were thickly beset with little grey tubercles, hard to the touch and projecting from the surface of the collapsed organ, and with larger patches of consolidation which appeared to be partly caseous. In the spleen was a tuberculous nodule as large as a little pea, and in the liver a few small tubercles were just visible. In the portal fissure a small caseous lymph gland was seen. The bronchial glands, as well as one situated just over the manubrium sterni, were enlarged , and on section showed irregular star-shaped caseous centres. The mesenteric glands were tuberculous, and the disease appeared to be more advanced in them than in the bronchials. One group of glands was fused into a mass an inch long, containing three caseous centres, each roughly a quarter of an inch in diameter. The mucous membrane of the large intestine showed several raised spots, each with a central depression. These intestinal lesions were subsequently proved, as we shall see, to be tuberculous in nature. They, together with the lesions in the mesenteric glands, were taken to be the primary lesions, and the pulmonary disease was believed to be secondary and caused by tubercle bacilli carried to the lungs by the blood stream. The kidneys were normal, and so were the mouth, pharynx, and submaxillary lymph glands. Tubercle bacilli were seen in smears made from all the lesions, including lungs, bronchial glands, mesenteric glands, spleen, and
GENERAL ARTICLES.
143
liver; but they were present in s mall numbers only; none were found in a preparation from the discrete grey tubercles in the lung, and very few e ven in the caseous portions of that organ. They were m ore numerou s, however, in th e caseous matter of a mesenteric gland. I t is recorded that they were short. After hardening and preserv a tion in glycerine solution for several years the organs of this cat were re-examin ed and sections were cut for histological investigation. Inspection with the naked eye again showed that the lun gs were closely beset with tubercles and patches of conso lidation. Under the stereoscopic bin oc ular microscope (X6) many of the consolidated areas could be seen to be composed of a number of small er tubercles which had coalesced. In spite therefore of the patchy appearance
FIG.
I.
Miliary tuberculosis of lung of cat, showi ng caseation commencing at the centres of the larger tubercles. x 30.
of the lesions at the time of death their distribution does not seem inconsistent with a hrematogenous origin. Microscopic examination of a portion of the hardened lung containing only the smaller and more discrete tubercles showed that , in spite of their grey and transluscent appearance when freshly seen with the naked eye, caseation was actually occurring in the centres of many of them ; while in some the presence of two or more such centres supported the view, already put forward. that the larger tubercles were the result of the fusion of several smaller ones. Under the high power objective the central areas of some of these little tubercles were seen to be structureless, and were stained a pale pinkish colour after treating with hrematoxylin and eosin; while around each caseous area was a darker zone stained with hrema-
GENERAL ARTICLES.
toxylin, and containing fragments and droplets of nuclear matter. This may be called the caseating zone in contradistinction to the more caseous central area. It is in such a zor.e that tubercle bacilli may be expected to be found in greatest abundance, and it is here one should more diligently search when they are scanty. In some animals, such as a duck of whose tuberculous organs I have sections, and, occasionally, the monkey, they may be so numerous here as to form a ring, visible, after appropriate staining, to the naked eye. In the tubercles of other animals they may be hard to find, as in the very acute pulmonary tuberculosis of a calf artificially infected with tubercle bacilli, or generally, I think, in the pig. In this cat only an ,occasional bacillus could be seen.! When found they were of moderate length and some of them were granular. The peripheral zones of these tubercles presented a marked .alveolar structure, showing that they had grown by setting up a ,pneumonic process in the infundibula and air cells immediately ,around them. These alveoli were filled with epithelial cells and polymorph and other leucocytes. 2 In this region, too, were occasionally to be seen characteristic erythrocytes contained in blood vessels which seemed to be alveolar capillaries incorporated into the tubercle, and, contrary to what one reads about the blood
GENERAL ARTICLES.
145
lymphocytes. There were no giant cells or signs of caseation. A few rather short tubercle bacilli were seen. The most notable thing about these tubercles was that they were surrounded by, or rather, I think I may say, their peripheral zones were composed of, tissue arranged circumferentially-like a capsule in course of formationand in which were so large a number of red corpuscles as to make these zones appear in sections stained feebly with hrematoxylin and strongly with eosin
FIG.
2.
Tuberculous lung of guinea· pig, showing a blood-vessel cut across in the middle of a tubercle.
A culture raised in 1912 from one of the caseous mesenteric glands was dysgonic in mode of growth. The separate little colonies which appeared on the original egg cultures were, indeed, definitely raised and nodular, but subcultures on this medium appeared as a dull, barely visible layer of growth, with a surface like that of ground glass. It was noted, however, that the bacilli were rather long. Four large rabbits injected with emulsions of tuberculous tissues (mesenteric glands two rabbits, lung and spleen one rabbit each) died of general tuberculosis in from fifty-two to eighty-six days. In addition two rabbits were injected with a culture, but these died prematurely of some intercurrent disorder with only early signs of tuberculosis. Cat No.2, like its fellow, was more severely affected in the lungs
GENERAL ARTICLES.
than anywhere else; and there were un mistakable signs that the oldest tuberculous lesions were connected with the intestine, for in a mesenteric gland was a hard calcareous nodule of the size of a small pea which was dry and crumbly when cut, and which shelled out cleanly from the surrounding glandular tissue. Another mesenteric gland had a smaller nodule as hard as a stone. The lungs appeared to be the seat of more recent disease. They were, as we have said, affected severely; tubercles were scattered throughout their substance, and in places these had become confluent, forming consolidated areas reaching, in the base of the right lung, to as much as half an inch in length. In the centre of one of those nodules was a small cavity. Sections of the less affected portions of the organ showed little tubercles tending to become confluent. Their structure was like that of the tubercles of Cat I. The peripheral zones were alveolar, the centres sometimes caseous. Sometimes several caseous foci were seen in one nodule, indicating coalescence of as many tubercles. The lung was congested and showed petechial hao:morrhages. Red blood cells were seen in some of the air cells outside the tubercles. In the peripheral zones of the tubercles there was often blood, not distributed uniformly but in patches. Sometimes it appeared to be in alveolar capillaries which had become incorporated into the tubercle, but often the red blood corpuscles were mixed indiscriminately with other cells, obviously the result of hao:morrhage. Even in the caseous areas there were a few collections of red cells. , The bronchial glands appeared normal, except for one gland close to the trachea which contained a small tubercle. Liver, spleen, kidneys, and cervical glands were free from obvious disease. Tubercle bacilli were found in the caseous material and in the contents of the cavity in the lung. It was noted that in the latter situation they were unusually long. Tubercle bacilli were found also in a mesenteric gland. A young rabbit was injected with an emulsion of the caseous nodule from the lung of the cat. It was killed four months later and found to have severe tuberculosis of the lungs and some tubercles in the kidneys. Cultures were raised from this animal and proved to be dysgonic. I njections were made from them into three rabbits. One received '01 milligram me intravenously; it died of general tuberculosis in twenty-five days. The other two, which were injected subcutaneously with '1 and 1 milligram me respectively, died of general tuberculosis, the former in sixty-eight and the latter in forty-seven days. I n these two cats, then, the tubercle bacilli were proved to be of bovine type, and it can hardly be doubted, in view of the pathological evidence, that the infection had started in both of them in the intestine or mesenteric glands. The probability that they were infected by cow's milk is, of course, almost overwhelming. Bovine tubercle bacilli are usually held to be relatively short, and it is perhaps worth recalling that in the notes of these observations the bacilli, though sometimes described as short, are on three occasions stated to have been long, and on two occasions beaded, and
GENERAL ARTICLES.
147
this not alone in the tissues of the cats, but in artificial cultures on egg and serum made from them, and on one occasion in the organs of a rabbit infected from the cat. I do not know whether a tendencv to grow to greater length than usual has been observed in tubercl~ bacilli obtained from other cats, and on this account these observations may be worth a brief notice. But t do not attach much importance to them because of the great variation in length and other morphological characters which may be seen in any given strain of tubercle bacilli under different circumstances of growth. I have recently seen them very long in a rabbit. Cat No. l-A third cat was the subject of a partial investigation. Its kidneys alone came into my hands. I believe there was very
FIG.
3.
A tubercle in the liver of a cat, surrounded by a highly vascular zone, in which the red cor· puscles stand out as conspicuous dark objects; stained with hrematoxy lin and eosin.
x
160.
little obvious disease elsewhere, for had there been I do not think the veterinary surgeon who kindly sent me the kidneys would have omitted to include the other organs also. The kidneys differed much in size, one being six times as large as the other; the former was as large as a bantam's egg, and weighed 27~ grammes, and the latter no bigger than that of a thrush, and weighed 4! grammes. The larger kidney was, apparently. normal. Of the smaller, the pelvis was full of curdy pus containing some granular tubercle bacilli. This material was sown on egg medium and injected intra peritoneally into a rabbit and subcutaneously into a guinea-pig. Both animals died of general tuberculosis with the K
14 8
GENERAL ARTICLES.
usual lesions, the former in I I2 days and the latter in sixty-six. It is recorded of the tubercle bacilli in the tuberculous omentum of the rabbit that they were short. Unfortunately the record of the cultures has been lost; but from the animal experiments alone there can be no doubt that the strain of tubercle bacilli with which the cat was injt cted was, like those of Cats I and 2, of the bovine type. If these three cases be added to those collected by Griffith, it brings the number of tuberculous cats in which the type of tubercle bacillus found in the lesions has been determined to sixteen. In all these cases the bacilli were typical examples of the bovine type, and of full virulence for the rabbit.
LAMB
DYSENTERY.
AN ACCOUNT OF SOME EXPERIMENTAL FIELD WORK IN I925 AND I926. By T. DALLING, M.R.C.V.S., Langley Court, Beckenham, Kent. SECTION 1.
Artificial Reproduction of Lamb Dysentery. FURTHER experimental field work in lamb dysentery infected districts in Northumberland and the south of Scotland has been cond llcted during 1925 and 1926 to investigate the methods of the artificial production of the disease and the possibility of protecting lambs against its natural occurrence. A considerable amollnt of time was spent each spring in the field, and a temporary laboratory was set up in Northumberland. The first records of the successful artificial reproduction in lambs of a disease clinically identical with the naturally occurring condition, were in I923, (I) when the feeding of young healthy lambs with (a) intestinal contents of naturally infected lambs, and (b) a mixture of cultures of B. welchii and B. coli, apparently gave rise to the disease. During I925 and 1926 we have carried out experimental work on similar lines, and in addition have investigated the results of the introduction of similar substances into young lambs by routes other than the mouth. In I925 twenty-five young healthy lambs were used. They were procured from clean farms on which lamb dysentery has never been known, and on removal from their mothers they were kept at the field laboratory, cow's milk forming their diet during experimentation. In I926 lambs born from thirty Cheviot ewes were used; these ewes carne from a farm and flock in which lamb dysentery is unknown; they were removed a few days prior to the beginning of