APPENDIX AA Report on the Occurrence o£ Respiratory Anthrax and Haemorrhagic Anthrax Meningitis following the Intrusion o£ U.S. Military Planes over Northeast China (ISCC/5) INTRODUCTION Following the invasions by American airplanes which disseminated insects and other objects (houseflies, anthomyiid flies, wolf spiders, ptinid beetles and feathers) carrying anthrax bacilli, there occurred suddenly and successively in some invaded areas of Northeast China a hitherto rare disease-anthrax infection through the respiratory route. The following report concerns the dissemination of insects and other objects carrying anthrax bacilli by American airplanes, the sudden and successive occurrence of anthrax infection through the respiratory route in Northeast China, and finally, discussion and conclusion. CHAPTER I Insects and Other Objects Carrying Anthrax Bacilli Dropped by American Airplanes Feathers (1) At 11 a.m. March 11, 1952 when more than four hundred inhabitants of Pei-ching Village, Chang Shan (i.e. the 5th) District of Antung Hsien were holding a meeting, village chief Wu Ching-ming and a farmer Chiang Wen-ch'ang first saw three American planes flying fr.om northwest to southeast. Soon afterwards they observed a greyish object dropped from one of the planes, slowly falling toward southeast. Men and women of the whole village were immediately mobilized to search for that object. A large amount of feathers was found at the shan region, south east of the village. Two days prior to this, the villagers had been engaged in. catching insects in the same region for two days without seeing any feathers. The feathers collected wel"e examined by bacteriologists Hsin Chiin, Ching Kuan-hua and Chao Cheng-lin and were found to carry anthrax bacilli (Document AA-1). (A) - 361 -(2) At 12:52 March 12, 1952, eight American planes invaded K'uan-tien Hsien. One of the planes dropped a cylindrical object-a bacteria bomb. The remnants of this bacteria bomb were found by a school boy, Li Ssu-chien of K'uan-tien Middle School in the afternoon of March 21 in the maize field of Lou-ho-t'ao outside the east gate of K'uantien city. Feathers were found in the vicinity of the bomb fragments. (Please refer to App. V.) Bacteriological examinations of the feathers carried out by bacteriologists, Hsieh Shao-wen (Samuel Zia) and Chang Nai-chu, revealed anthrax bacilli. (B) Anthomyiid flies and wolf spiders Anthomyiid flies and wolf spiders were also found in the vicinity of the remnants of the bacteria bomb in the maize field at Lou-ho-t'ao, outside the east gate of K'uan-tien City. Specimens of the flies and spiders were collected by ·entomologists, Prof. Liu Ch'ung-lo and Dr. Ma Shih-chiin, and were identified by Dr. Chen Sicien .H., Director of the Laboratory of Entomology, Academia Sinica, Assistant Professor Lu Pao-ling, Peking College of Agriculture, and Professor Wang Feng-chen of Tientsin Army Medical College. The flies and spiders were examined by Dr. Hsin Chiin, bacteriologist, and were found to carry anthrax bacilli. (It may be also mentioned that anthrax bacilli were also isolated by bacteriologists Hsieh Shao-wen and Chang Nai-chu from anthomyiid flies and wolf spiders disseminated at Tsingtao by American planes.) (C) Houseflies At 10 p.m. March 14; 1952 ·American airplanes invaded Ssuping area. In the afternoon of March 17 Wang You-ts'ai, an inhabitant of San-ho · Village, First District, Ssuping City noted large numbers of flies at San-tao-lin-tze outside the village. He called together Ch'iu Jung-sheng and Chao Sheng-tien to burn up as many of the flies as they could with hemp stalk. They reported the incident to their group leader Ts'ao Meichiu, who mobilized 6 more members to search out and exterminate a great many more. The chief Chao of· the Hsiao-hung-tsui-tze police station happened to pass by the spot at that moment and witnessed the above facts. Next day (March 18) at 3 p.m. Liu Chi-an and Hsii Chung-lin, sanitary inspeetors of the city, went to the spot. They searched and found large numbers of flies which were duly destroyed by some 40-50 persons mobilized by the District Government. The flies· were identified by the entomologists Prof. Ch'in Yao-ting and Dr. Feng Lan-pin to be houseflies Musca 'Vicina. From such flies - 362 - anthrax bacilli were isolated by the Epidemic Prevention Station of Ssuping city. This was confirmed by bacteriologists Hsin Chi.ln and Cheng Keng. (Documents AA-2 & AA-3) · Ptinid beetles In the evening of March 20, 1952, Lu Li-tsun, an inhabitant of Peichiao-ch'ang Village, Liu-erh-pu District of Liaoyang Hsien heard the noise of airplanes flying over the village. His sister-in-law also heard the noise; she went out of the house to look for, 'but could not see any plane. Aecording to the Air Observer Corps, two American· planes invaded Liaoyang area at 6 p.m. of that day and again at 6 :30 p.m. on March 27. At the time when Lu Li-tsun heard the noise of airplanes, Jen Wan-ku, a militiaman of Pei-chiao-ch'ang Village was on his way to the 4th group of inhabitants on patrol duty. He saw about 160 meters away on the southeast a red object of the size of a thermos bottle dropPing from the air above the houses of Chang Chia-feng, Wang Wen-ch'ang and Huang Yii-ch'eng. The object exploded when it was about 3-4 meters above the roof of the houses producing a feeble noise and an offensive smell. At the same time Wang Yung-ch'ang, an inhabitant of Ah-lao-ch'iao also saw the red object from a distance of about 700 meters away on the southwest.. Wang Hua-ming, a member of Wang Wen-ch'ang's family saw, through the window, the red object falling in front of their gate when he was sitting on his kang (brick bed). He rushed out of his room but the red object had already disappeared. He went back to his room again and lighted the lamp and saw numerous insects on the outer surface of the window pane. On careful inspection numerous insects were found on the ground and on the outer side of the walls of the above mentioned three houses. Action was .then taken to catch and burn these insects. Similar insects were found on other houses in the village. Next day the District received reports on the discovery of similar insects in neighboring villages and towns. Up to March 28th, these insects were found in 86 villages and towns including the town of Liu-erh-pu. An-shan city which is not far from Liu-erh-pu was also found to have such insects. The area in which these insects were found covered 30 kilometers from east to west and 20 kilometers from north to south. According to the District Government, such "red -object" as described above was also seen to have fallen in places and similar insects were found also in the fields. The weather then was still very cold. The earth froze at night, melting only in the day time. The insects were identified by entomologists Liu Ch'ung-lo and Lu Pao-lin, as Ptinus fur. Bacteriological examination by Drs. Hsieh Shao-wen (Samuel Zia) and Chang Nai-chu, proved that these insects carried anthrax bacilli. (Documents AA-4 & AA-5). (D) (E) Entomological Identification (1) Housefly (Musca vicina Macquart) The fly, specimen No. 13033, discovered in San Ho Village of Ssuping on March 17, 1952, has been identified as housefly, Musca vicina Macquart, by entomologists Ch'in Yao-ting, Professor of National Medical College, Shenyang, and Feng Lan-pin, Lecturer of the same College. This species of housefly belongs to Family Muscidae, Order Diptera. The characteristics of this species are: Four black longitudinal bands on the mesonotum. In the male, width of front about one-fourth to onethird that of the compound eye, the abdomen light orange in co1or with a central black stripe dorsally. In the female, width of front slightly narrower than the compound eye, and abdominal tergite orange in color, with grayish yellow tomentum. The identification is based on Li and Feng (3), Tokunaga (1) and a series of publications by Patton (11, 12). · This species of housefly is widely distributed. Outside of Asia, it is the commonest species in Hawaii (2). It has been recorded in China ( 4, 13). Its habits resemble those of the common housefly (Musca domestica Linn.), breeding in such media as horse dung and excreta, garbage and decayed organic· materials. There may be many generations a year, varying with the climate of the locality. The adult flies invade houses. (see Document AA-2) (2) Ptinid beetle (Ptinus fur Linn.) : The beetle, specimen No. 362-1 (2007), discovered in Pei-chiao-ch'ang Village of Liaoyang on March 20, 1952, has been identified as ptinid beetle, Ptinus fur Linn., by Prof. Liu Ch'ung-lo of the Department of Entomology of Peking College of Agriculture, and by Assistant Professor Lu Pao-lin of the same institution. This species of beetle belongs to Family Ptinidae, Order Coleoptera. Its characteristics are: A pair of moderately yellowish brown subtomentose cushions on the pronotum. Punctures on elytra arranged parallelly in serial rows, also fine brownish setae on the surface. A white hairy marking near the base and apex of each elytron (see Document AA-4). The identification is based on Reitter (20) and Hinton (18). Ptinid beetle has a wide distribution. According to Hsin (15) and Kuan (16), it has been recorded in China. (3) Anthomyiid fly and wolf spider Details are given in the Report on the Calcareous Bacteriological Bomb Dropped by U.S. Military Plane at K'uan-tien. (App. V) . ...,.,..,, 364 - Bacteriological Examinations (F) Methods The methods for the isolation of anthrax bacilli are briefly given here. Details are obtainable in the Documents at the end of this Appendix. (1} L For isolation of bacteria from 'insects: The insect is first washed in sterile normal saline and cultures are then made of the washing fluid. The insect is then ground up in a sterile mortar with sterile saline. The suspension of the ground 1nsect is cultured and inoculated into white mice. The media used are plain agar· plate, blood agar plate, .s.S. agar plate and cooked meat broth. 2. For examination of feathers: The feather is washed in sterile normal saline. This saline is then centrifuged at 3000 revolutions per minute for 20 · minutes. The sediment is used for culture and animal inoculation as mentioned above. 3. For the examination of · human post-mortern material: The material is divided into 2. parts, 1 .part is used for direct culture and the other part for .animal inoculation... When organisms· are first isolated from any material, further steps are taken for identification. Bacillus anthracis is preliminarily diagnosed if the morphology a:rid staining quality of the organisms, appearance of colonies, manner of growth in the broth, the presence or absence of motility arid pathogeniCity for white are identical with those of that organism. Further steps include the test on other laboratory animals for pathogenicity, biochemical characteristics and immunological studies. When all the findings are identical with those of the anthrax bacillus, the isolated organism is finally identified as Bacillus anthracis. Diagnostic Criteria . . 1. Preliminary identificati aJ t!l "" .a""'"' Cll aJ c; Colonies on agar plate Morphology Growth in meat broth Motility Milk Coagulation Test H2S I El·"'til Q) U,l aJ :;;: 'A"' aJ 0 ctS (,) Goat's red blood cells Rabbit's red blood cells Guinea pig's red blood cells Lactose Glucose Inositol - Dulcitol Salicin White Death of animal Mice Death of d 0 :.3 co til ctl d < + + + Arabinose .§ - - Sucrose Xylose 0 + - 0 Mannitol 1:l.O ·a ell I - In dol o"' 355 48004B 4.8003 17006 FeaFeaWolf Anthothers Spiders myiid thers flies K'uan K'uan K'uan Antung Tien Tien Tien Peiching village April March March March - -- - + - - - + + + - + - + + + - + - - - + - + + + - - -- + -- -- I- II - ·+ + + + + - + - - - - I Died I Died r Died I Died I Died I Died Died Died Died Died Died Died + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + ---+-. ,-_-f1-· ++ + . + + 366 + + - ++ TABLE II. RESULTS OF :SACTERIOLOGICAL EXAMINATIONS I Case No. Name Chii Chan-yun Manching Railway worker March 19 March 22 Anthrax of respiratory system District Occupations II IV Wang Wei-LiuTien Tze-pin shih Ch.eng-ho Shenyang An-shan An-tung Pedicab House Farmer driver wife March 20 April 11 April 16 March 25 April 14 April 18 Anthrax Anthrax: Anthrax of respira- of respira- of respira· tory sys- tory sys- tory systern and tern and tem and Anthrax Anthrax Anthrax Meningitis Meningitis Meningitis I Date of onset of disease Date o:f death Pathological diagnosis Grayish white colonies. Not transparent. Edge uneven and curled hair like. Gram positive large bacilli with square ends and in chain form. Spore formation at center Flocculent precipitation Non motile Colonies on agar plate Morphology Growth in meat broth Motility + + + + + + + + + + + + + + + Death of· animal Died Died Died Died Death of animal Gelatinous change at site of injection Enlargement of spleen Congestion and enlargement of liver Congestion of lung Culture of heart blood Sr.near from internal organs Died Died Died Died + + + + + + + + + + + + + + + + Milk Coagulation test H.s Indol 0• "' Goat's red blood cells :Z:: Guinea pig's red blood cells m'fil ,!:;> Rabbit's red blood ce:P,s Lactose Glucose Maltose Mannitol Sucrose Inositol Xylose Arabinose Dulcitol Salicin t: ..... '\U ·O Whitej Mice '5 (.) 0 .:1 m .s::s Precipitation Test - 367 - + + + + + + + + + + + + + b) There is fermentation of glucose, sucrose and maltose. Acid is produced without gas. Milk is cof!.gulated. 'c) No hemolysis on the red blood cells of goat, rabbit and guinea pig, d) The liver or spleen of animals died from anthrax is up with saline and then boiled and used for precipitation with the filtrate as an antigen, a precipitation test (Ascoli test) is made with anti-anthrax diagnostic serum. The result is positive. (3) Results of Examination The Tables I and II. of strains of organisms isolated are given in CHAPTER II Sudden and Successive Occurrence of Anthrax Infection through the Respiratory "Route Case 1. Chii Chan-yun, male, 55 years old, a. railway foreman the Man Ching Station ;and· raihvaymen :J:..i" Tso-hsiang and Liu Chung-ko on March 16, 1952 todk part in catching and killihg flies at a place one and one half kilometers to the north of the Man Ching Station. (In the night of March 14 American airplanes invaded Ssuping area including M;an Ching Station;) · · : · On March 19, Chii Chan-yun became ill with fever, headache and aching in the limbs. On March 21 he was admitted to the Railway Hospitai when he also developed cough, nausea, vomiting .insomnia, mental confusion and rigidity. of Examination of sputum revealed the presence of Gram positive encapsulated large bacilli. White blood cell count 28,000. Death occurred at 2:45 p.m. March 22, 1952. Autopsy was done by Dr. Sung Teh-yu of the Epidemic Prevention Station ·of Ssuping. There were congestion, oedema and small hemor· rhages in both lungs. Increased consistency was felt in the upper lobe of both lungs. Hilum lymph glands were enlarged. Bilateral pleural effusion and pericardia! e:ffusiop were present. Ulcerative hemorrhagic spots were seen on the mucosa of small intestine. The tip of the appendix showed hemorrhagic inflammatory process. The mesenteric lymph glands were however not enlarged. Bacteriological examination was done in the Epidemic Prevention Station of Liao-hsi Anthrax bacilli were isolated from thelung, liver and spleen. The organisms were re-examined by bacteriologists Hsin Chun and Cheng Keng and the original diagnosis · - 368 - of Bacillus Anthracis was confirmed. The pathological diagnosis of respiratory anthrax was confirmed by pathologists Professors Wu Tsaitung and Li Pei-lin. (The clinical symptoms indicate that the meninges were involved but the skull was not opened for examination at autopsy because of the refusal of the family.) (Document AA-7 and Document AA-10) Case 2. Wang Tze-pin, male of 47 years was a tricycle-rickshaw driver in Shenyang. He became ill on March 20 with general malaise. Next day he was confined to bed. On April 22 aching in legs, general weakness, upper abdominal discomfort, nausea and headache were felt. He was slightly better in the morning of April 23 but got worse by the afternoon. Until 6 a.m. April 24 he was still conscious and could move about. However since then he sank progressively into coma with restlessness. Neck was rigid. Kernig's sign was positive. Death occurred at 9 a.m. April 25. Autopsy was done by Prof. Chu Feng-ch'un and assistant Wang HungIieh of the National Medical College with the diagnoses of hemorrhagic anthrax meningitis, anthrax bronchopneumonia of right lower lobe, peribronchitis, interlobular cellulitis, suppurative hemorrhagic anthrax lymphadenitis of hilum glands, pulmonary oedema, pleural effusion, pericardia! effusion and multiple punctate necrosis and ulceration of intestinal mucosa due to anthrax infection. (No enlargement of mesenteric lymph glands.) The pathological diagnosis was confirmed by Professors Wu Tsai-tung and Li Pei-lin. Anthrax bacilli were isolated from the brain tissue, heart blood and spleen by bacteriologists Chu Chi-ming and Liu Shih-ming and confirmed by bacteriologists Hsin Chun and Cheng Keng. (Document AA-9 and Document AA-10) Case 3a. .Wei Liu-shih, female of 32 years, was an inhabitant of Anshan City. She was repeatedly engaged in catching and killing insects (Ptinus fur) dropped by American airplanes. In the evening of Aprilll she felt general malaise. Next day headache, chilliness and fever were noticed. Condition became worse on April 13 and she was confined to bed. Cough, chest pain, shortness of breath and vomiting developed. On April 14 mentality became confused with delirium, restlessness and She had attacks of convulsipn. The. neck 'Yas markedly rigid. Kernig's sign was positive. · Dry and moist rales were ecies of housefly in Ssuping should .be later than in Tsinan.. Row.ever, large numbers these flies were discovered in the :field at on March 17th, 1952. This is definitely abnormal. I. II. Ptinid beetle: This species of beetle is a pest of stored products. According to the reports of Hsin (15), Li (14), Patton (19) and Cotton (17), . under :natural conditions;· the beetle frequents· warehouses, granaries, - 371 - :flour mills and other factories and storage houses for and plant products. It damages stored grains, :flours, furs, leathers, etc. It is also a well-known museum pest, destroying especially dried spe. cimens of animals and plants. This species has one to three generations a year, varying with the temperature. In China, it usually passes the winter as larva, but occasionally a few adults may also survive the cold. It takes three and a half months to complete a generation. The adults are active in the night and retire into hiding during daytime; they alsl) have the habit of feigning death. When the circumstances under which ptinid beetles were discovered at Liaoyang are compared with their behavior under natural conditions, the following four points are noteworthy: 1. Under natural conditions, ptinid beetles should be found at places such as warehouses, especially those for storing grains, :flours, furs and leathers, or any other place for storing such materials. However, at Liaoyang and the other localities these beetles were found not around store-houses, but outside ordinary houses. They were found not only on the ground and walls outside the houses, but also in the fields. This is definitely not a natural phenomenon. 2. All the publications mentioned above have pointed out that ptinid beetles are nocturnal. However, at Liaoyang, these beetles were discovered in daytime at places where they do not normally ·appear. This is at variance with the normal habits of these beetles. 3. On May 26th, 1952, Ma Shih-chun, Assistant .Research Member of the Laboratory of Entomology, Academia Sinica, and Lu Pao-ling, assistant Professor of the Peking College of Agriculture went to Pei Chiao Chang Village. The purpose of their visit was to investigate from entomological· point of view whether at the time of the discovery of these beetles was there any possibility of appearance in large numbers of ptinid beetles under natural conditions at that place. They found that neither was there any storehouse for grains, furs, leathers, or other substances, nor was there stored in the houses of the people any furs, leathers, or large amounts of grains or other materials on which ptinid beetles might develop. They have come to the conclusion that it is impossible for ·large numbers of ptinid beetles to appear at that place under natural conditions. r 4. After a large number of ptinid beetles ·had been discovered in Pei Chiao Chang Village of Liaoyang on March 20, 1952, Feng Lan-pin, Lecturer of National Medical College and Assistant Li Shaohua investigated further whether in the neighboring areas of Liao- 37'2 ...._ yang were ptinid beetles appearing naturally. They searched in the vicinity of Shenyang and Liaoyang such places as granaries where these beetles might appear, but they were unable to :find any. This shows that this species of beetle had not appeared at that time under natural conditions. Thus it is concluded that the appearance of large numbers of ptinid beetles at Liaoyang is unusual and moreover, that these beetles could not have migrated or transferred from the neighboring areas. It is very evident from the four points mentioned above that at that time no ptinid beetles had appeared at places where they· could naturally occur, while on the contrary they suddenly occurred in abundance at places such as Pei-chiao-chang Village of Liaoyahg where natural conditions did not permit their appearance. Therefore, from the entomological point of view, the occurrence of ptinid beetles at Liaoyang and Anshan is entirely unusual. Finally, it is observed that the unusual occurrence of the house flies and ptinid beetles, as mentioned above, was preceded by the intrusion of U.S. military planes into those places. (B) Bacteriological Consideration. As mentioned in the literature. (21, 23) different strains of anthrax bacilli may show differences in certain biochemical reactions. This is amply confirmed by personal observations of Prof.. Cheng Keng in this country, especially in the speed of sucrose and salicin fermentations. But the biological and biochemical features of the 10 strains of anthrax bacilli isolated from feathers, insects and human autopsy material are entirely identical. This speaks for the common origin of all the organisms isolated from Ptinus fur, housefly, feathers and the clinical cases of anthrax infection. In 1894 Heim (25) reported the :finding of pathogenic anthrax spores on the body surface and in the excreta of Ptinus. Cao (22) experimented with the ova of housefly (Musca do1nestica). The surface of the ova was sterilized. They were then hatched in the flesh of an animal died of anthrax. Anthrax bacilli could be found in the larvae, and, in the adults; for at least 9 days after hatching. As far back as 1869 Raimbert had experimental proof that the fly could carry anthrax bacilli (27). Nuttall (26) mentioned Bollinger as having caught flies from a cow died of anthrax and isolated anthrax bacillus from the digestive tract of the flies. These flies were inoculated into 2 rabbits which later died of anthrax. In· 1912 Graham-Smith (24) reported the feeding of - 373 - larvae of housefly with foods carrying anthrax bacilli. The organism could be isolated from most of the adult flies. The above works indicate that the Ptinus fur and housefly could be used to carry anthrax bacilli. Some control observations have been made in our laboratory (See Appendices E and F). In June 1952, 3106 flies of various kinds were collected from Shen-yang including Fannia scalaris, Muscina stabulans, Lucilia sericata, Sarcophaga sp., Calliphora sp. etc. F-rom such a large number of flies no anthrax bacilli were isolated. At the same time 13 specimens of feathers from Shen-yang and 3 specimens from Kuantien were examined. No anthrax bacilli were found in these local specimens of feathers. No control studies have been made for Ptinus fur since it was impossible to find a local specimen. The control observations would further indicate that the isolation of · anthrax bacilli from insects, (Ptinus fur from Liaoyang, housefly from Ssuping, anthomyiid fly from Kuantien) wolf spiders (from Kuantien) and feathers (from Kuantien and Pei-ching Village of Antung) is not merely an accident. That B. anthracis could be used as a bacteriological weapon is well known. Rosebury and Kabat (28) put it on the top of the list of organisms for air-borne infection because: "The anthrax bacillus is one of the most thoroughly studied bacteria. Its properties make it an obvious possibility as an agent in warfare." "B. anthracis is surpassed by few micro-organisms in infectivity for animals, and by none in host range." Zelle et al (29-32) in Camp Detrick carried out extensive studies on B. anthracis as a bacteriological weapon. They reported on the selection of strains, nutrition studies and the · use of chemicals to enhance the invasiveness of the organism. Some variants were obtained especially suitable for infection through the respiratory route. The virulence of the organism could be artificially increased for the purpose of bacterial warfare. From the facts mentioned above it is not difficult to unravel the plot of American government of disseminating B. anthracis in order to wage bacteriological warfare in Northeast China. (C) Pathological Consideration In the 5 cases of anthrax infection the etiological diagnosis was established either by culture or by the demonstration of the organisms in pathological sections. Clinically there were manifestations of respiratory infection in every case. Case 1 had congestion and oedema of both lungs with increase of consistency in upper lobes, enlargement of hilum glands and bilateral pleural effusion etc., lesions en- 374 - tirely similar to those of case 2 and case 3a. Case 3b had anthrax bacillus bronchopneumonia though the bronchial lymph glands were not enlarged. In case 4 the lungs were evidently increased in weight and the bronchial lymph glands were enlarged. All 5 cases had lesions in the respiratory system. There was no cutaneous lesions in any one of these. Although hemorrhagic ulcers were found in intestines in case 1 and case 2 but the mesenteric glands were not enlarged so the intestinal lesions could not be primary in nature. They were probably produced after the swallowing of bacteria in the sputum or secondary to septicaemia (34) shortly before death. · So we conclude that all the 5 cases were anthrax infection through the respiratory route (36). 4 of the 5 cases had proven lesions of hemorrhagic anthrax meningitis. The extensive diffuse subarachnoid hemorrhage and the lack of striking cellular reaction were characteristic of anthrax meningitis (33-40). In case 1 the brain was not examined at post-mortem but clinically the patient also had headache, vomiting, coma and nuchal rigidity, pointing to the presence of meningitis. The isolation of anthrax bacilli from heart blood, liver, spleen, lung, brain and other internal organs proves the presence of septicaemia. The death was evidently due to a combination of hemorrhagic meningitis and septicaemia. Anthrax is primarily a disease of herbivorous animals. The susceptibility of man to B. anthracis is comparatively low. So anthrax infection is rare in human beings (33, 37, 39, 41). The mode of infection in the human disease is principally contact with diseased or dead animals or their hides and excreta harboring anthrax bacilli. The usual form of anthrax in man is cutaneous infection. Infection through the respiratory route is extremely rare (35). Under natural conditions respiratory infection of anthrax is also related to animal hides and wool, so it is called wool sorters disease. In the 5 cases mentioned in this report no history of contact with such things could be elicited. Anthrax meningitis is a very rare disease. Lin and Chen (38) saw 2 cases from 1937 to 1946. They searched the world's literature from 1927 to 1940 and came across only ll.papers, mostly of single case reports. Most of the cases in the literature started as infection and the meninges were secondarily infected. Since 1940 such reports became even scantier in the literature. In 1947 Shanahan et al (42) reported 1 case, a rug worker who had anthrax meningitis following a malignant pustule of the upper lip. In China post-m6rtem and clinical statistics from medical colleges and their teaching hospitals likewise indicate the rarity of - 375 anthrax meningitis. The Shanghai Medical College had 1178 autopsies from 1928 tol952. The China Union Medical College performed 3942 autopsies from 1916 to 1952. In the National Medical College at Shenyang 1093 post-mortem examinations were done from 1940 to 1952. Not a single case of anthrax meningitis was found among the total member of 6213 autopsies in the above mentioned three institutions. Professor Wu Tsai-tung saw only 1 case of anthrax meningitis in 21 years of his career as a pathologist and the patient he saw had definite contact with hides before the onset of the disease. Therefore there is no doubt that this disease is very rare in this country and abroad. In Man Ching, Shenyang, Liaoyang, Anshan and Eastern Shuang-shan Village, Chang-shan district of Antung Hsien anthrax of skin was rare in the past. Anthrax infection through the respiratory route and anthrax meningitis had never been observed. In the teaching hospitals of National Medical College, Shenyang from 1949 to June 1952 only 1 case of skin anthrax was seen among 1,400,000 out-patients. means that anthrax is very rare in Shen-yang area. In the Liu-erh-pu district including the town of Liuerh-pu and 35 villages, with a total population of 61,372 persons in 14,029 families no case of anthrax has ever been discovered in the past 10 years. In Anshan City, from Jan. 1950 to March 1951, among the 760,212 patients of all the public and private hospitals and clinics, not a single case of anthrax was observed. Anthrax was rare not only in man but also in animals. In Shenyang only 6 cases of animal anthrax were recorded in the past 5 years but has :not been seen in the past 3 years. According to the records of the Veterinary Hospital and the personal records of the veterinarians at Liu-erh-pu anthrax has not occurred among 4646 domestic animals under their care in the whole district in the past 10 years. A similar result was obtained on investigation into the animal diseases in Anshan. In the recent years, there was also no anthrax among domestic animals in Man Ching station and Eastern Shuang Shan village. This is not just an accidental occurrence, it is the result of widespread use of prophylactic vaccinations. ··' Conclusion ( 1) Anthrax was a rare disease in man and animals in Man-Ching, Liao- yang, Anshan, Shen-yang and Antung Hsien in the past. But from March 19 to April 16, 1952, within a short period of less than one month, cases of anthrax infection through the respiratory route occurred in succession. This is an extremely unusual phenomenon. - 376 - (2) B. anthracis was isolated from insects and other objects. diss.eminated · by American airplanes. The same organism was obtained from brain and other internal organs of persons dead from anthrax infection. The biological and biochemical properties of B. anthracis from insects and pathological materials were entirely identical. (3) All the 5 cases of human anthrax infection had manifestations and lesions of the respiratory system indicating that the infection was introduced through the respiratory route and produced fatal results through septicaemia and hemorrhagic meningitis. (4} Summing up the facts mentioned above, we conclude that the human anthrax infection was produced by the bacteriological warfare of the American government. References Entomology (housefly) Entomology (Ptinus fur) Bacteriology Pathology 1-13 14-20 21-32 33-42 Entomology: (1) M. A. Tokunaga 1943 Medical Entomology Vol. II, page 853-1410 Clinical and Experimental Society, Osaka. (2) Essig, E. 0., 1942 College Entomology, New York. MacMillan Co. (3) Li, H. H. and Feng, L. C., 1951, Morphological studies of the common housefly, Musca vicina, in China. Pek. Nat. Hist. Bull., 19 :278-284. (4) Meng, C. H. and Winfield, G. F., 1938, Studies on the control of fecal-borne diseases in North China. V. A preliminary study of the density, species make up, and breeding habits of the house frequenting fly population of Tsinan. C.M.J. Suppl. II.: 483-486. (5) .......... , 1941, Studies on the control of fecal-borne diseases in North China. XIII. An approach to the quantitative study of the house frequenting fly population. A. The estimation of trapping rates. Pek. Nat. Hist. Bull. 15: 317-331. (6) ...•..•... , 1941a, Studies on the control of fecal-borne diseases in North China. XIV. An approach to the quantitative study of the house frequenting fly· population. B. The characteristics of an urban fly population. Ibid., 15: 333-351. - 377 - (7) .......... , 1942, Studies on the control of fecal-borne diseases in · North China. XV. An approach to the quantitative study of the house frequenting fly population. C. The characteristics of a rural fly population. C.M.J., 61A: 18-19. (8) .......... , 1943, Studies on the control of the fecal-borne diseases of North China. XVI. An approach to the quantitative study of the house frequenting fly population. D. The breeding habits of the common North China flies. Ibid., 61A: 54-55. (9) .......... , 1943a, Studies on the control of the fecal-borne diseases in North China. XVII. An approach to the quantitative study of the house frequenting fly population. E. The food preference of common North China flies. Ibid.,. 61A: 104. (10) .......... , 1943b, Studies on the control of the fecal-borne diseases in North China. XVIII. An approach to the quantitative study of the house frequenting fly population. F. A preliminary study of the life history of Musca vicina Macquart and Chrysomyia megacephala Fabricius. Ibid., 61A: 161-165. (11) Patton, w. S., 1932, Studies on the higher Diptera of Medical and veterinary importance. A revision of the species of the Genus Musca, based on a comparative study of the male terminalia. I. The natural grouping of the species and their relationship to each other. Ann. Trop. Med. & Parasit., 26: 347-405. (12) .......... , 1933, Studies on the higher Diptera of medical and veterinary importance. A revision of the species of the Genus Musca, based on a comparative study of the male terminalia. II. A practical guide to the Palaearctic species. Ibid., 27: 397-430. (13) Wu, C. F., 1940, Catalogus Insectorum Sinensium Vol. V, Diptera and Siphonaptera. Peking: Fan Mem. Inst. Bioi., 524 pp. Entomology (Ptinus fur): (14) Li Feng-sun, 1952', Economic Entomology of China Vol. II. No. 1 Hunan Agricultural Series. (15) Hsin Che-liu, 1951, Pests of Grains and Their Products in China Page 138 Commercial Press Ltd., Shanghai. ......... 378 - (16) Kuan Chih-ho, 1952, Pests of Stored Grains and Their Control, Na- tural Science Vol 2. 73-81. (17) Cotton, R. T. 1947, Insect Pests of Stored Grain and Grain Products. Burges pub. Co., Minneapolis, Minn. (18) Hinton, H. E. 1941, The Ptinidae of economic importance. Bull. Ent. Res., 31 : 331-381. (19) Patton, W. S. 1931, Insects, Ticks, Mites and Venomous Animals of Medical Importance, Part II Public Health. H. R. Grubb. Ltd. (20) Reitter, E., 1911, Die Kafer des Deutschen Reiches in Fauna Ger- manica. Bd. III. Bacteriology: (21) Breed, R. S., Murrey F.G.D., Hitchins, A. P. 1948, Bergey's Manual of Determinative Bacteriology, 6th edition, Williams and Wilkins Co. Baltimore, pp. 719-720. (22) Cao, G. 1906, Sui passagio dei germi a traverso le large di alcuni insetti. Ann Igience sper., 16, 645-664. Cited from Steinhaus, E. A. 1947 Insect Microbiology, Comstock Publishing Co., Inc., Ithaca, New Y01·k, p. 53. (23) Eurich, F. W. and Hewlitt, R. T. 1930, Medical Research Council. A system of Bacteriology in Relation to Medicine, London, Vol. 5, Chapt. 10, p. 448. (24) Graham-Smith, G. S. 1912, Forty-first Ann. Rept., Local Govt. Bd., 1911-1912, Suppl. Rept. Med. Office, 304-329, 330-335. Cited from Steinhaus, E. A., 1947 Insect Microbiology, Comstock Publishing Co., Inc., Ithaca, New York, p. 54. (25) Heim, F. 1894 Du role de quelques coleopteres dans la dissemination de certain cas de charbon. Compt. Rend. Soc. 58-61. Cited from Steinhaus, E. A., 1947 Insect Microbiology, Comstock Publishing Co. Inc., Ithaca, New York, p. 55. (26) Nuttall, G.H.F. 1899 On the role of insects, arachnids, and myriopods, as carriers in the spread of bacterial and parasitic diseases of man and animals, a critical and historical study. Johns Hopkins Hosp. Repts. 8: 1-155. - 379 - (27) Raimbert, A. 1869 Recherches experimentales sur la transmission du charbon par les mouches. Compt. Rend. acad. Sci., Paris, 69: 806-812. Cited from Steinhaus, E. A., 1947 Insect Microbiology, Comstock Publisping Co. Inc., Ithaca, New York, p. 53. (28) Rosebury, T, and Kabat, E. A. 1947 Bacterial Warfare. J. Immun. 56: 7-96. (29) Young, G. A. Jr., Zelle, M. R., and Lincoln, R. E. 1946, Respiratory · pathogenicity of B. anthracis. I. Methods of study and observations on pathogenesis J. Inf. Dis. 79: 233-246. (30) Zelle, M. R., Lincoln, R. E. and Young, G. A. Jr. 1946 II. Genetic variation in respiratory pathogenicity and invasiveness of colonial variants of B. anthracis. Ibid. 79: 247253. (31) Lincoln, R. E., Zelle, M. R., Randles, C. I., Roberts, J. L. and Young, G. A. Jr. 1946 III. Changes in pathogenicity due to nutritional modifications. Ibid. 79: 254-265. M. R. 1946, IV. Chemical-biological (32) Young, G. A. Jr. and synergism Ibid. 79 : 266-271. Pathology: (33) Anderson: Pathology, C. V. Mosby 1948. p. 241. (34) Aschoff: Pathologische Anatomie, Achte Aufl.age, Erster Band, Jena, Gustav. Fischer, 1936. s. 158. (35) Boyd: Textbook of Pathology. 5th -edition, Lea and Febiger, Philadelphia, 1947. p. 189. (36) Henke u. Lubarsch: Handbuch der speziellen pathologischen Anatomie und Histologie III/1. Erster Teil Berlin. Julius Springer, 1928. s. 806. (37) Kraus u. Brugsch: Spezielle Pathologie und Therapie innerer Krankheiten. Berlin u. Wien, Urban u. Schwarzenberg. 1919 s. 473. (38) Ling, C. C. and Chen, Y. S. 1948: Chinese Medical Journal 66: 4:n434. (39) Moore: Textbook of Pathology, Saunders, 1947. p. 229. (40) Muir: Textbook of Pathology, 3rd edition, Wm. Wood 1933. p. 722. (41) Panton and ¥arrack: Clinical Pathology, 5th edition J. & A. Churchill, 1945, p .. 107. (42) Shanahan,· Griffen and von Aversberg, 1947. Am. J. Clinical Path. 69: 319-322 . I .- 380 - DOCUMENT AA-1 REPORT ON BACTERIOLOGICAL EXAMINATION .OF FEATHERS 1. Specimen No.: 17006. 2. Source of specimen: sent from Peiching Village, Antung City. 3. Date received: March 15th, 1952. 4. Kind of specimen: Feathers. 5. Procedures: The feather was put in a mortar, washed and ground. with sterile physiologic saline. The washing fluid was injected intraperitoneally into 2 mice, each receiving 0.5 ml., and was cultured on plain agar plate and blood agar plate for bacterial isolation. Smears were also made for microscopic examination. (1) Under microscope, no bacteria were seen on the smears. (2) The cultures made from the above-mentioned washing fluid on agar plate and blood agar plate resulted in growth of grayish white, irregularly edged and rough-surfaced colonies. Stained smears revealed large-sized bacilli with square ends, lined up in chains. There were centrally placed spores in the bacteria. Gram positive. No motility. Pure cultures were made from this kind of colony. (3) 0.5 mi. of the above-mentioned washing fluid was inoculated into the peritoneal cavity of two mice. One of the mice died after 24 hours and the other died 36 hours afterwards. The spleen smear was stained and examined under microscope. There were found encapsulated large Gram positive bacilli with square ends. Cultures yielded the same kind of bacteria. (4) Examination of the pure cultures. I. Morphology: (1) . Cultured on plain agar plate and meat broth, the bacteria are large bacilli with ·square ends, lined up in chains, with centrally placed spores. No motility. Gram positive. (2) Direct smears made from heart blood. liver and spleen of the dead mice and guinea pigs inoculated with this bacteria showed Gram positive encapsulated large bacilli. - 381 II. Cultural characteristics: (1) turbid. Plain broth: Growth with flocculent sedimep.ts, . broth not (2) Plain agar plate: Grayish white colored opaque colonies with irregular edges and rough surfaces. Under hand lens the periphery of the colony was curly-hair in appearance. (3) Gelatin: Stab culture grew like an inverted fir tree. Very slow liquefaction of the gelatin. (4) Blood agar: No hemolysis. (5) Milk: Coagulated. III; Biochemical characteristics: It fermented glucose, maltose and sucrose without gas formation, but did not ferment lactose, mannitol, dnlcitol, inositol, xylose, arabinose and salicin. Hydrogen sulfide negative. Indol reaction negative. IV. Hemolysis test: 2% suspensions of red blood cells in normal saline were prepared respectively with blood from rabbit, guinea pig and goat. Bacteria suspension (1loopful in 1 ml.) was mixed with the red cell suspension and kept in an incubator for 2 hours and control test was done with B. subtilis. The results are shown in the following table: Suspension of bacteria isolated 2 Rabbit Guinea pig Goat V. B. S'ILbtitis (control) No hemolysis Hemolysis No hemolysis No. hemolysis Hemolysis Hemolysis Pathogenicity test:· (1) Mouse: Died in one day after subcutaneous injection of %, loopful of the bacteria, and in three days after injection of 1/100 Smears of liver, spleen and heart blood showed the presence of encapsulat.ed Gram·positive large bacilli with square ends. (2) Guinea pig: Died in two days after subcutaneous injection of 1/4 loopful of the bacteria. · Autopsy findings: gelatinous exudate at the site. of injection.. Spenic enlargement. Smears of liver, spleen and heart blood showed the presence of Gram positive encapsulated large bacilli with - 382 - square ends. General and biochemical characteristics of the bacilli isolated from these organs were identical with those isolated directly from the feathers. VI. Immunological characteristics : Ascoli test. The liver and spleen of mice and guinea pigs died after inoculation were ground separately,. and their respective suspensions were made. These suspensions were immediately boiled for 30 minutes in water bath and filtered through filter paper. Precipitation reactions were carried out with these _filtrates and diagnostic anti-anthrax serum. Control matreals included the liver and spleen of healthy mice and guinea pigs and also normal rabbit serum. The results are as follows·: Boiled Antigen Boiled filtrates o:t visceral organs of healthy animals --·--,um Mice ·------ I Guinea pigs Mice ' Saline Guinea i Diagnostic anti-anthrax <+> <+> (-) (-"i (-) Normal rabbit. (-) (-) (-) <---> (-) serum serum. Conclusion: Bacillus anthracis isolated from the specimen of feathers. by: Ching Kuan-hua, M. B. Assistant Prof. of Bacteriology, National Medical College, Shenyang. Chao Cheng-lin, M. B. Assistant Prof. of Micrology, Harbin Medical College. Reexamined and Reported by: Hsin Chun, M. D. Chief Technical Expert of Northeast Epidemic Prevention In" stitute. Date of Report: April 25, 1952. DOCUMENT AA-2 REPORT ON ENTOMOLOGICAL IDENTIFICATION OF HOUSEFLIES Serial No. of specimen: 13,033 I Original No. of specimen: j Date received: March 22, 1952 Circumstances of discovery: At 10 p.m., March 14, 1952, American military planes invaded the area of Ssuping municipality. On March 17, inhabitants of San Ho Village, Ssuping municipality found large quantities of :flies at San Tao Lin Tze, outside the village. ·-- Results of identification: Scientific name: Musca. vicina Macquart (Diptera, Muscidae) Common name: Housefly. Comments: Houseflies usually inhabit the vicinity of houses and animal enclosures. In the Northeast they do not appear outdoors until May. Their sudden discovery in the cold weather of March in open fields must be attributed to the activities of American aircraft. Remarks: Identified by: Ch'in Yao-ting, Professor of Biology, National Medical College, Shenyang. Feng Lan-pin, M.B., Lecturer, Department of Medicine; National Medical College, Shenyang. Date: March 24, 1952 DOCUMENT AA-3 REPORT ON BACTERIPLOGICAL EXAMINATION OF HOUSEFLIES Date of receiving specimen: March 18, 1952, at 5 p.m. Kinds ·of specimen: Houseflies. · Source of specimen: Ssu-ping City. Procedure and Results.: 20 flies, crushed with a pair of sterile forceps were used for the following. bacteriological tests. 1. Direct smear: Large Gram-positive spore-bearing bacilli with square ends were seen. 2. Culture on agar plate: After incubation for 18 hours, there was a growth of grayish white colonies with rough surfaces and irregular edges. When examined with a hand-lens, the edge was in the form of curled hair appearance. These colonies were taken for further examinations as follows : A. Direct smear examination showed Gram positive bacilli with square ends and arranged in long chains. Hanging drop examination showed no motility. B. Culture in meat broth: After incubation for 18 hours, there was flocculent sedimentation ·at the bottom Of the tubes. The supernatant liquid was clear. Microscopic examination of the direct smear revealed also Gram positive bacilli in long chains. Hanging drop examination showed no motility. C. Culture in peptone water revealed the same findings as in broth. No indol formation. D. Intraperitoneal inoculation with the suspension of the pure culture into 3 white mice and 3 guinea pigs 0.2 ml. of the bacteria suspension was used for each mouse and 0.5 ml. for each guinea pig. They died one After another from 26 to 42 hours. On autopsy; smears made from the spleen revealed the same kind of Gra!ll-positive E. Cultures of the heart blood of' these dead animals yielded pure groWth of the same bacteria • .Preliminary . Diagn,osis: ·BaciUus .antl'tracis isolated from the hou$efl.ies .. Examined by Chang Yu-chia Ssu-ping Health Station Date of report: March 23, 1952. ' '·' • i ' I ' - 385 • • • , . ,. · , REPORT ON BACTERIOLOGICAL IDENTIFICATION 1. Nq. of specimen: 13033 ,_, 2. Source of specimen: A strain of bacteria isolated from the house. flies at Ssu-ping, sent by Anti-epidemic Station of Ssu-ping. 3. Date Recejved: March 30, 1952. 4. Procedures: The following examinations were done after receiving the specimen: I. Morphology: Gram-positive large bacilli with square ends and central spore formation. Lined in the form of a chain: Hanging drop examination revealed no motility of the organism. II. Cultural Characteristics: 1) Plain agar plate: The colonies appeared rough and opaque with uneven edges and curled-hair appearance. 2) Meat broth: Growth with flocculent precipitation. The supernatant fluid clear. No pellicle formation. 3) Blood agar plate: The appearance of colonies was the same as on the plain agar plate. No hemolysis. 4) Gelatin media: Stab culture showed inverted iir tree,like growth in 24 hours, while liquefaction took place gradually several days later. 5) Milk media: Coagulated III. Biochemical Characteristics: A pure culture of this strain was used for the following examinations. The results were: Indol reaction (-) Lactose (-) Glucose Maltose Sucrose Dulcitol (-) Mannitol Xylose Inositol Arabinose Salicin Hydrogen sulfide <+> <+> <+> IV. (-) (-) (--:-) (-) (-) "(-) Hemolytic Reaction: Three 2% saline suspensions made respectively: with the red blood cells of goat, guinea pi¥ and rabbit; Each of these suspen!dons was then mixed with the .bacterial susp_ension (1 loopful/ml.). They were incubated for 2 hotirs and examined for hemolysis. Control examinations were made ·with Bacillus subtilis. The results were as f9llows: - 3.86 - suspension Present specimen B. 81Lbtitis no hemolysis hemolysis no hemolysis no hemolysis· hemolysis 2% R.B.C. Rabbit Guinea pig Goat hemolysis Pathogenicity Tests: A saline suspension was made with the present specimen., of bacteria (2 loopful/ml.) .Into each of the two white mice, 0.2 c.c. of the suspension was injected intraperitoneally, and into each of the two guinea pigs, 0.5 'c.c. were injected subcutaneously. Mice died in 30 hours and guinea pigs died in 3 days. Autopsy on these dead animals revealed enlargement of both liver and spleen. From smears of the heart blood, spleen and liver, Gram positive square ended large bacilli with capsule formation were . observed. On cul, ture, same kind of organism was obtained. VI. Serological examinations: A piece of liver and spleen of the dead guinea-pig was each ground separately and· diluted with· saline. After boiling for 30 minutes, Ascoli precipitation test was carried out with the filtrate. The filtrates of the liver and spleen of a healthy guinea-pig were used as controls. Boiled organs of healthy animals Boiled anti:getns a Liver Anti-anthrax diagnostic serum for Ascoli test Normal rabbit serum ·.., Spleen Liver Spleen + - - + - - Saiine - ' Conclusion: Bacillus anthraeis confirmed. Hsin tCh:iin• 'M.D. Chao 'LintM.B•.r•.. c., · Reported····· by · Rsin · Chilif "1\I.D., · · Exalhi:Q-ed· ·' •,, I' .• ·'' Chief Technical Expert of Northeast Epidell}ic Prevention Institute. Date of report: ' April 15, 1952. \ ·,., 387. " DOCUMENT AA-4 REPORT ON ENTOMOLOGICAL IDENTIFICATION OF PTINUS FUR Serial No, of specin!.en: Chien-362-1 l Original No. of specimen: Fang-023 I Date received: April 6th, 1952 Circumstances of discovery: Discovered on March 20, 1952, at Liaoyang.. Prior to the discovery, the inhabitants of the place had seen a reddish object dropped from the air. Large numbers of insects of this species were discovered immediately afterwards outside houses on the walls and in the fields. Results of identification: fur Linn. (Coleptera, Ptinidae) Scientific .name: , Common name:. Ptinid .beetle . . Comment: Ptinid beetle is a· pest of warehouses. Under natural conditions, they· are ·found mostly in storehouses, granaries, :fl.our mills, or other places for manufacturing or storing animal or plant products. In the present instance, large nu:inbers were suddenly dis· covered outside houses on the walls, in the :fields. This is evidently unusual Remarks ,r:Y. : i i i i ' Identified by: Liu Chung-lo, B.A., Ph.D, Director of the. Entomological Research Institute, · Peking College of Agriculture. Lu Pao-lin, ,M.S., Assistant Professor. of Department of Entomology, Peking College of Agriculture. Date: April .30, 1952 ' -.-. 3,$8 - DOCUMENT AA-5 REPORT ON BACTERIOLOGICAL EXAMINATION OF PTINUS IflJR Serial No.: 362-1 Date received: April14, 1952. Kind of specimen: 6 ptinid beetles (Ptinus fur) Source of specimen: Liaoyang Method of examination: Six ptinid beetles were washed in 5 cc sterile physiological saline. The washing fluid was inoculated on plain agar plate, blood agar plate, S.S. agar plate and cooked meat broth. The beetles were then ground in a sterile mortar with 5 cc of a mixture of serum and meat broth and the suspension was plated on different media as before. Results of examination: These plates were incubated for 24 hours. On the plain and blood agar plates, 2 kinds of colonies were observed. One was rough surfaced, elevated and grayish white in colour with an irregular edge. The diameter of each colony was 2-3 mm. Smears made from these colonies revealed Gram positive bacilli, with square ends, and lined up in chains. The other kind of colonies was round in shape, also elevated and greyish white in colour, but the surface was smooth and the edge regular. Smears revealed Gram negative short bacilli. On the S.S. agar plate, there was growth of colorless and transparent colonies. In the cooked meat broth there was a turbid growth with production of gas. Smears revealed both Gram positive and negative. bacilli. Gram positive bacilli From the blood agar plate the bacteria were transferred to serum broth. Observation made 6 hours later revealed no motility. The bacteria were then again inoculated into the following sugar tubes and special media. The results were as follows: I. 1. Bouillon tube-growth with flocculent sediments. 2. Milk-coagulated. 3. Methylene blue reduction test-negative. 4. Sugar fermentation reactions: Fermentation took place in the tubes containing glucose, maltose and sucrose with production of acid but no gas. There was no fermentation in the tubes containing lactose, mannitol, dulcitol, inositol, xylose, arabinose and salicin. - 329 - 5. No production of indol or HsS. 6. He:p:J.olytic test-no hemolysis. 7. Animal inoculations: A 24 hours' pure culture on agar slant was washed with 5 cc of .sterile physiological saline and 0.5 cc of the suspension ·was injected subcutaneously into a guinea-pig. The animal died in 24 hours. Autopsy revealed the spleen enlarged to about 3 times of its normal size. At the site of injection there was gelatinous exudate. The smears made from the heart blood and local exudate both showed Gram positive. encapsulated bacilli. Culture of the heart blood revealed the same kind of organisms. Meanwhile the bacterial suspension was diluted to 1: 10 and 1:100, and 0.3 c.c. of each preparation was injected into 3 white mice. They all died within 20-30 hours. On autopsy, the spleen was found enlarged to 4 times of its normal size. On the surface of liver and spleen, there were hemorrhagic spots. Direct smears made from the peritoneal fluid, liver, spleen and the heart blood revealed Gram positive encapsulated bacilli with square ends and cultures revealed the same kind of bacilli as before. A 24 hours' pure culture on an agar slant was again washed with 3 cc of physiological normal saline and 0.5 cc of the suspension was injected subcutaneously into a sheep. The animal died in 44 hours. Direct smears made from the heart blood, meninges, lungs: and spleen all showed Gram-positive bacilli with capsule formatiozL From the bacterial cultures the same kind of organisms was obtained. 8. Ascoli's Test: Positive II. Gram negative bacilli (1) Blood agar plate: very small, grayi:sh, elevated colonies of pinhead size. No hemolysis. Edge regular and surface smooth. China blue plate: Transparent, pinkish, well grown. Semisolid agar: showing motility. (2) (3) Indo! (-), Citrate ( +), Liquid base, turbid, Reduction of nitrate(+), Methyl red (-),No liquefaction of gelatin, V. P. test(-). (4) in_3 days. _ (5) injection (0.5 cc) into a white mouse-no death Ferrn..e:titation· Reactions·: - 390 Glucose, maltose-Production of acid but no gas. Lactose, sucrose, mannitol, xylose, arabinose, dulcitol, salicin and inositol-no fermentation. Conclusion: . 1. BacillUs anthracis isolated from Ptinus fur. 2. Gram-negative bacilli, not belonging to the "intestinal group." Examined by Wang Chin-tung Reporte(l by Hsieh Shao-wen (Samuel Zia), M.D. Professor of Bacteriology Chang Nai-chu, M.D. Assistant Professor of Bacteriology China Union Medical College. Date of Report: April 23, 1952. 391 ._..., DOCUMENT AA-6 REPORT ON BACTERIOLOGICAL EXAMINATION OF AUTOPSY MATERIAL FROM TIEN CHENG-HO 1. No. of specimen: 168 2. Source of specimen: Antung .3. Date received: April 19, 1952. 4. Kinds of specimen: Heart blood, spleen, lung, kidney and brain of Tien Cheng-ho. 5. Procedure: After receiving the specimens, smear examination, isolation culture and animal inoculation were immediately carried out. The following results were obtained. (1) Microscopic examination of the stained smear: From the brain, spleen, lung and kidney, large Gram-positive bacilli with square ends W.ere found. (2) Isolation culture: (a) The heart blood, spleen, lung, brain and kidney were streaked separately on plain agar slants. After 24 hours incuba· tion at 37°C, large grayish white colonies with uneven edges and rough surfaces were seen. Microscopic examination of stained smears made from these. colonies revealed Gram-positive and spore· bearing large bacilli. The morphology of the bacteria in the colonies from various cultures was identical. (b) The heart blood, spleen, lung, kidney and brain tissue were inoculated into meat broths for enrichment. Microscopic examination of the stained smear on the next day revealed large Gram-positive bacilli. Isolation of culture was made on plain agar plates and morphologically identical bacilli were obtained from various enrichment media. (c) The brain tissue was made into a 10% emulsion with normal saline and 0.2 ml. of this emulsion was injected subcutaneously into a white mouse. The mouse died 12 hours later. At autopsy, hemorrhagic spots were seen on the heart, liver and spleen. Microscopic exap1.ination of the stained smears made from these organs revealed encapsulated large bacilli. The same kind of bacilli was obtained from the agar slant used for isolation culture. - 392 ... (3) Ascoli precipitation test: A piece each of liver and spleen of the dead white mouse was minced separately with 5 mi. of sterile normal saline. Each of the suspension was boiled for 30 minutes and the supernatant clear fluids were used for Ascoli precipitation test against anthrax diagnostic serum. The results were positive. ( 4) Pathogenicity to guinea-pigs. The bacteria isolated from the brain tissue was made into a suspension (a slant of culture was mixed with 10 ml. of saline) and 0.5 mi. of this suspension was injected under the abdominal skin of a guinea pig weighing 750 gms. The guinea pig died 95 hours later. Autopsy :findings and bacteriological culture: Gelatinous and bloody exudate were seen at the site of injection. The heart, liver, spleen and lung were congested with hemorrhagic spots. Microscopic examination of the stained smears made from the above mentioned organs revealed Gram-positive encapsulated large bacilli with square ends. Isolation cultures made with various organs on the agar plates grew large colonies with curled hair-like ·edge. Microscopic examination of the stained smears revealed Gram positive large bacilli. 6. Conclusion: From the heart blood, brain, spleen, lung and kidney, Bacillus anthracis was isolated. Examined by Sun Ching-ch'ang, Laboratory of Antung Health Bureau. Date reported: April 25, 1952. REPORT ON BACTERIOLOGICAL IDENTIFICATION 1. No. of Specimen: 38040 2. Source of Specimen: A strain of bacteria isolated from the heart blood of Tien Cheng-ho, sent by Laboratory of Municipal Health Bureau of Antung City. 3. Date Received: May 17, 1952. 4. Procedure: The following examinations were done after receiving the specimen: 393 -... I. Morphology: Gram-positive large bacilli with square ends and central spore formation, lined in the form of a long chain. Hanging drop examina. tion revealed no motility of the organism. II. Cultural Properties: 1. Plain agar plate: The colonies appeared rough and . opaque with edge uneven and curled hair-like. 2. Meat broth: Growth with flocculent precipitation. supernatant fluid clear. No pellicle formation. The 3. Blood agar plate: The appearance of colonies was same as those on the plain agar media. No hemolysis. 4. Gelatin: Stab culture after 24 hours incubation produced inverted fir tree-like growth which liquefied gradually several days later. 5. Milk: Coagulated. III. Biochemical Properties: A pure culture of this strain was used for the following biochemical examinations: Indo! reaction (-) Lactose (-) Glucose Maltose· Sucrose Dulcitol (-) Mannitol Xylose Inositol Arabinose Salicin Hydrogen sulfide <+> <+> <+> (-) (-) (-) (-) (-) (-) IV. Hemolytic Reaction : Three 2% saline suspensions were made respectively with the · red blood cells of goat, guinea pig and rabbit. Each of these suspensions was then mixed with a bacterial suspension (1 loopful/ml.), incubated for 2 hours and they were examined for hemolysis. Control examinations were made with Bacillus subtilis. The results were as follows : suspension Present specimen .2% R.B.C. Rabbit Guinea pig no hemolysis no hemolysis no hemolysis I Goat - 394 __... Control test with B. Subti!is hemolysis hemolysis hemolysis v. Pathogenicity Tests: A saline suspension was made with the present specimen of bacteria (2 loopfuls/ mi.). Into each of the two white mice, 0.2 ml. of the suspension was injected intra peritoneally, and into each of the two guinea pigs, 0.5 nil. was given subcutaneously. 1Mice died in 18 hours and guinea pigs died in 48 hours. Autopsy on these dead animals revealed enlargement of both liver and spleen. From smears of the heart blood, spleen and liver, Gram positive square ended large bacilli with capsule formation were isolated. On culturing, similar organisms were obtained. VI. Serological Examinations. A piece each of liver and spleen of the dead guinea-pig was ground separately and diluted with saline. After boiling for 30 minutes, Ascoli precipitation test was carried out with the supernatant clear fluids. The liver and spleen of a healthy animal were used for control. Antigen , Boiled antigen Boiled viscera of healthy animal Liver Spleen Liver Spleen Anthrax diagnostic + + - Serum of healthy rabbit - - - precipitation serum Conclusion: - Normal saline - - Bacillus anthracis Confired. Examined by Cheng Keng, Sc. D., ·Professor of Bacteriology, D'epartment of Veterinary Medicine, College of Agriculture, National Nanking University. Reported by Hsin Chiin, M.D., Chief .Technical Expert of Northeast Epidemic Prevention Institute. Date Reported: June 2, 1952. - 395• - DOCUMENT AA-7 REPORT ON BACTERIOLOGICAL EXAMINATION OF AUTOPSY MATERIAL FROM CHU CHAN-YUN 1. Specimen No.: 7. 2. Source of specimen: Ssu-ping City. 3. Date received: March 23, 1952. 4. Kinds of specimen: Spleen, lung and liver of Chii Chan-yiin, a small piece of each. 5. Procedure: (1) Microscopic examination of the direct smears made from the liver, spleen and lung revealed large Gram-positive bacilli with square ends and capsule formation. (2) Isolation culture: The· liver, spleen and lung were streaked separately on plain plates. After incubation at 37•c for 24 hours, there...grew large with curled hair-like edges. Microscopic . . . of the smears from these colonies 1.'1evealed largEl'Gram-positive bacilli with centtally placed spores. They showed no ing drop .. ( 3) Animal inoculation: The spleen was made into a 1:10 suspension, 0.2 mi. of which was injected into the abdomen of a guinea pig. The guinea pig. died 24 hours later and on autopsy, its liver and spleen were enlarged, with congestion and hemorrhage. Large Gram positive square-ended bacilli were found in these organs under microscope. were enlarged, with congestion and hemorrhage. Large Qram positive square-ended bacilli were found in these organs under microscope. ( 4) Ascoli precipitation test. A piece of the spleen in the human specimen and a piece each of the liver and spleen of the dead guinea pig were cut with scissors into fragments in physiologic saline. The suspensions were boiled for 20 minutes and the supernatant fluids, after filtration, were tested fo:r precipitation reaction with diagnostic anti-serum for anthrax. The results were all positive. Conclusion: Bacillus anthracis was isolated from the liver, spleen and lung specimens. Examined by: Chang Yii-chia, Ssu-ping Health Station Date reported: March 26, 1952. 1 396 --- REPORT ON BACTERIOLOGICAL IDENTIFICATION 1. No. of Specimen: 13042 2. Source of Specimen: A strain of bacteria isolated from the ·lungs of Chi.i Chan-yiin at Ssu-ping, sent by He.alth Station of Ssu-ping City. 3. Date Received: May 8, 1952. 4. Procedure: The following examinations were done after receiving the specimen: I. Morphology: Gram-positive large bacilli with square ends and central spore formation, lined in the form of a long chain. Hanging drop examination revealed no motility of the organism. II. Cultural Properties : 1. Plain agar plate. The colonies appeared rough and opaque with edge uneven and curled hair-like. 2. Meat broth: Growth with flocculent precipitation. supernatant fluid clear. No pellicle formation. The 3. Blood agar plate. The appearance of colonies were same as those on the plain agar plate. No hemolysis. 4. Gelatin: Stab culture after 24 hours incubation produced inverted fir tree-like growth which liquefied gradually several days later. 5. Milk: Coagulated. III. Biochemical Properties: A pure culture of this strain was used for the following biochemical examinations: Indol reaction Lactose Glucose Maltose Sucrose Dulcitol Mannitol Xylose Inositol Arabinose Salicin Hydrogen sulfide (-:---) (-) (+) (+) (+) (-) (-) (-) (-) (-) (-) (-) IV. Hemolytic Reaction: Three 2 %. saline suspensions were made respectively with the req blood cells from a goat, a guinea pig and a rabbit. Each of these suspensions was then mixed with a bacterial suspension (1 loopful/ml.). They were incubated for 2 hours and examined for hemolysis. Control examinations were made with BaciUus subtilis. The results were as follows: 397 - . : Present specimen R.B.C. suspeusion Cohtrol"test· with B. su.btitis · Guinea pig no hemolysis no hemolysis · hemolysis hemolysis Goat no hemolysis hemolysis Rabbit V. Pathogenicity Tests: A saline suspension was. made with the present specimen of bacteria (2 loopfuls/1 ml.). I,nto each of the two white mice, 0.2 mi. of the suspension was injected intraperitoneally, and into each of the two guinea pigs, 0.5 ml. were given subcutaneously. Mice died in 20 hours and gui:q.ea pigs died in 48 hours. Autopsy on these dead animals revealed enlargement of both liver and spleen. From smears of the heart blood, spleen and liver, Gram positive square ended large bacilli with capsule formation were observed. On culture, same kind of organisms was obtained. VI. Serological A piece each of liver and spleen of the dead guinea-pig was ground separately and diluted with saline. After boiling for 30 minutes, Ascoli precipitation test was carried out with the supernatant clear fluids. The liver and spleen of a healthy were used for control. Boiled viscera of healthy animal Boiled antigen um Liver Spleen Liver .· SI>leen + + - - - - - Anthrax diagnostic precipitation serum Serum of healthy rabbit · Normal saline - Bacill'U8 anthracis Confirmed. Conclusion: Examined by Cheng Keng, Sc.D., Professor of Bacteriology, Department of Veterinary Medicine, College of Agrictl,lture, National Na11king University , ' ' Reported by Hsi;n Chiin, M.D., Chief Technical Expert of 'Northeast: Epidemic .. ·''Preyention · Institute. · ' . ., . ',, ,'i' .• Date of Report: May 20, 1952. :' . ... . DOCUMENT AA-8 ;, ., " REPORT ON BACTERIOLOGICAL . EXAMINATION OF AUTOPSY MATERIAL FROM 'VEl LIU-SHIH 1. 2. 3. 4. 5. No. of specimen: 498 Source of specimen : An-shan. Date received: April 16, 1952. Kinds of specimen: Brain tissue of Wei Liu-shih. Procedure: Right after receiving the specimen, isolation culture and animal inoculation were· carried out. When suspicious colonies were found, they were taken for pure culture examination and animal inoculation. Result: From the brain tissue, Bacillus anthracis. was isola:ted. 6. Examination of the Pure Culture: (1) · Morphology: Gram positive spore-bearing bacilli with square ends, those from the animal body showed the presence of capsules and those from the culture media were lined up in long chains. No motility. (2) Cultural Properties: Blood agar plate: Big and rough colonies with uneven edges, non-trans.parent and non-hemolytic. · Plain agar slant: Rapid growth. Rough surface. Semitransparent. Bouillon: Flocculent growth at bottom, supernatant fluid clear. (3) Biochemical Properties: Milk-coagulated. Gelatin liquefied with inverted fir tree-like growth. (4) Pathogenicity in animals: White mouse (18 gms.)-Subcutaneous injection of 0.3 mi. Died in 16 hours. (5) Immunological Properties: Ascoli' precipitation test-positive. Bacillus anthracis was isola ted from patient's brain· tissue. by Liu Shih-ming, Technical Expert, Dairen Health Research Institute. ·Reported by Chu Chi-min_g, M.B., Ph.D., · · · Chief Technical'Expert, National Vaccine· and Serum Institute, 'Peking. · 7: Conclusion: Date of report: April 27, 1952. - 399 ij.EPORT ON SUPPLEMENTARY EXAMINATION OF BACILLUS ANTHRACIS 498 1) Biochemical properties: It fermented glucose, maltose, sucrose with production of acid but no gas. It did not ferment salicin, lactose, ·:p:J.annitol, dulcitol, inositol, xylose and arabinose. H.S- negative Indol- negative . Hemolytic test : No hemolysis of the blood cells of goat, rabbit and guinea pig. 2 Present Specimen Control Specimen no hemolysis no hemolysis hemolysis hemolysis no hemolysis hemolysis .. .. Goat Rabbit Guinea pigs 2) (B. Subtilis) Pathogenicity test on guinea-pig: A loopful of the pure culture was mixed with 5 mi. of normal saline. 0.5 mi. of this suspension was injected into each of two guinea pigs subcutaneously. Both animals di-ed in 4 days. Autopsy findings: At the site of injection, there was gelatinous exudate. The liver and spleen were enlarged. The lungs were congested. There were .Gram positive. square-ended and encapsulated large bacilli in these organs and B. anthracis was isolated from the heart blood. · · 3) Liver and spleen of dead guinea pigs were used for As coli precipitation test-result positive. Boiled normal tissue control antigen . Anti-anthrax diagnostic serum for Ascoli test Normal rabbit serum I Liver + i - 400 - Liver Spleen - - Saline - Conclusion: Bacillus anthracis confirmed. · Examined by· Cheng Keng, Sc.D ., Professor of Bacteriology, Department of Veterinary Medicine, College of Agriculture, National· Nanking University Date of Reported by Hsin-Chlin, M.D., Chief Technical Expert of Northeast Epidemic Prevention Institute. June 1, 1952 ....... 401 _. DOCUMENT REPORT ON BACTERIOLOGICAL EXAMINATION OF AUTOPSY MATERIAL FROM WANG· TZE-PIN 1. 2. 3. 4. No. of specimen: 422. Source of specimen: North District of Shenyang. Date received: March 25, 19.52. Kind of specimen: 'Brain tissue, heart blood and spleen of Wang Tze-pin. 5. Pro.cedure: Right after receiving the specimen, isolation culture was carried out. When suspicious colonies were found, they were taken for pure culture examination and animal inoculation. Result: From the heart blood, brain tissue and spleen Bacillus anthracis was isolated. No other organism. 6. Examination of the Pure Culture. (1) Morphology: Gram positive spore-bearing bacilli with square ends; those from the animal body have capsules and those from the culture media form long chains. No motility. (2) Cultural Properties: Blood agar plate: Colonies big, rough, non-transparent, not hemolytic and edge uneven. Plain agar slant: Growth rapid. Surface not smooth. Semitransparent. · Bouillon: Flocculent precipitation at bottom, supernatant fluid clear. (3) Biochemical Properties: Milk-coagulated. Gelatin-liquefied with inverted fir tree-like growth. Pathogenicity to animals: White mouse (18 gms.)-Sub· cutaneous injection of 0.3 ml. Died in 36 hours. (5) Immunological properties: Ascoli precipitation test positive. 7. Conclusion: From the heart blood, brain and spleen Bacillm anthracis isolated. Examined by .Liu Shih-min, Technical Expert of Dairen Research Institute. Reported by Chu Chi-ming, M.B., Ph.D. Chief Technical Expert, · National Vaccine and Serum Institute. Peking. (4) Date of report: April 8, 1952. REPORT ON SUPPLEMENTARY EXAMINATION OF BACILLUS ANTHRACIS 422 1 Biochemical Properties : Fermentation tests: It fermented glucose, maltose and sucrose with production of acid but no gas. It did not ferment salicin, lactose, mannitol, dulcitol, inositol and H 2 S-negative Indol- negative Heyolytic test: bit and guinea pig: No hemolysis on the blood cells of goat, rab.. B. subtiii8 B. anthracis Goat Rabbit Guinea pig (control) hemolysis no hemolysis no hemolysis no hemolysis hemolysis hemolysis • 2 Pathogenicity test on guinea-pig. A loopful of the pure culture was mixed with 5 ml. of normal saline. 0.5 ml. of this suspension was injected into 2 guinea pigs subcutaneously. Both guinea pigs died iu 3 days. Autopsy findings: At the site of injection, there was gela· tinous exudate. The liver and spleen .enlarged. The lungs con.. gested. There were Gram positive square-ended and encapsulated bacilli in these organs. B. anthracis was isolated from the heart blood. 3 Liver and spleen of dead guinea pigs for Ascoli precipitation test: positive. Boiled antigen um Anthrax diagnostic preclpitation serum Serum of healthy rabbit Liver + ...... Boiled viscera of healthy animal Spleen Liver + ...... - ·- 403 - - Spleen -- saline • - - Conclusion: Bacillus anthracis confirmed. Examined by Cheng Keng, Ph.D., Professor of Bacteriology, Department of Veterinary Medicine, College of Agriculture, National Nanking University. Reported by Hsin Chtin, M.D., Chief Technical Expert of Northeast Epidemic Prevention Institute. Date of report: June 1, 1952. - 4:04 - DOCUMENT ·AA-10 AUTOPSY REPORTS ON FIVE FATAL CASES OF ANTHRAX INFECTION CASE NO. 1 Chii Chan-yun male age: 55 Name: Residence: Man-ching Station, Ssu-ping City, Liaohsi Province Occupation: Railway worker (8 yrs.) Summary of Clinical History: Present illness: In the morning of March 19th, 1952, he began to have fever, headache, and aching of the extremities. On March 21, he had nausea, vomiting, general malaise, heaviness in the head, pain in the joints and insomnia. In addition, he had cough and the sputum was found t< contain encapsulated large Gram positive bacilli. White blood cell count. 28000. After admission, he vomited repeatedly. Later he was discovered to have rigidity of neck and mental cloudiness. He died at 2:45: p.m. March 22, 1952. Date of autopsy: March 23, 1952, 24 hours after death. Summary of Autopsy Findings: External examination: Body moderately nourished. Finger nails <;yanotic. Superficial lymph glands not enlarged. Skin, no important change. Peritoneal cavity : No abnormal findings. Pleural cavity: There is accumulation of about 1000 ml. of fluid in: both pleural cavities. Under the left pleura there is ecchymosis. Pericardial cavity: There is an accumulation of about 100 mi. of fluid. Heart: No abnormal findings. The heart is in the systolic stage. Lungs: Show edema ·and congestion, and there are prominent hemorrhagic spots in the left lower lobe and apex. In the left apex, an old bean-sized tuberculous lesion is found. Hemorrhage in the right lower lung is not marked. The upper lobes of both sides show areas of consolidation. Hilar lymph nodes enlarged. Liver: Normal in size and color. There is a rice-sized hemorrhagic. spot on the upper surface and a millet-sized hemorrhagic spot over the' lower surface. . Spleen: Enlarged. Size 20 x 12 x 5 em. Bluish gray in color. · Stomach and duodenum: Gastric mucosa shows areas of ecchymosis and that of the pyloric region shows hemorrhagic spots. On the duodenal . mucosa there are erosions. 405 SmalL Intestine: There are 8 ulcerative hemorrhagic areas distribut.. ed over the mucosa of the jejunum and ileum. The size of the bigger one is about 1 em. in diameter and the smaller one is about 0.5 em. in diameter. Appendix: There is hemorrhagic inflammatory condition at its tip. M esenterie lymph glands: Not enlarged. Pancreas: There is also an area of ecchymosis, about 2 em.. in diameter. Bacteriological examinations: Bacillus anthracis isolated from both liver and spleen. Autopsy performed by: Sung Teh-yii., Director of the Ssu-ping Health Station. Liu Chan-yuan, Physician of the Ssuping Health Station. Comment: From the clinical records, we may conclude that the patient had a severe generalized acute infection. Cough, leucocytosis and the finding of large Gram positive bacilli in the sputum clearly point to anthrax infection of the respiratory tract. Bilateral pleural effusion, hemorrhagic. spots on the visceral pleura of the lungs, pericardia! effusion, consolidation of the upper lobes of both lungs, enlargement of hilar lymph glands and splenic enlargement establish the diagnosis of pulmonary anthrax. The isolation of Bacillus anthracis from the liver and spleen speaks for septicemia. Clinical symptoms such as loss of consciousness and rigidity of the neck indicate meningitis. The ulcers and hemorrhagic spot in the mucosa of small intestines are probably produced by bacilli swallowed with the sputum, but as the mesenteric glands are not enlarged, it is apparent that the intestinal lesion is not a primary anthrax infection. Reported by: Wu Tsai-tung, M. B. Professor of Pathology, Nanking University Medical College. Li Pei-lin, M. B., Ch. B., Ph. D. Professor of Pathology, National Medical College, Shenyang. CASE NO. 2 male Name: Wang Tze,..pin Residence : Shenyang. Occupation: Tricycle-rickshaw driver. age: 47 Summary of Clinical History: Present illness: At 10:30 p.m. on March 20, 1952 the patient began to feel general discomfort. On March 21, he rested in bed. · His · ap· 406 petite .remained good and he was still able to move about. On March he experienced soreness of legs, general malaise, discomfort in the upper abdomen, nausea and headache. In the morning of March 23, he felt better. But at 5 p.m. he experienced constricting sensation all over the whole body and distension over the upper abdomen. He was mentally clear until 6 a.m. March 24, when he gradually developed mental cloudiness with aphasia and restlessness. He was then sent to the First Municipal Hospital at 9 a.m., where he was examined with the following findings: Temperature 36·c, Pulse imperceptible. Comatose. Pupils dilated with no reaction to light. Bulbar conjunctivae congested. Lips cyanotic; Mouth tightly closed. Heart sounds indistinct. Neck rigid. Kernig's sign positive. The patient died at 9 :35 a.m. on March 25, 1952. Autopsy was performed 5 hours after death. Summary of Autopsy Findings: The body is that of a stout man. Nutritional status good. External examination of the body, including the skin, revealf:l no abnormal findings. The right pleural cavity contains about 1300 mi. of yellow clear fluid and the left contains about 500 ml. In the pericardia! cavity, there is ap.:. proximately 100 mi. of light yellow clear fluid. Hea1·t: Weight 340 gm. No important pathological changes. Lungs: Right lung weighs 490 gms. The visceral pleura is smooth, except the portion near the mediastinum which shows definite gelatinous oedema. On palpation of the lung tissue, no consolidation is found. But on.the cut surfaces, thickening of the peribronchial tissues is seen. The interlobular tissue of the lower lobe also shows the same thickenings with edema. The parenchymatous tissue of the lung shows no remarkable change, except for edema of varying degrees. The bronchus and the bronchioles show mild degree of congestion but no erosion or ulcer. The tissues around the bronchus and the lower end of the trachea and the· adjacent lymph glands form a hemorrhagic mass measuring about 5 X 3 X 2.5 em. The surrounding hilar tissue of the lungs is edematous. Left lung weighs 340 gms. No important pathological change. Under the microscope, the tissues of the hilum of the right lung and the lymph glands show .marked infiltration with polymorphonuclear leucocytes and diffuse hemorrhage. In a small bronchus, the following changes are noted: Desquamation' of epithelial cells, edema of bronchial wall and infiltration with polymorphonuclear leucocytes.. The lung tissue, besides marked edema, shows no important changes. In the sections stained with Gram method there are large Gram positive bacilli in the wall Qf the small bronchus as mentioned above and in the neighbouring lymph - 407 - glands. In the interlobular tissue of the right lower lung there are marked edema and mild infiltration with polymorphonuclear leucocytes and mononuclear cells. Intestines: In the upper portion of the jejunum there is a beansized hemorrhagic area with an ulceration seen in its center and red discoloration around. On the mucous membrane of the colon, there are also more than ten isolated pea-sized hemorrhagic spots numerous in the ascending colon. Under mie1·oscope, the mucous membrane of the intestine shows fibrin and polymorphonuclear leucocytic infiltration with· a number of Gram positive square-ended bacilli. The lymph tissue of the wall of intestine is normal with no polymorphonuclear infiltration. Mesenteric lymph glands normal. Brain: The dura mater shows no change. The blood vessels on the surface of the brain congested. There is marked diffuse hemorrhage in the subarachnoid space. The vessels at the base of the brain show no sclerotic change. The vessels in the meninges of the spinal cord are con. gested. Under the microscope, there are diffuse hemorrhages and polymorphonuclear and mononuclear infiltrations· in the subarachnoid space. The small vessels inside the brain are congested and in the perivascular tissue there are 'polymorphonuclear and mononuclear infiltrations and many foci of hemorrhage. The lumen of a blood vessel extending from tE.e meninges into the brain parenchyma is found to be full of bacilli. In the wall of that vessel there is polymorphonuclear leucocytic infiltration with perivascular hemorrhage. In the Gram stained sections many Gram positive square ended bacilli are found in the subarachnoid space. The liver, spleen and other organs show no important changes. Bacteriological culture: Bacillus anthracis was isolated from tbe heart blood, brain and spleen. Diagnosis: Hemorrhagic anthrax meningitis, anthrax bronchitis, peribronchitis and interlobular cellulitis of the right lower lobe. Purulent hemorrhagic anthrax lymphadenitis of the hilar lymph glands. Pulmonary edema with pleural effusion. Pericardia! effusion. Multiple necrosis and ulcerations of the mucous membrane of the intestine due to anthrax bacillus infection. Autopsy by Drs. Chu Feng-chuh and Wang Hung-lieh, Department of Pathology, National Medical College, Shenyang. Reexamined and confirmed by: Wu Tsai-tung M. B.. , Professor of Pathology, Nan, king University Medical College. Li Pei-lin, M. B., Ch. B., Ph. D. Professor of· Pathology, National Medical College, Shenyang. - 408 --- CASE NO. 3A female Name: Wei Liu shih Residence: Anshan age: 32 Summary of Clinical History: Present illness: The patient had repeatedly joined the work of catching and killing insects disseminated ·by American airplanes before contracting the present illness. In the evening of April 11, 1952, the patient felt slight general discomfort. On April 12, she experienced headache, chilliness and fever, but still carried on her household· duties. On Apri113, the symptoms increased in severity so that she became bedridden and had cough, chest pain, dyspnea and vomiting. Then, only with great difficulty was she able to go to the Second Combined Clinic in the Tieh-tung District for treatment. After she returned home, the symptoms became progressively worse. On April 14, she was sent to Anshan Municipal Tieh-tung Health Station where she was found in a state of mental confusion, delirium and restlessness, with labored respiration, frequent convulsions, imperceptible pulse, a pale face, cyanotic lips, tightly closed mouth and dilated pupils. Light reflexes of the pupils were sluggish. The neck was very rigid; Kernig's sign positive. Moist rales in the lungs were detected on both sides of the chest. She died at 11:50 p.m. on April 14. Autopsy performed 21 hours after death. Summary of Autopsy Findings: The body is that of a middle-aged woman, normally developed and moderately well-nourished. External examination shows no abnormal findings. Each pleural cavity contains about 1000 ml. of slightly turbid fluid orange in color. The pericardia! cavity contains 150 ml. of orangecolored fluid. Heart: Weight 230 gm. No pathological change. Lungs: On the back of the lower lobe of the left lung, there is a :firm area which measures about 4 X 3 em. in size. The cut surface appears to be dark red in color, within which there is a fan-shaped area measuring 2 X 1.5 em. and appearing grayish yellow in color. The bronchial mucosa is slightly congested. Microscopic examination reveals necrotizing pneumonitis and the presence of Gram-positive bacilli. The hilar lymph nodes are markedly enlarged to the size of a pigeon's egg. On section, hemorrhagic lesions can be seen on the cut surface of these lymph nodes. Microscopic examinations reveal hemorrhagic lymphadenitis and the presence of Gram positive square-ended bacilli. Liver: Congested. Spleen: Enlarged, weighing 400 gm. It is very soft in consistency. Post-mortem changes are very prominent.· - 409 - Gast1·o-intestinal tract : Shows no abnormal findings. Uterus: Gravid, containing a fetus measuring 15 em. in length. Brain and spinal cord: On the surface of the cerebrum in the subarachnoid space there are diffuse hemorrhages. In some areas the sulci and gyri can not be distinguished, being completely covered by blood. There are also diffuse hemorrhages on the surface of cerebellum and the base of the brain. Purulent exudate is not found. Diffuse hemorrhages are also present in the subarachnoid space of the spinal cord. The meningeal vessels a:r:e extremely congested. The blood vessels of .the base of the brain show no arteriosclerosis or other changes. Microscopic examinations reveal extremely prominent hemorrhages .in the subarachnoid space, being filled with large numbers of red blood cells. There is also infiltration of polymorphonuclear leucocytes and a small number of large mononuclear cells. In Gram-stained slides it is found in the a.bove-mentioned hemorrhagic areas numerous large Grampositive bacilli, some of which are arranged in the form of bamboo, morphologically identical with_Bacillus anthracis. Other organs: No significant changes. Bacte1·ial cultu're: Bacillus anthracis was isolated from the brain tissue. Diagnosis: Hemorrhagic anthrax meningitis, necrotizing anthrax pneumonitis of the lower lobe of the left lung, hemorrhagic anthrax lymphadenitis of the hilum. of the lungs, edema of the lungs, bilateral pleural effusion, pericardia! effusion and acute splenic tumor. The autopsy was performed by Drs. Chiang Ying-kai and Kuo Cheng. teh, of Pathology of the National Medical College, Shenyang. On reexamination of the pathological material we confirm the above diagnosis. Reported by : Wu Tsai-tung, M. B., Professor of Pathology, Nanking University Medical College. Li Pei-lin, M. B., Ch. B., Ph. D., Professor of Pathology, National Medical College, Shenyang. CASE NO.. 3B Wang· Shu-chih. Female Age: 23 Name: Residence: Liu-erh-pu, Liaoyang County. Occupation: Primary school teacher. Summary of Clinical History: The patient was healthy in the past, without any chronic illness. After the American planes had dropped insects, she participated in the - 410 - work of catching and killing insects. She worked tilllO p.m. every night. Two days before h.er death (April 6, 1952) she felt dryness in the throat with hoarseness of voice. At times she had headache and pain in her joints. She was still able to carry on her usual work until 9 a.m. on April s, when she lost her consciousness while she was in the toilet. This was discovered by her colleagues and she was moved to her own room. Her respiration was dyspneic, with white frothy discharge mixed with blood escaping from her mouth. Light reflex lost. Fingers cyanotic. She died at 10 :35 a.m. on the same day. Summary of Autopsy Findings: Time of autopsy: 24 hours after death. The body is well developed and well nourished. There is no abnormal findings on the surface of the body. Chest cavity: There is no excessive fluid in both· pleural cavities. The anterior surface of the right lung shows fibrous adhesions. The heart is 290 gms. in weight. The pericardium is smooth and shining. A few hemorrhagic spots are seen on the surface of the left ventricle. The endocardium is stained red by hemoglobin. The valves are normal. Both ventricles slightly dilated. Myocardium is soft. The anterior surface of the right limg shows fibrous adhesion, while the rest of pleura shows no changes. The posterior portions of both left and right lungs show marked congestion but no definite consolidation. Microscopically, besides the edema in most of the alveoli of the lungs, there is also polymorphonuclear leucocytic and monocytic infiltration in some of the alveoli of the right upper lobe. The mucous membrane of the bronchioles is detached, and in the lumens of the bronchioles there is cellular exudate. In the Gram stained slides, scattered large Gram positive' bacilli are seen. The two ends of the bacteria appear square. Inside: some alveoli and vesesls, such large bacilli are also s.een, but in areas without inflammatory changes no such bacilli are seen. Liver: Capsule smooth. Cut surface shows prominent cloudy swelling. A few Gram positive bacilli are also seen under the microscope in the hepatic sinuses. Spleen : No important changes. Gastro-intestinal tract and .mesenteric lymph glands: No change. Other organs : Also no remarkable change. Brain and spinal cord : There is diffuse hemorrhage in the' subarachnoid space of both brain and ,spinal cord. On the coronal section of the cerebrum, hemorrhages are also seen beneath the ependyma of the left and the 4th·ventricles. The artery of the base of the brain shows no sclerotic changes. - 411 - Microscopically, diffuse hemorrhage can be seen in the subarachnuid space. But the infiltration of the meninges by . polymorphonuclear leucocytes is very slight. In Gram stained slides, the small vessels of the brain and meninges contain Gram-positive bacilli. No bacteriological culture done. Diagnosis: Acute hemorrhagic anthrax meningitis. Bronchopneumonia of the right upper lung. Pulmonary edema. Autopsy performed by Drs. Chao Wen-tou and Wang Hung-lieh. Department of Pathology, National Medical College, Shenyang. . The autopsy findings point to acute hemorrhagic anthrax meningitis, bronchopneumonia of right upper lobe and pulmonary oedema. In the pathological sections large Gram positive bacilli with square morphologically identical with B. anthracis were found. So the diagnosis of anthrax infection is beyond any doubt. Reported by: Wu Tsai-tung, M. B., Professor of Pathology, Nanking University Medical College. Li Pei-lin, M. B., Ch. B., Ph. D., Professor of Pathology, National Medical College, Shenyang. CASE NO.4 Age: 44. Male. Tien Cheng-ho. First Section of the Eastern Shuang-shan Village, 5th District of Antung County. Occupation: Farmer. Clinical History: In the morning of April 16, 1952, after sending out 3 cartloads of manure into· the field, he suddenly experienced chilliness. He immediately returned home and rested. The symptoms at that time were generalized joint pain, fever and mild headache. Food intake was as usual. On April 17, symptoms became worse, he was still able to take food, but vomited twice. On Apri118, after dawn, he became unconscious. Both hands were. tightly clenched as inI spasm. A, local doctor named Lin Hou was sent for. Body T€mp. was 38.7·c. No definite diagnosis was made. He died on the same day (April 18) at 12, noon. Name: Residence: Gross Findings on Autopsy: Time of autopsy: April 19, 5:00 p.m. (29 hours after death) at the home of the deceased. The body is that of a male. Livor mortis over the back prominent. No generalized edema. No enlargement of lymph glands. Pupils 0.5 - 412 - em. in diameter. Conjunctivae pale. During the removal of clothes some fresh red fluid comes out from the nostrils. No fluid in the peritoneal cavity. Peritoneum .normal. Stomach markedly dilated. Gastric mucous membrane smooth. Under the serosa of the greater curvature of the stomach, there scatter a number of hemorrhagic areas about the size of finger tips. The surface of intestine shows no particular change. Mesente1·ic lymph glands not enlarged. Liver weighs 1200 gms. Surface smooth and grayish purple, showing postmortem autolysis. Cut surface dark purple with· also autolytic changes. Gall bladder shows no inflammatory changes. Spleen weighs 200 gms. a little bigger than a palm, measuring 7 finger-breadths in length and 5 finger-breadths in width, dark purplish red in color. Surface smooth, cut surface was purplish black. The tissue also shows evidence of autolysis. Each kidney weighs 150 gms. The right, kidney is 6 fingerbreadths in length and 3 finger-breadths in width. Surface smooth and dark red. Cut surface dark purple and the tissue also shows post-mortem autolysis. The left kidney shows similar findings. In the pleural cavity, there is no inflammatory exudate. The parietal pleura shows no adhesions. There is 15 mi. of dark red fluid in the pericardial cavity. Epicardium rich in fat. The heart is in a relaxed state with a blunt apex. The myocardium also shows postmortem autolysis. No other changes seen. normal. Weight 250 gms. Lungs dark bluish gray, and cut surface purplish black showing hemorrhagic changes. The right lung· weighs 850 gms. the left lung 700 gms. The hilar lymph glands are about the size of a thumb. Cranial cavity: Dura mater normal. Under the leptomeninges, there is diffuse hemorrhage. Subarachnoid space filled with blood. Cut surface of brain showed no hemorrhagic spots .. No inflammatory exudate in the cranial cavity. I In summarizing the above findings, it may be said that the body shows very marked postmortem changes, hemorrhages in subarachnoid space, in serosa of stomach and in the lungs and there is enlargement of the hilum lymph glands of the lungs. Results of Bacteriological Examination: (1) Direct smears of the brain tissue, lungs, spleen· and kidneys stained with Gram method reveal numerous Gram positive large bacilli. (2) From bacteriological culture and animal inoculation of the heart blood, brain tissue, lung, liver, spleen and kidney Bacillus anthracis was isolated. - 413' - Based on the above findings, the diagnosis is that the patient died of anthrax bacillus septicemia. Examined by Sung Wei-yi, Superintendent of Liaotung' Municipal Hospital. Kao Chuan-li, Physician of Antung Municipal Hospital. Wang Chun-lin, Laboratory Technician of Antung Municipal Hospital. The organism · isolated from this patient was reexamined and con:firmed as Bacillus anthracis by bacteriologists Hsin Chtin and Cheng Keng. The clinical manifestations fit in with the diagnosis of anthrax septicaemia and meningitis. Post-mortem examination revealed lesions of hemorrhagic mening-itis. The gnding of B. anthracis from direct smear and culture of internal organs would confirm the diagnosis of septicaemia. There was increase of weight of both lungs. Cut surface showed congestion, hemorrhages and oedema. Hilum glands were enlarged. The primary focus was evidently in the lungs and the infection was introduced through the respiratory route. Reported by Wu Tsai-tung, M.B. Professor of Pathology, Nanking University· Medical College. Li Pei-lin, M.B.. Ch. B., Ph.D. Professor of Pathology, National Medical College, Shenyang. ·-· 4 U - SUMMARY OF 5 CASES OF ANTHRAX INFECTION Chi.i Chanyun 2 Wang Tzepin 3a Wei Liushih Male Male Female ,Female Male 55 47 32 Railway worker Tricyclerickshaw driver House wife 23 Primary school teacher April 6 1952 44 Farmer 1 Case number Name Sex Age Occupation March 19 March 20 April 11 1952 1952 1952 A.M. Night Night March 22 March 25 April 14 April 8 1952 1952 1952 1952 at 2:45p.m. at 9:35 a.m. at 11:50 p.m. at 10:35 a.m. 24 hrs. 5 hrs. 21 hrs 24 hrs. after after after after death death death death Date of onset of disease Date of death Time of autopsy + + + + + - + + + ....Ill Meningeal +' + u + - + :1: + + + + + Fever a Headache ....0 General aching Cough IJ d Ill E Nausea and vomiting irritation 'E ;=: Coma <.1 Ill :a d q:: Abdominal pain Pneumonia or bronchitis Pulmonary congestion and oedema ' Enlargement hilum glands Pleural effusion ....'lj Pericardia! effusion 0 'd Hemorrhagic meningit,is Ill A. Intestinal ulcer Enlarged me. senteric gliUlds ,, .. + + + 29 hrs. after death + + + + - + + - - + + + .+ + + + + ' + + ·+ + + ..... - ' - .. + + + + + - - Aprill8 1952 at 12 noon + - + ,_ .... April 16 1952 -· - ;! ·Skin. aathrax: .. 3b 4 Wang Shu- Tien Chengho chih i .. + -- ··-· 4li: - + ,. I ... + .. ,. . .- - + .. : + ...... - ' - +' -. ---- •, .. , DOCUMENT AA-11 Epidemiological Investigation of Five Fata1 Cases of Anthrax Infection Case No. Name I I_ 1 I Chli Chanyun 55 Age \ Sex Residence I Male I / I Wang Tze-pin Wei Llu-shih I / Man-ching Station nearby Ssuping / _ _ aa 2 I 47 Male I 32 ·r / City di"Stric; of Sh•nyongl I I City district of A""h'n l I 3b Wang Shuchih --23 I Female Anthrax o! Anthrax o! resp. system, resp. system, anthrax (brain and meninges not meningitis examined). 1- - - - -: · - --- Anth1:ax of resp. system, anthrax meningitis Liu-erh-pu Town of Liaoyang Hsien History of · No No contact with furs or leather ·-=-='------+-------+_____, History of using new tooth-brush or shavingbrush Not used Not used No - - - ho · I 44 Male - East Shuang. shan Village, 5th District An tung l-0-c_c_u_p_a t_i_o_n_-\-_R_a i_l_w_a_y_-71- T-r -ic_y_c_lc- -House wife worker rickshaw school driver t eacher Disease deDurin g exter- During How infected During exexveloped terminntion mination of termination ptinid bee- of ptinid beeo:f flies drop- aiter the dissemination tles dropped tles dropped ped by by American by Americnn by American American airplanes of airplnnes airplane airplnnes insects and objects in the Shenyang District Pathologiaal diagnosis 4 _____, During disposal of the feather dropped by American planes - · - -- -1 Anth1·ax of resp. system, anthrax meningitis Anthrax ol respiratory system and anthrax meninl!itis No No Not used Not used - Not used - --------- --;----- -) Although he History ot Only a donwas a farm· contact with k ey in the diseased far- district of No tnrming animal in the neighbourhood er, no history of con· ming animals Manching Station, not tact sick No Any domestic No No No No animals suffering from anthrax . - ·Any neighbours sufferBe!ore and alter their death, no neighbour sufl'ercd from any type ing from ot nnthrax anthrax No No No No l Disposal of the corpse I .I By incineration after autopsy 416 Fig. 1. Chart showing the course of the four American planes intruding over Lung-WangMiao and Pei Ching Village on March 11, 1952. - - - -·· 1; ( ..3J Jt Fig. 2. Chart showing the localities of PeiChing-Tzu and Luan-Shih-Shan-Tzu in Antung where American planes dropped feathers on March 11, 1952. l Fig. 3. Samples of feathers dropped by American planes. Fig. 4. Chart showing the course of an American plane B-26 intruding over Ssu-Ping and Man-Ching on March 14, 1952. ,J .i Fig. 5. Chart showing the course of two American F-86 planes intruding over Liu-ErhPu on March 20, 1952. ;. J;l : 1' .t ak t j•!l :: F -UG !#! 1{ x · ii5 -rf"A ·1/r '5L ® Fig, 6. Chart showing the course of two American F-86 planes intruding over Liu-ErhPu on March 27, 1952. lll 0 0 0 l I " Ji "·\ ..... ,. I l I i ,i\p \ <•··!, ·.._jfl •-·" J J' I /I I /, f .• '' ' Fig. 7. Map of Liu-Erh-Pu, Liaoyang Hsien. ".FJ '£ 1 .z 3 ,., 4 s ::£ G '\ l'l) ,· / :,, ,....... / 0' ·If:]·= L6 Fig. 8. Map of Liu-Erh-Pu in Liaoyang Hsien w here American planes dropped objects. (Explanation: 1. Location where the falling object was seen; 2. Village Government; 3. Location where Jen Wan-Ku discovered the objects; 4. House of Jen Wan- Ku; 5. Location where Wang Yung-Chang discovered the objects; 6. House of Wang Yung- Chang at Ah-Lao-Chiao. E ij .l. 0 £·1 .X ffl F-f,(; tu1 1t ;v@ '1 \"T ..;- 7-ldb Fig. 9. Chart showing the course of two American F-86 planes intruding over Sha-HoTzu, Shen-Yang area on March 7, 1952. j_ ]:} i\< +.;. EJ i·JJ. F -(lfi .rl.r) 1': 1'f. :?LIB l!'ig. 10. Chart showing the course of two American F-86 planes intruding over FengChi-Pu, Shen-Yang area on March 13, 1952. Fig. 11. Bacillus anthracis stai ned with methylene blue. Fij,{. 12. Fig. 13. Colonies of B. anthracis. Smear of the spleen of an infected mouse, showing capsule formation of B. anthracis. Fig. 14. Cut surface of the left lung: Hemorrhagic lymphadenitis in hilar region due to infection by B. anthracis (Case 3A). / Fig. 15. ....... Bronchial mucosa (Gram stain) of a case of bronchitis due to B. anthracis (Case 2) showing numerous anthrax bacilli. F'ig. 16. Purulent and hemorrhagic lymphadenitis of the hilar lymph node due to B. anthmcis (Gram stain) showing large numbers of anthrax bacilli (Case 2). Fig. 17. Hemorrhagic meningitis due to B. anthracis. The cerebrum s:!lows extensive and diffuse hemorrhages in the leptomeninges (Case 3A). Fig. 18. Hemorrhagic meningitis due to B. anthracis. Coronal section of the cerebrum showing diffuse subarachnoid hemorrhages (Case 3B). ,. I' ' ' .. Fig. 19. Hemorrhage meningitis due to B. anthracis. Besides diffuse hemorrhage, there is also inflammatory cellular infiltration in the leptomeninges (Case 2) . Fig. 20. Hemorrhagic meningitis due to B. anthracis (Gram stain). Large numbers of Gram-positive anthrax bacilli in the leptomeninges (Case 2). ••• .l<'ig. 21. A • small blood vessel in the brain containing B. anthracis (Gram stain) (Case 3B). - -il- . 1"'11 In <1 IGO II\ r CJ $111Hfl; ···· · ·-·-·--------· I SKI\---··- L _____ Fig. 22. Plan of Man-Ching Railway Station and its neighbourhood. I< 'JV. . 7:.ltli ... J'":, l t•, Fig. 23. Statement by Prof. C.H. Hu that there has been no case of anthrax among 3942 autopsies since 1916 in the China Union Medical College (formerly P.U.M.C.). I I ' - ' ... l -. .iJ. , '.11) ... --.;... "'{. - . f ..'· :!:.. I + - 7h. li J Tt ,_ .... :rf ;. 1-tt r I I } ::. i) .,.,. -- 1Jf :2. ...... 4:- f; · . ,; • . ' \ ' ""'tf <-·1 ' I Fig. 24. Statement by Prof. C.Y. Ku that there has been no case of anthrax among 1178 autopsies since 1928 in the Shanghai Medical College. ' The Figs. (25a, 25b, 25c) are concerned with Zelle's research work on bacteriological warfare cited in Zinsser's "Textbook of Bacteriology", 9th. ed., 1946. Z!JVSSER )S TEXTBOOK OF B .A C T E ·R. I 0 ,L 0 G Y 'l' llt: 0}' 1\ ,\('T t :K itii .OC:V AXU J;l \l: ... o:-.l:t, :O. t' \o:(' ll· l l ' Tlll'W1\ l' Y ,\ l'\ 11 I:S' I• ; :t 1101 /.') S1' J'UJ.:XTS ,\XU FOil OY TO 'I'Hl; Pitt'. \'1-:S 'fiUS 01-' .\ X II ITIIJ.U.' llt-:.\I.TH HEI' 1SJ.:I1 llY DAVID T. SM ITH , M : D . 11\ ' Jli\t .'II0\ 10 \ ,tH..\' .A"\D l•ft()t't:...!\fiH Of :O.It-.IIH :JNE !Hl)m Ullo\\'I ' HHn Ot' ]) 0 N A L 0 S. r.I A 1\ Tl N, M . D : , Ill Ill P. ri. \•Ill \ 1, :-.;'l't\' l , ).JhOI('IN I AS I> l'lil\1.\C llt;A I.1'11 A:O.:O 1\Af 11· HIOLO(.'li', IA'I I'Ht WEl'SOH I'll" \11'\I \ 'E I\.SITY SCIIOOJ. 01' !o. ti: I)IC. I't: NO lH.IAl\' F . CONAI\T , ·PH.D. I'HOFI ''till f)l ' }.1\C:ni.(K!Y ASU J)I IL.l' I'TI1 {OX 1.1•,\\t;}. nn:.: uzo.; l\' \: 11,;1'1\' St'I II W' tl . Of' !o.II.O: rtiCI"f-:. \1:\' 1\Y A. POSTON. l ' \ l l\l ' t ' lllll 1.._ )ti\f. II : IIIOI .OI . \ , !JIIldl l •!\ 1\t: IISII\' ,_u toOI . HI 'II 'OWt"'l. I ·A lNC. :-iE\\' YOIIK Fig. 25 a. t:ACll .l.l,; ·\ \'1'1111\G< 1'-P \\1111\\\ pl1,1i1. 'lll•IUI. ttiut! kwilli: llu ·H·(\IP', tlw 111111 He!-iiMancc .-· Ht·t HH"'' n( iJ .. j .. l'\1tt·llto h ,, ... ,.f,t tll t' tln•tfl"' 1,, ... ;\ 1'1' d... , ,,. ·,nl h\ill ,I fjl\ 1::11 lt IH• IJllll+' tl'- ... f!t'lh uf .1 h'IIIJU ' t ;.tlliP' ftotm.dillJ t. ll•t• •. t11 '11\ilu tu no·u l lbr tlwtni ,,d ,, . ..,j .. ! ,IHt il !llh q, j., , ...... f·J!li.ll (:"-It in. ("' p.llft .. f.. nu-.. l•.!' t•·ritt. ••I IWI tlh• .. f nf :""\ l 1.. iu tlrir1\ tnimak... -. f.IIP f••l 11!,111\ \I 'H I " llilhltll{ J•.H \•'J!d,t l t\r· ... p•·ll''" 1!11·if \litl1ilit\ I•• k•·pt 111.n \t th•Uft l :t t1tl \\ biJ,. liwt' ' ,1n .. 111 /h1 • ..It'f 1 witl '" ' .f,n \•f ntd\ 1,... ,,,... I" th• it1 t t\." tht""·· ('." rn"''' ,. !toblinuu,., 1 :200U. qr ;llll"' 111 fut t l mi-(trtlr!i' ll ir•t! in mn-..itnuh, cf,,,., .. , , :i I''''' ,.,·nt. penicillin, .:;a 1u·r '"nt. und streptomytln. •n pt•r ' ' 'ttl ,,f !he· Hlf,•, lhl .m im;,)!f ! \Jil11·r rJ nJ .. V nriabi Hty. - \ ' irukui i\H tlu!i x h,w\lli prnclwr Hllll!h dtl i •llunit• .... IJ'"'" \ ind•'hl vr l\1 111\ lflllrnt. "111tHtth (:-\I . ntu• +lid I \1 I. illlll ;.!4 •tlitlinl I L 1 furfll' dt·· JiJ:l'n. Uu t<;,,,ti.t. I'J.l t : Jl'.''! h ,IK o-.t'nl'iltlt·/4 t I tJ ht' .. >,jll;. \ ar in!'"' llwt rtf' tt''' J1j,_ .,».arl..w1 l\t' H' , .. 11n · t,,!l } .ltl.q•tnl r'• :"l'!.l'IU!'.lJ. .'!!!''I... 'J)u• ,tHt' llllflll'd ... Voltlt· h i'.lr- II' Ut h\ nl!ti\,!11111[ t\w .. tH .11 ,t I1'111Jk'l .llt111' u£ 12c. tu 1'\ ( :. \H ' It' .t.-.p!!rn:,!L'llttlf:-o, 'Ji w i.t! hn\1 ,., •·r. t lnl ill tho • \ 1lt<
  • oSi 8rit. J, Exp. P•t'll ., 1939, 20:11W, ' A. M. A""· d• f l,$l, PMtchr. 19-W, ?O:I29 i 1945, 71138.1; 12:58, Can:tpt. r &nd. bi•1. d. 1877. 2:2'17. _ . - . CHYKY, and Uim.o. Mht . .. d... cntJN-te., 18114. - - •ncl W01.11'0UIO:IIt. Milt. •. 4. l. c....u.-.... 1181. r. ........ c. J. In/m. fJi• .. 19-10, 67 :193. :zor.. )lcCOLo.nocu , K.. aod Aoam:ac, P. 11... J. Clia. PW... 1!141. 17 •1Sl. Afn a.tn, E. S., Soon, E. A , Not., II. A., MAD1!'1 , S. H., and T. f. l.lrunUul ol., NtsN' '-'TU, W: J. . /. ht/rtc.l, J.>;, .. 1929, 44:73. 1 } \sr&u", CHAirU£Jtr ..H tD, Md AnrJ. G. u.. 1881, 9"l. 1-'ut. LAI'tllEM. '1'1trtcl}ahr f. Dtat.w:l.e AlrJ .. 1855, 8:103, ltul. f. llahlu/111., I AL1.. 1929, 110 :102. S<;UAft , ......_ , _ _ .. _.,...,....., . S3:37 l R.•• ,... • • ·' Fig. 25 c. The following Figs. (26a to 26k) show the reseach works of Zelle and his co-workers on the production of a variant of B. anthraci" ad,apted for respiratory infection (J. Infect. Dis. 79, 1946). , Journal of lrlfectio·us. Diseases t:.. un.ltJ I Q0-1 b_v IAt JoAn R«1tftlln ur JIHW7l!.Jil'l'• WI! 1.11\M IIURROWS · J)/IN H. CAMPBW. ·G. M. DACK ·It W. HARRISON CLAY G tHJH' • STEWART A. KOSER • KARL F. MEYER C. PHIWP •IIlLER · F. 0. NOV\' 79 . 1946 ··: I'UULI>IIHJ TilE JOHN JtOCKU'BI..LD Of TI!E UNIVIUI'I'r OP ,cHICAGO . MI!MORIAL FUND l'lll<'!\(!0 · ILLJNO\S · U.S.A. Fig. 26 a. ·. : Tfl E LIN! VERSITY OF CHICAGO PRESS ., .. • .. RESPIRATOR\' I'ATIIOCEN ICITY 01:· Ut!Cil.l.li S .1 N111 R.IIC!S I. MP'rJIODS (ift" GrOitGJ< ,\ . VOW JR.., (r.AI'l'. VC, K. ZI .I.I .F., ( LT.(J <;,) th S• ) • •\\1> (t,T. RAU•u E. 1.1NCot 1NTROt.WCTmN .. In J.!t'Otraf. variation in Of tlliY spccirs fl't1111 3 lwr"ditr. C:n\f Barilltu unthrncir opotc• will pres•·nted in !his and sub· H•qtll·nl papers. The mcthoda oi iuvt•Stig'ntiOII US<'rl thrtJU!{hOUt tl1c ics nnd llliflCrvations un the gcmsis of anthra"- Ifill Lt: in tlu· j>rcot·hl pllpcr. A V<;ry et)mplf•t(: tn·:ttit...! on a.nlhr:\'< h.l-, been prt·f''-' nt..·d by Solrernhcim' w!ti<'h includc'-<>wr L,OOO 6pecific oo t>ncc·n t.l.!l "''II mention of a sinnlnr p:\pus withnul spt:·c:ific ol the disuoe in· tltt! nnd lf\(lrf> work c•n r,• .. piratc•ry anlhrax :lrl: considt!rt'fl. lluclont"'' fuund ir vo••ibl:, w infrct miC•!. J,tuinea and rnbbit& bv iCtg tltt:!se:. r1nirnnlr. l() iulw.lt:. ;,llllhrJ >: . pon·:,. This W3B (Mt·c l: ntlt•rk:nl who \\ t19 abh: to in.lt:cL shee-p ir• til t! :,,11/lt.' TJ1i.'i iwport:uH t '"'tH'k s hnwtd that infec·,ion mu ld lw c•tnhl islwd tl• et al. •-• Thl'sc L'Ondudt·rl that while it was edrncOiill :tniln.a1s wlth anihrJ.x infection of tht.· lu ng w;u, l•:l'ih aftt·r rh• lttn\:, h.ul hr·t·n by iull :tlation of t"hlnrin,· The rcaulr.. in tht· prd·t·nL t•·\)Wr fur the IOO!>!t p..trt wilh nf till' l·,trJj,:_-rwork<.·rs. Huw"-'\'t'r, the llll'thod u( JAl!')Ure Clnpln)'td. \\ hich l"··rmih rt·.l· acrura.l f• mt•.t::-un.:nh·nt l•l clo:<.aKc, and tlw analysi$ of the Unrn ru:tk\.· it JHI'!i..ctiblt· 1n cornp:1re t he rt:..spir::u ory JMthoKtnici"· of Wffen:nt' .11HI tlw suttt:('(Jll· H.t.'("t!ivtd lor (IUhJirAtil)tJ )\IM f5, 1H6. • C..'lrl'lrd nut nt C:U'I'Ip f)fttrick, Frtlder- id.:, Maryland fr'•IU Dtl""tD1btr 194,3 thtwgh Au$;,U i t 11)-iS. 1 1\oll•·, W., on(.·d til withd raw a ,..:u n plc Th · uuu rh• tl•H!tl fvr fht: dch·nni nation o t \.11 111 1 ltld. {,,J < r .. 1...., lh \ \!'I t ' IJl,jl!t• fl.j,;Jr, sUfllWIIt"iu n.., \\ vr•· uf llh.' COll C\.:II I r:H inn Q( ••• .1.1 .air tn the .\ !1 ·thrr \\ .t:, tu a me re:llr\' m.,nom'" ,•r tor t'flnt nJIIing the llf t dtll!l l .r ·Ill'' If• I \\ith flll'll!il\,d,Jt <.Uf• I'C I 1(•1 r lth•T 11 r •'ltit·;lf!l•ll •; \\'••n· 'l j>t·ui n::,:-. f nr ,:pt·l,,h'a •I •• .. , 1 )\ 1'1 •lo ·•tilt r l•l .til ll/1 l'oorJil.illtl liiiJl t •ull i•!• 1\L.' Wt•u,l \':lht·ll'l 1wl uli."•'l • •. i'• \ r Fig. 26 c. • 246 I.J!OkGF r\ . Y <>tJSG, jR., R, An •·xposun· nwthod for producing t•\.pt·ritncnlal re::.;r'lira tory unthrax itt ()( :mim.lltt is rl csrri iJt!d . This t•x pnsun• lt•chuic pltt'i statistical tn·atnhmt C\f tht· n.•:m lting dnta makt·s pn&'iiblc ·quJn tit.;t ti \'t' t·umparison of the n· lntjve pa th oge nic·ity or d ifTt•rt·n r thra:\ and nf tht• com· 11arativc foiUht't.'ptihility of ani1n1l SJH:'t it•i\. 1\ RAI.PR E. follow to:: Attivc hypL·rcmia with M:JilH' cauSed by int'r<·a,.-d capillary r.ermeability; pr(\'R'Iln· o£ h3d lli in all vct.tk'ls. Jncl to somt t•xtt• n 1. in th<· .tl \leola r walls. The urigin.II or invasion of the cc.HJ id nul Uc rlch·r· mined hi.tologically. Invasion of tlw hosJ by iuh.>lt-d ,.,.. thra.x iR Sh(IWU lt'J thrtl\ll:(h th,•lymphat.ic •y•wm. studiC1J m;l(ll.· ou Lhl' or- prcs.·nt in the luug• anrl i"<'fl· hronchinl lym ph nod,·• th,>t highly pa thc.tgt>nk ,tn: mnr<.· invnsivc than spon.:to of moctcor;at rP•pir;t· lory p.lthogt•nidty in that Ut ·r .tr-:: prt!scnl in number, it, t in· llronchi.ll lyrnph nodl':-\ ;tnd an: ·tl'kl mort.: able to in rht• it-.•·lf. ·\d:n0\li,'(J5tffti·nl 'l"ht' .\uthnt" i .h '" l"\llff•·• tlu ir fm· 1lu. ,1 · nf .\. J.• MMn iu H\Uch nf thi• .... Fig. 26 d. : R !·.SI'Il II PATli(,GhNJCJTY A.t\& (,1•::-.;.FTh \'ARlA"tfUl\' lN ..,,.., 01< C"QI.ONJAL VAfO.\NTS OF 8. ltNTII/jCIS \1. R. Zbl.t.o-: (LT.(J.G.} KALl'llll. UNCOLN (L'J'. 11SNR), ANT> •EORG!l A. YOUNG, JR. (CAPT. VC, AV>) MATEftL\1.5 AND Vou iatinn in morphologir.ill and phy· .. thill'iH"lC.: d Stirt\ Of bac;tcria is .. •·osnmou ph•·t11Hl11!JWn. Rcvit!WS h. vc lon·n publi,lwd uy ll :ulley. ' ·' In .• nu·rcl.,tion bt·f·n observe-:\ bctwN·n \lruh·JH•· .lntl ,·olt,ny morphology with h• '-HIIutth Ptlony tyfk,·S t·xhibitinp; t.l11• ·.•rt·.tl•-r \lfUit•IH'•·. H, )\\'t·vt•r, BacillrH ontl:rcui., i!-1 n•gardtod as an ,.,,.·pti•ul i11 tiMr tiH· Nmfx> th ,,j till!-> np• dt·.., h.'"'' lwcn found to IH• \ irnknt 1h·HI tlw norrn.tl fonn-. !\uH).l.t'!.;lt·t·:t ha!:l rt:vit•wrcl 1h1• t·.trlit·l' and hns cl.u3silit·d n1lolli.tl \ ,tdanb of TJ. tm tfirnciS ilttCJ "0 111l' 7 ;\unge!oitf'r ronciU<.!I''S ll•.tl 1111 nr lit tlt• rull't•l;tliun Ut:twtcn tulnll)' t)"j•t· .tllrl \iruh'IH't' oc...:urs in H. 11 "tflr,ll i\ -.inn.• Ill' H'd bQ lh viru, h IIi ,tnd ;1 \'lfuh·nl rult urcs of the sanh· .tit hougl 1 nwk· 1 ( 1!t 111 j, 1 j 1 Ylw· S(• pniut Hit.: ,J l•f it, srudi1·d t:u llurcs of dif. f··rinJ! hut of • l) I lir 11J lh l' !lciii H: All wllures atudicd Wt'I'C. d"riwrwi:tt• indic3. l Cd. 'I h(· 1'111 l.O% pep!;C'I"' 0.6% {lJSP t ,' 1.\II•''.Hb O.OOIJO.\ F.,.<;O, •HI.U 0.0002 M CnCI, · 611.0 ne mcdiun1 w:HI eterifiz:clf at 110 C (Qr 20 min· ult:a, the being aterili.:.ed 51.1);\nltt·ly nntl udtkd aseptimllr t(') the rcn of 1ht> ml'dium. :;pore 6USpeu(iOnl \n·rt chef'\:cd for v.tri· atioo by obo;rrving upvrnnJs of 2SO UllOI' smra.rrd nutrif>nt tlg:.'lr: plalc!i. All v;llions of rnlony murpholt)ky am1 rpp':' _ ulllizing- mndc \\!th ,,{a 11 u1gnilico;ui•;u of 9X Tlw rncrhtlfl to rt·'>pirtlfi'\r}' owthrax. and trfol lll\f•nt It 1'\' 1' l>t:cn _ 1!1 duutl , 1• till' lllllll i H'r or J. lnfccl. o;,, 40: I. lnfccL Pi•. 60: ll9. t. W. ]. 1?29, J. Infec t. Oi•. 44 : 1.1, l, t;tcin, C. ·D 1944, Ant. J, Vt:t. Rc!'... S: .•8. J. in iipOft':\ rlu· !Y'f !0 •} \dtit·h ,,ill the h1rS miuull·,_ It HI SO l)r SO% h:t hal rt"!!pir.IINy .h I.R I. ...d 5()% IIH)f'ht!i f )' ,!fl\(ll!g auim:t/!' 10 l''\jl1.11o\Ht', :\1!11 3· •>•1 lrrmqh of 16 .lll i· for .1 7 I, Jl.,dh· .. , Phtlip 1927, 1, fl ,,d\e)'. Philip t9 H, uwr ho.l.; fli i!hJ.,uro- of f('Sj)ir:-ttory p.lthn· t\11{'11 piratory tctlt!l Wl'l'c l!•tti\,·tll•u ptEILik:tlinn june:. \\'.,,k ,·,,ni ... lcml .1 1 C'.rtnl fl llctriclc, Frederi ck. \!,l!d.tuol fnm• "'-plt'llllK'r, l9,4, through (Jf br lilt'r ;>{ t.tl t·nlmtial ''•td.l nt . ., '1\ t; n' mil'trb{:Of"' p,1t h l">gt•l}lt'lly Ulld /1',- 1/l l/11 , ... j \ 1d u/ /1 ;/uJitrrlril . y(·,,.,, (MJ/idll) o.oot "'M,.so, ·4H,O o.oooz .. •. rrv.o tOI0/1\' 1.\'IH' ;'lJ UJ SUlXU· • lit:-.1 p.lpo-r' , 11(·\oy>, 0.8%;\u('Oiit' 0.25% 0.03 M Kll,I'(J, O.ll.l• K,I!OP, Pt', T lw prt·r,l'J11 p;tJ.M'r pre·on t hl! rurrc l.l1l cmn pO!h ihn ot thi> IUC·d ium is yj(it-1\n· )n·l\\t·i PPY medium v.ere grown in in the third p.1pcr of fH.'ti t"l. • lsola tfu. l by Or. D. W. H•·:ul••r.. llll .tml IJr ·l. £. rt)llowing Il l\ of J.Oi · mal pill&:l!;M. • 5. Yo\liiJ, G. i\ ., Jr., M. H. und R. E. Lin· cofn 19-46, J. Cnrecl. Oia. 19 : 1.U--2.J6. 6. lintolll, R. E., M. R. 1.ell(', C. I. Runlilcs. ]. L. Robert<, nml d. A. \·ou"l!'· Jr. 1946, J, lnlec1. p;,, 79: 251-265. Fig. 26 e. ;1 :; i11•li• 1ti11 • t•t·nit it,. j., '-'OIIlt' 'iLrt I'\ I'IH'I'"o d l•·; ;, mt·•:t n•ll·• •d \', hi• h 't"llnn y tHor .. clo llH'Ih't•s in rt·:>ptra wt'n· oh--c•rvNl he · t '' f•tn t·ult of tlw sa me- rn1ony I Y!'H'. 'I ll(· nf tht! difff"n•nt-cs W.l.o; •·qual to that 11bs!·rvt.'d hd\\1'<'11 ,.,1rinnt 1)'1'""· !hone<·, it app<·ars Lh011 whift. PI !Iii -. Ill. lN DUI: 'f(J :O.:l/TI\; H). !'JHSflll I l' \:-PU·'( 1.:.NS.1 • J . H.IHII R1' ", )A ,\ ! • ffEOIH;v ,\ , EnvironnH!Ill mny affct·t th<'. ceJJ.s sclt'Ctivrly l)' fX; S of with qucnt changes in the gt,\nNic con:-.titu· tiou of thr JK)pulation, (2) by induring tcmpurary a ltern lions tht· of the cell, Or {.l) hy both. or Most of the lil<•rn t\lrc dcnlinl( with tht effect. of nutrition of b:ltlcria un l{cnicily is cont:l·rncd with changes that occur after colonia l variation is oln·iousJY mnnifcstcrl or nflc-r tr:wsfer fol' llhlny gencr:\tions. in path- aft<•r serial nrc likely lube the n( changes in the \o nstitutioll or tltc culture sinre ti<:ri.tl provides a.mplt• opportunity for· F.t•lection of lllUt.wt t) J>t'S of diff<•J'cnt 1 whi ch lll •\)' or Ill:\)' nn f d iff<'r in colrmy uwl'pholo}!y. Fur showJl rhar Phylo- pk, J\JeNt·\\ 1 SlfHltUfii of lnw llHJrt· \'irull' nt :tfh·r ir 1111 vinlh•tHt' lu.•t.' JllH' in a syntlwt dhun nitro · gu1 lw \\',J.'> t'c \!'J \'1 \!.,.I .. ,,f iu h r II'>' I i\ it )' which wa..:. h·tiiiH•r.u y .\fl\ 1 .thou t PI lransfen1 a nrr<.lidon!"l u\ldt·r which variant jf( .1hlt· to llll 'f'h:llli - t.'•llly t Jldl';th' cultun·h or \irllh..'Jii'l' (run) tlh· tll'iJ.:in;d ru ltu n: \d th luw virn i< Tln.t!'- 1 tht• innl·:JS<• in ,,,u.; rhu · on ly to m.·· d ;d a :-.hrm t·tl I hut cuJ· th·.ttir llt n f Pi tltl•r Ph .\'IOmrmru lrwlrj!l · ,, ., lion. TIH.'n · .tn otht r n·,-)(,rt"i Ill l h t• lilcr'olHn· of t' han f.(t''" in lht· pathogt·nidty or l>d(' h-rial l'!tlt lll'l'k foJ . lowing cuftivntion upon m• diums for tinu· . :trc also t'!\JII 'dJl tt·tHs in hit-h rhangt!5 in \\ t 1'1' t>h ...•·n·,·d .,hcf nf b.l•'lt·ri., in ht•lh pbntand <\nim.ll •u by c·ult:v.tlion in tht• prt ·f'(·Jwt· of .nlli.,, .l,lk.i. !u \'irlu.!lly ,dl SIH h l'' "I"'! tlt<:ll'l•'l>lf•f h• ll(·t·n rlr ,, fht' I.).Jlhto,:• •I r c! 11,.1'\ •'ld d·t; h.t\' l'tro•ollt '•' ,.f \' I''" ' t ,,f tlti \l• f lt'tl If'. •• • ri• II l. l.int.·oht,R. J.-, J•tliJ,j . ... -.... h60·ll7 1. 7"•tlr, M. rc l'Ji l, l lnf• l'l tJ t !51 S \ ' oung, G. t\ J: ., ).I ft Zdk .t1 •.t I< r· I in co)() 1946, j . lnft·('t . 1Ji9. 79: 2J.\ :?H•. Fig. 26 g. , I. ·sporcl prodtl<"Cd in 1'1'\' (p<·pa•t•) nll'diY or DSI' mvdiuma, pathoge11iclty is Thill loa& of pathogenicity is about J. fold. . . 2. When added to a basal medium in Fig. 26 h . E XI'ERI'I ENTAt. R llsl'IRA1'0Ie c!Tcr.l. of l'.S L Wt p,tth o-gen icity . Include-d in tlw:w u\a tt: ri a ls Ubnilter's pas\l·, Puerto Rico invert nwl:.\";,t.'t., d rit."'d IJn ·wt:r's yeas t, plaamcr.lhl, ' ' :\ I M 111itc'' }'t.'dium in which "a;tlu" h:l.d bt'C 11 omitlt't..' plfl lh.: S. J T lw f.H' H W ur .. rs in corn liqtlut t', ttblHg (h·r rca.,.,·d ,q J}lt '.H' to lw· wa h ·t The: loss .genic tl{)Qrea, in i:-. nnt to tlu.• 7. ln mediun11 con tainlng gluco.e, J; t lh·r.d o \\ t h r ond iti no s of th e M nSO, is necessary lor high ll h tli\llll . T lw t.·h•Whl' in p.. ttl:wR,,•nic it y {)(> th. tu J 1 t H rt•ll nc:r t a ;'Cfl tnp.Uli t·d hy ,·olonial tio11, , t th l i"' nnn- lwrt·(Hta rr . $ . T Jw ous 8. Corn t hei r c·flt-ct on ui pn KtttC(•c\ in CS L.-fr,·c m('i.liu ms i ... 1, :..!". th;111 th .tt tlf prod uctd in ( mH.Ii unt R. H own·<•r, -.p11r•· .. ,,,Pd tiH d in l ' S I.-fn·t• 1\h.:rliumfi . 1110n iH\',biH: iJ I l·XflO · ht !It· ·· o l r r'·!-i pi r.ltory pa tho- !o't'tlil·ity 11 1.1H prndll(t•d in C\)L- tH I\tai uing (J. Tin· .u loli tiim tfw rt':u lil y ft:r · •n•·nt,thh c. ul nm t'(\1\lpuunrl:'!, ity. Ca.C.1,, MIJSQ,, ZnSO, nud no influence on p:tth<.>genidty in lhc medium• uBtld and did not interaf:t wit.h MnSO,. liquor or .oalta produ<:e during the growth of t.he cell and formation of t he The addition Q( the¥41 .factors to sporC su;$pensiona itt their "b"<'nce does not a.Jier pathoge nici ty signitkautly . !1. The signfbnteol tbiauon·genctic environmentally indu<:t:d alteration in pa thogenicity to palbogcni ci t y of bacterilil, culture• is di !:i ltl..'fl W\c.' , • Fig. 26 i. I Ill \lh l\ ThP infl'lll'llf"t• o( .\1 (\Jill Pt. II \l it•! y of rht·mic,d ,,( tht• dh·.tSt.' :t \':\1 im tht· t hra \ lla!-1 \.x:cn r•·rxu lt•d. Mus t of tht>st' l!tudi cs "-t' rt' IIJ.tdt· in ;.m :tlu·mpt to t•\'itl•·n\''' .of th<·ra- ' to[ \\.ht.' l\ ,dim, ft·l' litlll. "hirh t1hnw tlw 'arit·tr ,Jf '' hir·h courH· «)f tiH· C:tdc.u·l ''ill :ulvr·rs..:ly ir,rcct<'t•atcd •ul!lnhal uf lmd :1(•\'l;•t<•. Likc wiS<>, Snnart•lli' !hnwcd rlwt tht' in jfctiou of nr&i'rtic, quinin<·, lact ic ;teid, snc1ium nuclentr, g lucoR<\ pt·plol\t', so· diu.n hyp- uj .tntlnax !;po rt.·S. The adjuv.ll\ts Wt•rt· uM·d in ('i.)ltt:t•nlriltioiHi small rnnugh to to I he IH'hil. Similarly, llru•kCL' tl<•n1<' digitonin and snpvnin, •.•llts.t• no appaH.'nt \\'•,rk \ l,,r,\ r t ;1im·d lO: UJ">. r. t.nunm . l·xp . ltl ,tuth r••.. : In rh i"i (Mp1..1 . niTl.'\:l COJ\t t 1111.1 qu fli.dt•nll\' i\lorr- n•( 't·ntly. \'t•lu t•t .d.t. :-;ho\\ I! th:tt <·hlori m· prtili hpO:;l.'S lllit't• aucl ).tlli nt·.t :lit' mininJ.d wit h Kn. 2 ·v.h iu }'n·viously itl llnuni;,l'd ra hhir,. :n\lhrtl \, tlll'r•· ill 11\kl'tt·d tinns lowt•f't ·tl uM.•rl iu pr·ulit! val;tl' fQr thr tlh· tn"atnwnt ()( aninM is inft·c·h•d wlllt l[o\\l!\.t·r·, '! .-.. dl'l· .t \';l rid" dt · or rhcmic;l)t"', nf lh·.t\'Y mt·(,, Js! have ht·t·n to ln\\ 1·r tlw nutural t't·-.;i::.ta nc:l· to ,.llllh r.t\. inrc·,· ti nn th•t:thlhilwd h)' :;piratory t' \Jlll'i\tn· I ht· ol tilt: S} t•rlt·( ,..., ohs.crn·d will ht · digcu:P;t·d 111 n· l.ttion to th e •HHttn· of natuml rt·!'> i:Hilflt't' to n:.-pir".'\tt•rr inft•c·· tamrwith /1 . ME'tlt{)ll'i 'llw IIH' tlwd & \I'll"! in lilt 'o(· .. \1 '• n• t ":'4''' ti.lll y 1h,.. $.\1\lr ll' nupl")'''' pn·vi• ttJ ••ty' t lilt' ilu·mi.·,lh; Uiol'll 1\l'rr lll'hulil."tl fllllll v;H)'it1).!: u( :onl111inu frOftt I ,,( 'i.ht.' J udm · h1.•'fll in tlw l'" I)(I>IUn' ('ba mhor. 01 hrr u ....-.1 tn p rod ucr tlu· rltH,tl ,:.( :ton!brnll:: SIJOf\':0 f' lwmi•• 11 "' ' .'" fit 1rnnined l)y •.. volumN ul t'hr forrncrl iu lfw dt1\111hn h)' 1111';11111 Cif t' UIIi)U MrUpk:I'JI. Qu.'lt\ · ut.tli\" ' ··uloriuw Jti,· wt"rt• pla8t- Otl llw tr.tppt•d ( Shh"t' the l'otton samplt•rl!< tr.lf,l"'d 1lw ..,,.,..,.. .. , •Ju.uuha tiw platr "'''l'l' tH;,hlc frQ111 th<" 1liluw d•emicorl tolut\Otl 11• tht r:un(t'tl1M1ic.tl o( theantln•L"to:'-Jlf"''!) in th<- duunh..·r 1"1t.tUrl. · As >1\.--.cril.ctl in firat of n-•tMI.IHif'Y t.' XI)Q&Iti'C wns IUf',\fi Url'tl in uf Rl! "·hic.•h i11 tht Dvtfi18'C uumlKT oi !lf)Ort'' (multi· plit.d hy to· •, in lilt.'( nr c•lou.J fur"' S-minuh• rtFopimhK)' au: of rltt• ··hc•mknl is takt.·n O'lllhi" .wt•m"... S. Vclu, II , P. Suulk; ouul fl . 104 ( Hull. ,\,·.ttl , r.,t,o,l. 12.!': l.'i'). . . It \' tlllll•'• C, \, J, .. /.dlo.,,n)•l IL 1· 19111, I fl tf, •'t. I •i - 7Q; ': }·' : :·· Fig. 26 j. !-ol lIs of >.!Its c•f ho•,\1) ll ll'i.t l' flctrillus i11 Wldd1 o11lcl '"" ,. owlc·thal dTt•<:! if 11sc1 r.1tdy rau:.<·s ,\nt lom• infc·rtion" in . . tud This ti<>llship !1('1\H·c·n tlw o·hcmic.ll" '"' t lo r". I\ iN rnoly A· is prl'oo:nh·cl :utrihuting thtl to clwmi<-al in .tetivatioll of til<' 1·nzyme M)'sto•ms, mainly those wliich dcpt·ndo·nt upon tlw of frvt sulfhydryl l(roups for their activity. ll is pointt·d out that studies of the: physiological effech of chemical compounds which exhibit synr·rglstic rein· lions with pathogenic. orgillliRmS mny be uf v.lluc in furtlwrinft thr• undl'rstnnding of tlw natur<' of niltur.ll n·slotanao to !-l. (x.\rifu· rlflfhrtll i.'> 11 l'h• RlllhOOI 'wish to thank Charleo Frazlrr r... 1\htkinK thr chtJUit'aJ dC'lt:"rn\lnJ;\liOOS atnd (or l Ute· rf•l.ut:-tl tn rhemh.u1 problem. enp n)Ul\tl'r'""' Fig. 26 k. Fig. 27. Witness Liu Chi?An. Fig. 28. Witnesses Wang Chiao-Ping (left), Sung Wei-I (right). 3.5. {if f: . Fig. 29. Witnesses Liu Chung-Kuo (upper), Sung Teh-Yu (lower left), Pan An-Ying (lower right). i. Fig. 30. Witness Wei Hung-Chin. Fig. 31. Witnesses Ho Ming-Chia (upper), Liu Ching (lower left), Chao Y?-Chin (lower right). Fig. 32. Witness Old Mrs. Liu. APPENDIX BB Hearings on the Incidents in Liaotung_ and Liaohsi Provinces Connected with the Dissemination of Anthrax Bacilli and Fatalities Arisiurrb Therefrom A) Depositions of eye-witnesses, 22nd. July, 1952 First Case narne age occupation 1) Wang Yu-tsai 56 farmer 2) Liu Chi-an 21 sanitary officer 3) Liu Chung-kuo 27 railway platelayer 4) Chang Yti-chia 28 medical student acting as bacterio·logical technician 5) P'an An-ying 32 doctor 6) Sung Te-yu 30 pathologist 7) Liu Chan-yuan 24 doctor, assistant to Dr. P'an. 1) Wang Yu-tsai found the flies in large numbers on the heaps of sand which he was engaged in carting, and recognised at once that this was abnormal for the season. (This was the: case in which the document 00004 spoke of "flies gathering" .. Criticism of this in the west earlier in the year had asked how the flies could be gathering if they were supposed tO> be dispersing. It was established that this was purely a. mistranslation from the Chinese text, the word should have been "gathered".) ' (M) For him it was a sudden and inexplicable new phenomenon,. he had never seen the like. before. (N) Assessed density 100-200 flies/sq. ft.; where less dense, still dozens. They could only, creep about and seemed unable· to fly. 2) Liu took about 40 people with him to the spot to; destroythe flies. Together they collected between 6;000 and 7,00() in an about 4 x 5 metres; outside this region there were only a few. .,--- 4:17 ,...... (0) There were no dungheaps anywhere near from which the swarm might have come. It was a sandy heath. 3) Liu Chung-kuo found quantities of flies on and beside the railway line when walking with his foreman Ch'ii Chan-yiin. They both thought that it was very odd to find so many flies in such cold weather, and he went back to the station to report, while Ch'i.t remained and started to collect the flies, picking them up with his bare hands. When Liu returned with help, he noticed that whereas before the flies had been spread out along a length of about one rail length, now they had dispersed to about two rail lengths. Liu and his mates poured petrol on straw and burnt all the flies up. Afterwards the sanitary chap sprayed him and said it was all right for him to go, horne. ( 0) The last train before they saw the flies had passed about an hour previously. , (M) They had had instructions to report any such phenomena to the stationmaster. (N) The, group who returned and finally destroyed the insects used forceps, improvised chopsticks, and masks. Ch'ii was the only one who used his bare hands, and he definitely did not wear a mask. 4) Chang Yii-chia, medical student, was acting as a bacteriological technician at Ssu-P'ing. He noticed that the abdomens of the flies which he received seemed very swollen. He crushed them with forceps and observed smears microscopically. Colonies on agar plates were typical of B. anthraC'is; were quite large, with rugose surface like masses of hair. Hanging drop preparations were also made. Inoculation of the culture into a guinea-pig brought about its death in 4·0 hrs. Recultured in bouillon, a cloudy precipitate was seen, containing the . bacilli. All results and material were sent on to Shenyang (Mukden) for confirmation. (Z) Agar colonies further described. (Mr. Chang said that he hoped very much that he would be ·able to return to his medical studies at Harbin Medical College · and complete them. The Commission offered him their con· ·gratulations on an excellent piece .of front-line scientific work, and wished him every success in his medical career.) =- 418 ' · 5). P'an. Ari-ying was the doctor who attended Oh'ti in his last illness, the course of which he described. (A) He knew of the connection which Ch'ti had had with the flies, and suspected that it might be anthrax due to them, but was not able to reach a definite diagnosis in time. (Z) There was severe dyspnoea with rales when the last visit was made a couple of hours before death. 6) Sung Te-yu described the results found at autopsy. 7) Liu Chan-yuan not called. It was established from several witnesses that there was no sheep-raising anywhere around the district of Man-Ching and Ssu-P'ing. Second Case Wang Yu-lan, sister-in-law of the pedicab Wang Tze-pin. 1) Wang Yu-lan said that her brother-hJ.-law was single and gained his living in and around Shenyang (Mukden) by plying for hire as the driver of a pedicab (bicycle-rickshaw). (A) Though she did not see him every day, she believed that he had engaged in the collection of· insects when in one of the suburbs where a fare happenep. to have taken him. She heard that they were small ones. Did not know whether he wore a mask or used any other precautions. Third case (a) name 1) Wei Hung-ch'in age occupation 33 forester, State employee 1} Wei Hung-ch'in had gone to Harbin on duty when he received a telegram saying that his wife was dangerously ilL He was very surprised because she was usually in good health. When he got back to. Anshan he found that she had been taken to the isolation hospital and had in fact died the night before. Friends who were looking after his children told him about her illness, which was similar to the other cases. She had . certainly. been out collecting beetles for several days previously.· Whether she. took full precautions, . including .a mask, he could not say. (She - 419 .,.....,. pregnant with a baby which she would have had if she had lived). Case (b) name 1) Lu Li-tsun 2) Jen Wan-k'u 3) Wang Hua-ming 4) Li Lien-chung 6) Wang Shao-chih 42 38 15 32 32 . 6) Mrs. Liu 57 7) 8) 9) 10) 14 14 Chao Yii-chin Liu Ching Wei Hua-nan Ho Ming-chia 11) Lai Yii-chung occupation age 25 farmer at Liu-Erh-Pu Home Guard sergeant schoolboy village constable chairman of local mercantile and industrial association old lady, landlady of the dead schoolteacher school-girl school-girl doctor husband of the dead school-teacher, himself teacher at a Teachers' Training College school-teacher, colleague of Mrs. Ho (Wang Shu-chih), 1) Lu Li-tsun heard .the planes about six o'clock in the evening. 2) Jen Wan-k'u saw an object fall from one of the planes as stated in the Report. (N) The object seemed to be about the size and shape of a large thermos flask, red in colour. As it fell, it turned flame-red or orange. The red colour could not have been the refl:ec;tion of the sunset on a metallic object as the sun had already set. (0) The noise of the explosion was very slight. The colourchange, if such there was, occurred at the same time • . (N) · · He smelt ·a disagreeable burning smell as if of burning · animal skin. or horn (or feathers), though he estimated the falling :object as about .160 yards. away from him. The :explosive puff occurred ·when it was from 9 to 12 yds. above· the· ground, and 3 to 4 yds; above the house-roofs.' , ·'There was no wind that evening. The main mass of insects was found underneath where the container had seemed fall; this"·point :fixed by three different people whQo Irad· seen it,faU from different directions. """'" 420 ..._ (M) He .could not say whether the brightness of the object was like that of a· paper lantern in which a_ light shines through a transparent exterior, but he did not remember thinking this at the time. · . .. S) Wang Hua-ming when lying on the k'ang in his .home, also saw the object falling in the twilight, and also smelt the smell. A first brief reconnaissance outside revealed nothing, but after he lit the lamp in the house, he noticed insects thickly gathered on the exterior of the window-panes. They were as big as a large rice-grain, and had a "hard shell" and two long feelers. Remembering what his schoolmaster had said about bacterial warfare, he assembled the family and neighbours, who collected as many as possible ·of the insects, using masks or handkerchiefs tied over the ·mouth.. · (N) The object which he saw falling was certainly not like a lamp or paper lantern. (It may be noted that the accounts of these two witnesses would not be incompatible with• the delivery of two self-destruc. · tive containers, one of which had a mechanism which went off too soon and burnt its load of feathers, while the other duly_ delivered its load of beetles). 4) Li Lien-chung described the collection of the insects. (M) He saw only one kind, namely beetles, just as the boy said. · 5) Wang Shao-chih was waiting, on the evening of 27th, Mar. for a meeting to start, and went out to hurry up some of the .members. While walking past a cooperative store he shone his flashlight on the wall, and saw many insects. He had never seen anything like so many in his life, nor had he seen any of that kind before. They had a "shell, six legs, and two wings". At the time of year insects were normally very scarce, for the snow was still largely unmelted. He guessed what the origin of these bettles was, and -coming back to the meeting-place, mobilised the members to collect arid destroy the insects . . (M)·. He recognised the beetle Ptinus from a photograph. 6). Mrs. Liu an old lady of nearly sixty, was the landlady of tlie school-teacher Wang Shu-chih, and looked after her child. Wang Shu-chih was very in participating in hunts - 421 __,. for insectsi and spent the better part of eight .or. nine days on· it. ·After that she fell ill. She went with her husband to visit parents, but did not improve hereafter, and finally collapsed in the toilet at the school where she taught. (A) Regarding the manner in which Wang Shu-chih and others colleCted the insects,she, herself, certainly had a mask and gloves, and they used twigs with sticky ends; but Wang Shu-chih was young and impetuous, and 1Mrs. Liu did not feel sure that she took all the precautions which she should have done, at all times. (Z) The surviving child, a little girl of 2, was in perfect health and so plump as to be hard to carry. ,-) Chao Yii-chin and 8) Liu Ching giving evidence together, said that they were in the class taught by Wang Shu-chih. At 9 a.m. on the 8th, April they went to the toilet and found Wang Shu-chih there in a state of collapse. She died 1lj2 hrs. later. (M) The two girls had helped to collect insects, one of them actually with Wang Shu-chih. She thought that Wang Shu-chih generally wore a mask. 9) Wei Hua-nan when called as a doctor to Wang Shu-chih after her collapse, saw at once that she was already beyond aid. He had never seen a similar case in 20 years' experience. He rang up the Department of Health about it a.t .10 a.m., and· the young woman died at 101;2. Having been acquainted with her previously, he could say that she had a good physical inheritance, was strong and healthy, and in an excellent state of nutrition. 10) Ho Ming-Chia a teacher at a Teachers' Training College, was the husband ·cif Wang Shu-chih. They had had an extremely happy married life 'for five years. Her health had always been excellent. Described what he knew of her illness. Whether she always wore a mask when collecting insects, he could not say. 11) Lai Yii-chung was a teacher in the same school as Wang Shuchih, and a friend of hers. She was informed of her collapse by the girls immediately after their discovery. She was struck by the fact that the dbctors 'said they had never seen a similar case. · '(P)' "here were no lesions, so far as she knew, i:h t'):J.e · mouth l - of Wang Shu-chih, her teeth were good, and she had not bought a new toothbrush shortly before her illness. (N) Regarding the precautions taken when insects were being collected, she had actually seen Wang Shu-chih working without· a mask. She herself had not gone far afield for insects, and had always worn a mask. Other friends tically always wore them, and cor :;idered it rather risky on the part of Wang Shu-chih not to do so. (0) Everyone in and around the school was a bit scared at these events. (The Commission did not close this session without offering its deepest sympathy to the bereaved). Fourth Case name 1) Wu Ching-ming 2) Chiang Wen-ch'ang 3) Wang Chiao-p'ing 4) Sung Wei-i age occupation 28 farmer and Constable of village 22 farmer and Home Guard sergeant 43 farmer and Constable of village pathologist 1) Wu Ching-ming, Constable of the village· of Pei-Ching-Tzu, near the coast of Liaotung,. said that the village people were holding a meeting after .assembling to extinguish a straw fire caused by a child accidentally at one of the houses Three American planes suddenly passed over, flying very low and very fast; the people took cover. He could not be sure of the type. (Other sources of information show that they were F-86 jets). He saw one of the trailing ones throw something out. Some said it was a bomb but he thought it was falling too slowly for that. It seemed to have a bright metallic colour and appearance, though it was hard to be sure, as the sun was shining (it was about 11 a.m.). It descended toward the Luan-Shih Hills. Chiang Wen-ch'ang and other Home Guard members went off to look for it, while he (Wu) rang up the District Government. Near what must have been the point of impact Chiang found great quantities of feathers which seemed to be those of hens, ducks and geese. The whole some 300 went ·OUt and worked for two hours population collecting them. He saw that some were kept for examination and not d.estroyed . .,_ 423 ,..... (N) The wi:rid was from the northwest on the morning .t}lat the .object was dropped. When the people got to the part of the hills where the feathers were, they found that the feathers were distributed over a triangular dispersal area, the height of the isosceles triangle measuring about threequarters of a· kilometer and the base rather less than half a .kilometer. It was supposed that the apex of the triangle represented the point of impact, since the feathers were most thickly distributed there, thinning out towards the base of the triangle gradually. The long axis of the triangle was in the line of the wind, the feathers spreading south,eastwards. No fragments of any container could be found near the presumed point of impact. No insects of any kind were seen among the feathers. Masks (or handkerchiefs) and chopsticks, had been prepared beforehand, and were used. Among the feathers some lumps of cotton-wool were found (the witness was not familiar with this, but called it "balls like the cotton :flower itself"). No remains of bird skeletons were found anywhere on the hills. (A) No trace of container, reaffirmed. No smell. All the feathers were small, downy, and moist; not as if from adult birds. (M) He thought he saw the (metallic) object burst when about 30 ft. above the ground, but could not be sure, as he was 1 km. away. The total volume of feathers collected was enough to fill 14 large lemonade bottles. Chiang Wen-ch'ang agreed in all details with the facts stated by the previous eye-witness, (M) but saw no explosioD;. ( 0) As the first to reach the feathers, with 1;hree .fellow· members of the Home Guard, he noted them carefully. They were quite conspicuous, because largely white. The wind was rolling them gently along, but dispersal was greatly inhibited because they kept on catching in grasses, twigs, and plants. . He confirmed the low height and great speed of the planes. ·Snow was still lying in the valleys of the Luan Shih Hills, and no trees were out anywhere. (Z) He did not touch any of the feathers,; nor let anyone else do so. All were downy. ( 0) No one in his· village or any other Chinese village that he knew of ever used feathers. for stuffing bed-mattresses o:r cushions. Any such things that they had were stuffed with cereal husks such as millet. They were not accustomed to soft cushions or beds. 3) Wang Chiao-p'ing, Constable of East Shuang-Shan village, knew the dead farmer T'ien Ch'eng-ho well. He generally had good health. Whe11 he heard what had happened he (Wang) was therefore very surprised, and went to enquira about it from Mrs. T'ien. Four days before his death T'ien had been in normal health, and had gone to his fields to manure them. These fields lay about a km. south of East Shuang-Shan village, and therefore not far from the northern end of the base of the triangular dispersal area. Seeing a few feathers, T'ien had picked them up and handled them, as something rather unusual, but then remembered that they might be dangerous and let them lie. Two days later he fell acutely ill. After his death others went ancl found the feathers just in ·the location he had mentioned, and burnt them. (A) T'ien had been warned of the danger, but forgot, and picked the feathers up out of curiosity. (P) The dead farmer always shaved at home. 4) Sung Wei-i described the course of the disease which he, as physician, had followed. It was clinically identical with the other cases. The autopsy, which he described, was concordant with all the. other pathological evidence. His findings of anthrax bacilli were confirmed by the bacteriological specialists of National Medical College, Shenyang (Mukden). (M) The Chinese . peasant does not generally carry a handker· chief. [The Commission did· not close this session without offering its deepest sympathy to the bereaved]. c·- :425 · - B) Questions Addressed after the Depositions ofthe Scientific Specialists (qualifications .in App. TT).1) Prof. Lt.): Pao-ling (entomologist) (N) The beetle Ptinus furl' is a pest of stored grain, leather, etc. It ought to occur only in granaries, museums, and places where dried stuffs are stored. Yet here it appeared in large numbers in the open air. Moreover, it' is a noc· turnal animal, yet at Liaoyang it made its appearance in the daytime. Furthermore, subsequent efforts to discover individuals of this species in granaries and other places where it would be likely to be found, in neighbourhood, completely failed. The whole phenomenon is therefore oecologically very peculiar. No figures could be obtained' as to swarm density, bt.:.t it was certainly considerable. The appearance of the beetle showed no seasonal abnormality. In size it is 3.4-4.4 mm. long . .The species is widely fotind in China, but it is nowhere a common insect. No previous records of any similar swarm of this insect are to be found. 2) Dr. Cheng Keng (bacteriologist) (Z) Emphasis is to be placed on the fact that whereas strains of anthrax bacilli isolated from various natural places show marked differences in the fermentation tests, especially with regard to behaviour towards- maltose and salicin; in the present case every one of the suspected objects tested (flies, beetles, and feathers) was found although collected from different places, to be carrying bacilli with the same fermentation charactenstics. This is a most unusual occur· renee. 3) Dr. Pai Hsi-ch'ing (pathologist and Vice-Minister of Health, Northeastern Region). (A) All cadavers of anthrax victims were cremated, although this is very contrary to traditional Chinese custom. Rela· tions gave consent at the special re-quest of the government. All possible additional precautions were taken to guard against infection. (N) As regards anthrax among workers in the famous Chinese pig bristle industry, there. had been no cases for a number - &2.'6 - of years, a fact which· was thought due to the widespread inoculation of the animals. So far, anthrax vaccine had not been available for man, but it might .well be that .it would. be desirable to institute further researches on it immediately: ·. . 4) Dr. Fang Kang ·(bacteriologist, Central Research Institute of Health, Peking) (information received later). Although in general opportunity did not permit of quantitative work on the various kinds of insects which were suspected of having been infected artificially, a rough estimation .was carried out on the Ptinus beetles from the present series of incidents. Six specimens of the beetle were ground up in 5 mi. of physiological saline and an inoculum of 0.004 ml. (one loopful) was transferred to each of several vessels of nutrient medium, with the aid of a loop of 3 mm. diam. There resulted from 15- 20 colonies of anthrax bacilli in each case, from which it was possible to calculate that each insect had carried between 3,000 and 4,000 spores or bacilli. C) Visit to the Pathological Laboratories of the National Medical College, Shenyang (Mukden). Prof. Dr. Li P'ei-lin (pathologist) demonstrated slides showing: (a) haemorrhages in sub-arachnoidal spaces (b) anthrax bacilli in and around meningeal blood-vessels (c) acute perilymphadenitis and peribronchitis (d). anthrax bacilli in hilum lymph gland (e) anthrax bacilli in mucosa of small intestine (f) necrotic foci of colon with anthrax bacilli (g) pneumonic lung tissue (h) anthrax bacilli filling up ,.a capillary in brain substance On behalf of Prof. Lu Pao-ling specimens of the three arthropods concerned: (a) Musca vicina (b) Tarentula sp. were demonstrated. (c) Ptinus fur It was particularly noted that the elytra of this beetle were exceptionally abundantly covered with chitinous bristles, which on breaking off could readily enter lJ.Uman respira.. tory passages, where they .would act as inoculation needles- 427 ....... D) Summary of Data on Selected Cases of Anthrax Victims. Dateo:f Death Case No. 22/3/52 24/3/52 14/4/52 8/4152 18/4/52 1 2 3a 3b 4 Name Ch'ii Chan-Yi.in Wang Tzu-Pin Wei Liu-Shih Wang. Shil-Chih T.'ien Ch'eng-Ho Occupation railwayman pedicab driver housewife school teacher farmer .. Mode of Infection flies (Musca. Vicina) ? beetles (Ptinus fur) beetles (Ptinus fur) feathers Note: In connection with the foregoing .it is of interest to note that in the "Textbook of Medicine" of Cecil & Loeb, 1951, it is said of pulmonary anthrax (p. 243) that "death may occur within 18-48 hrs." It· goes on: "Absence of the severe symptoms usually accompanying acute infections and the rapidity with which collapse sets in are characteristic features of this form of anthrax. Not infrequently the disease is suspected only because the patient's occupa· tion subjects him to inhalation of dust from hairs soiled with spores." The same work says of gastro-intestinal anthrax that the organisms are carried to the mouth. from external lesions. Here again the infection may be symptomless, collapse, cyanosis and apoplectic death terminating the infectoin within 1 - 3 days. Haemorrhages in myocardium and ,brain are seen on autopsy. Individual susceptibility to anthrax seems to yary con· siderably. The mortality rate may be as high as 40%, even if septicaemia and meningitis do not supe.rvene. The organism is sensitive to certain sulpha-drugs and antibiotics, if the necessity of giving them could be recognised in time. E) Frequency of Occurrence of Cases of Anthrax according to Autopsy Records ' 1) Shanghai Medical College Hospital, Shanghai 1928-1952 no. of. autopsies 1178 2) China Union Medical Hospital, Peking 1916-1952 no. of autopsies 3942 3} National M.edical College,· Shenyang (Mukden) 1940-1952 no. of autopsies 1093 · · ·In all these· records, there was not one single case of acute pulmonary anthrax acute haemorr:hagic .anthrax meningitis. or - 428 -