The effects of rebreathed air on tuberculous guinea-pigs

426

[June, 1924

TUBERCLE

AMERICAN SECTION. Under the Editorship of Dr. A. K. Krause.

THE EFFECTS OF REBREATHED AIR

O~

TUBERCULOUS

GUINEA-PIGS. By HENRY SE\YALIJ, M.D., and HERBERT GUTSTEIN, M.D. From the lieeearcli Department of the Nation.ii Jeurish. Hospitai jor Consuanptiocs, Denver, Colorado.

WI'l'HIN the past forty-five years many researches have been directed to the determination of the question whether air, which has been breathed and exhaled from the lungs, contains any excreta save the familiar carbon dioxide, which may render it noxious for respiration. While, numerically, investigators have been fairly equally divided in opinion, the weight of authority has undoubtedly negatived the notion that exhaled air contains any organic poison which renders it unfit for respiration. The discomfort experienced in crowded, unventilated apartments has been attributed to physiological reactions set up by combined high temperature and excessive humidity of the air; and lethal results, when they occur, have been explained as the outcome of the poisonous excess of carbon dioxide and insufficient content of oxygen. The researches upon which these opinions were based were for the most part carried out before the birth of the great conception, first suggested by Theobald Smith, that foreign protein matter introduced into the living body may so modify the tissues that they become dynamically hypersensitive to it. This immunological point of view has been nearly universally neglected or disparaged by students of ventilation, except for the familiar work of Rosenau and Amoss [1]. In a recent contribution [2J the present authors have described experiments upon guinea-pigs, which seem to show that exposure of these animals, even on a single occasion, in confined air highly polluted with the products of respiration, and with a CO 2 content of approximately 10 per cent., sufficient to cause well-marked dyspnoea, often "sensitises" the animals, so that on re-exposure, after an appropriate interval, there is a perceptible modification of respiratory behaviour. If these results hold, it is obvious that they imply such a modification of tissue metabolism, occasioned by the experimental conditions, as might, without ordinarily notably altering animal behaviour, greatly reduce the subject's resistance to the implantation and development of infectious disease. As a matter of fact, many of the guinea-pigs set aside after completion of the experiments described died within the next six months of infections. In an addendum to 0111' article the impression was conveyed that a large percentage of our fatalities was due to tuberculosis. Further observation made it clear that we were dealing with a "pseudo-tuberculosis," which was not restricted to the animals of our series. Further report will be

.Tune, 1£)24]

EFFECTS OF REBREATHED AIR

427

made of this disease, which produced abdominal lesions suggestive of tuberculosis, but which has thus far failed of transmission to normal guinea-pigs by inoculation. One certain case of generalised spontaneous tubercul osis occurred among the animals of our own group. Un fort unat ely, of sev eral of our animals SU CCUIlI bing during the writers' ab sence to a disease having the macroscopic lesions of tuberculosis, the specimens were not preserved . Further pursuit of th e problem of the relation of respiratory sensitisation to the dev elopment of tuberculosis will be detailed in another con tri bu tion. The foregoing investigation was on an accidental side lin e of an original . problem which may be sta ted thus: Admitting that rebreathed air, und er m ost unfavourable co ndit ion s of actual ventilati on, involves no perceptibly deleterious effects up on healthy human beings, what effect does it tend to have upon that large proportion of people who harbour latent tuberculosis or incipient clinical tuberculous disease'? From the point of view of the hygiene of school children this is a question of urgent moiu en t. \Ve have attempted to throw light on the problem through the three series of experiments to be presently described. For several reasons the male gu inea-pig was chosen as th e subject of investigation. Our apparatus was m odified from the form first devised by Brown-Sequard and D'Arsonval, and was described in a preceding article l2] . A glline'L·pig was enclosed in each of six large glas s air-high t hell jars, communicating with ouch other in series through glass and rubber tubing of about one-third inch hare. The jars, figs. 1 ana 2, measured, inside , about 10'[) in. in diamet er and 8 in. in hei ght. Each carri ed two apertures. 1 in . in diameter, on e hor ed near the bottom a nd the at Iw1' ut, the top , 011 oppos ite sides, for the reception of perforated ruhber st opper. Th e air eurru nt ente red by th e lower and emerged by the upper perforat ion . Each jar Uig. I) snt in u groo ve, an inch or more deep, t urn ed in It heavily varnish ell wooden block.

FIG . l. --D iagralll of struc ture and nrrnngement of individual jar.

A considerable depth of mercury in t ho groo\'o formed an air- tight seal for tho rim of th o jar, and the surface of t.ho metal was kept covered with u layer of l\lholnne to prevent volnt ilisut.ion. 'l'he guilHm-pig, t.ogoth or with vessels containing wat er and outs, rested on a movable platform elevu ted OIl {our posts a bout an inch

428

'£UBEROLE

[June, 1924

above the surface of the wooden pedestal. A device effective for cleanliness was a coarse-meshed galvanised iron screen. with edges turned down ~ in. and laid upon the top of the platform . A disk of blotting Impel' under the screen completed the adjustment, the movable parts being guarded by the projectinq edge of a metal band encircling the periphery of the platform. The terminal jars of the series were provided with thermometers. Air entered jar 1 through a tube, the glass terminal of which wa s fast ened vertically to the wall of t he room (fig. 2). This terminul, constricted at the lower end and partly obstructed hy wire above, held. It pith hall, the position or oscillations of which gave a very satisfactory index of velocity in the ventilating current. The gluss tube conveying the air to eueh jar projected well under the platform supporting the guinea-pig. so that the latter was effectually protected from draughts. The system of ventilation was actuated by a filter pump in an adjoining closet. communicating through IL hole in the wall. The ail' leaving jar (j bubbled through a small wash bottle, tbe tube emerging from which was attached to the pump and, by an offset, connected with a manometer.

FlO. 2.-Diagram of arrangement of series of jars.

In preliminary experiments it was determined that, with an air-flow of about 1'5 litres pel' minute, the pith ball in the afferent tube of the apparntus oscillated violently near the bottom of the tube, and that at higher ratea it ascended and hugged the obstruction 11t the top of th e tube. As the pith bull gave practicul information of the inflow of air into jar I, so the rate of bubbling in the efferent wash bottle was evidence of the outflow from jar G, and the stand of the manometer indicated the aspirating forco in action. In 0111' actual work the efficiency of our ventilation was determined and adjusted wholly by chemical estimation of tho content of carbon dioxide in JILl' fi, and frequ ently in the remaining vessels. The analyses were made with the aid of a modified Haldane uppuratus. and the air to he examined WI1.8 drawn from any jar by plunging a large hypodermic needle, connected with the I luldune instrument, through the efferent rubber tuho. It, should be obvious that, during an experiment, the animal in jar I breathed pure air while that in jar (j respired the exhalations of the five preceding members of tho series. In our preliminar-y experiments on normal gu iuen-pigs it was found that 11 high mortality 11tt.ended continuous exposure to ail' currents insufficient for ensy breathing : we therefore finally adopted a rate of vontilation under which, with a guinea-pig in each jar. the air of jar G showed continuously It C()~ content of approximately 1'5 per cent. At tho same time, no CO 2 could be detected in jar 1. Its content was therefore leSR than 0'1 per cent., the functional threshold of our upparutus, uml in JILl' 3 the CO 2 content averaged 0 '5 to 1'0 per cent. During tbo course of our ohservations the CO~ content in jar li occasionally. though seldom, ascended to over 3 per cent., and in jar a to more than 2 POI' cent. We will not dwell here upon the difficulties encountered at \ho outset, under slow rates of ventilation, (rom

June, H)24J

EFFECTS OF RISUREATIIRD AIR

42H

the accumulation of the water of respiration in the connecting tubes. Swabbing these with a mixture of melted wax and vaseline and the use of faster ail' currents seemed to remedy the trouble. The maintenance of our animals in a sanitary environment demanded a regular cleansing of the apparatus. This was usually performed every day, the animals heing removed to their cages for It few hours. They were, however, frequently kept continuously in the jars for :W hours, ant] on rare occasions for two full days, at the end of which time they apparently suffered from the environment. Healt.hv guinelt-pigs might proba hl y have been mn.intained for long periods under the conditions dcscribed : nevertheless there was, even with free ventilation, something inimical ill the conditions. This was most obvious in their psvchical behaviour. The aninmls tended to become dull, listless and depressed. \Ve suspect excess of CO~ as the most potent cause of this demeanour. On removal from the jars they would itt once regttin their normal lively behaviour. When, after inoculation with tubercle bacilli, the animals were far advanced in disease, timely removal from the jars was apparently often a life-saving procedure. As it was, the high mort.alitv among the subjects of the jar experiments, as compared with controls, is sullioient evidence that even with what would be cousidered fair ventilation and satisfactory cleanliness, there \Vas somet.hing deleterious in the life of confinement. Onr apparatus was established on a shelf at tho side of It narrow room, at one end of which was it window to tho open ail' and under it It steam radiator. The proximity to the latter accounts for the fact that the temperature in jar 1, within about two feet of tho rudiator. was habitually higher than in jar ii, some 10 feet removed from it. Routine temperature readings, ltS well as careful notation as to respiration, &c., of their clinical condition were taken he fore each removal of the animals from the jars. In jnr 1 the temperature was usuully 2:1 . to 2ii C., though it varied between extremes of ahout 20'" to ao" C. The (til' in jar (j was constantly and unexpectedly cooler by from 1" to if'thltn that of jar 1, except in summer when art.ificial heating was not in vogue: then the temperature of jar (j exceeded that of jar 1 by l'~ or 2". Even under an ample vsntilatinu current, after 10 to ~o hOHrH' occupation. it tiln. of moisture was apt to be precipitated more or less densely on the inner walls of tho last throe jars. FIRST

SImms

OF

EXPEHIMEN'I'S.

In om first series of experiments it was planned to inoculate, subcutaneously, It group of young male guinea-pigs with It small fixed weight of It culture of highly virulent tubercle bacilli of human strain. Six of these animals woro assigned to life in the jars, the same jar, of course, being reserved for its aSHigno(! occupnnt.. When a jar animal died it WItS replaced by that control animal which seemed in best physical condition. The virulence of tho bacillary culture used was such that one-billionth of :t milligram had heen found to cause glltndllhtr, but not general or pulmonarv, tuberculosis in the course of two to throo months. Our inoculations were 11\(\[10 under the skin of the depil:ttol1 loft inguinal region in male !-(uinea-pi!-(s of approximately 200 to 300 grrn. weight. gltch uuimnl reeeivod one-millionth mgm, of tho culture suspended in I 0.0. of sldt solution. For tho propurut.ion and injection of the wuter-clonr sUHpensions WI. 11m !-(roath indebted to Dr- 11..T. Corper, director of the laboratory. Tho perfection of his· tochnique is attested by the fact that when examined 3:) dltVS after their inoculation every guinOlt-pig showed murkod gllwdular enlargellIont~ in till> Idt groin with, in somo cases, ulceration of the skin. Three of our guinea-pigs were from It group inoculated on. No\'om~)or 28,. H121, and ih.o remainder on December n, H121. Of the 22 unirnuls WIth which the expert-

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TA.BLE I.-EXPERIMENT 1, LOT X. GUINEA· PIGS INOCULATED EACH WITH 0·0‫סס‬oo1 MOM. TUBERCLE BACILLI. ANIMALS CONFINED IN JAr:S WERE SUBJECTED TO REBREATHED AIR. ANIMALS 1 TO 8 WERE CONTROLS, AND WERE KEPT IN ANIMAL HOUSE.

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Trac heo bronchial nodes greatly enlarged. Guineapig in fair clinical condition

Guinea. pig in fair clinical condition when gassed

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This guinea-pig, missing 24.6.22, probably had died and had been thrown out. On 23.6.22 was noted as gasping having deep, respiration

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432

'l'UBERCLE

[June, 1924

mont started, on July 8, 1922, when it was concluded, we had full histories of 20 subj ects.

The principal data of this experiment are summarised in Table I. It will be noted that of the control group, none of which had occupied the jars, five guinea-pigs died during the course of the experiment and three remained alive, and in relatively good clinical condition, until killed by the inhalation of illuminating gas in a closed box on July 7, 1922. Six of the jar guinea-pigs died during the course of the . experiment, to which it seems proper to add a seventh, from jar u, which almost certainly had died and had been thrown out by the attendant by mistake. Five guinea-pigs remained alive in the jars when they were "gassed" on July 6, 1922,212 to 220 days after inoculation. Thus, of animals which died antecedent to the date of gassing on July 6 and 7, five out of eight controls, or 63 per cent., died within a period during which seven out of 12 jar animals, or 58 per cent., succumbed. These figures would seem to indicate that the environment of the jar animals had been more favourable to life than that of the animal room in which controls remained. However, such a conclusion would be wholly erroneous. 'I'he average clinical conditions of the jar animals, except in jars 1 and 2, was decidedly more intensely pathological than that of the controls. In comparing more definitely the two sets of animals it seems wise to exclude the guinea-pig that died first, four days after inoculation and after eight days in jar 5, because tuberculosis could not be assigned as the cause of death. Also, the first pig that died among the controls, 67 days after inoculation, should not have been included in the series, for it was evidently debilitated from the outset. Excluding these two subjects, we find that the average life of the four controls after inoculation was 185'5 days; while of six "jar" pigs, dying within the same period, the average duration of life after inoculation was l1u'3 days, an average excess 'of more than 69 days in the life of the control guinea-pigs. It has been mentioned that in replacing the deceased jar guinea-pigs from the controls care was taken, except in one instance, to select that control which seemed in best physical condition. It is also worth noting that during the major part of our observations antecedent to the introduction of a ventilating fan, the resthetic condition at least of the air of the overcrowded animal room left much to be desired. In pure air the control guinea-pigs might have done much better. Nevertheless, our jar guinea-pigs seemed happier and more lively when removed from the jars to their cage in the animal room. But the most significant evidence of a specifically deleterious effect of rebreathed air upon the lunga was revealed by the autopsies. In passing judgment on these records we realise that, in spite of much effort, our work was extremely crude. It is often difficult, even impossible, to allocate a series of lungs according to the extent of their tuberculous involvement. Comparisons are reliable only when very gross differences exist in the distribution and intensity of infection. and of W. S. Miller [4] have taught us 'l'he researches of Krause that the distribution of tubercle bacilli from the site of inoculation is measured within a few days 01' hours; that, indeed, the peripheral implantation of the transported bacillus and tissue reaction thereto, the

un

J une, H)24]

EFFECTS OF REURBATHED AIR

433

important thing, ea rly produces pr ofound and ge neral changes revealed to the micr oscopic but not to unaided VISIOn . We will reserve for the present discussion of th e condit ion s de te rm in ing th e fixat ion of the transported bacillus. It is a familiar fact that afte r subcutaneous or intrap er itoneal inoculation of t ubercle bacill i in guinea -pi gs t he in volvem ent of the lungs in disease is a late phenom en on. 'I' his latent per iod of pulmon ary disease is obviously largely determ ined by th e mass of infect ive ma terial inocul at ed. R eceiving one milli on th m illigram (0'00 000] mgm .) of bacill i, a guinea-pig of our jar series dying GH da ys afte r inoculat ion, sho wed no macroscop ic evide nce of pulm onary t uberculosis ; and one of th e controls, dying 155 day s a fte r inoc ulat ion, sho wed but doub tful posit ive pulmonary disease on ins pection. On t he othe r hand, in a piece of wor k ca r ried out by one of us seve n years ago in com pan y with Hegner and Po well [ 5J , in a group of 41 guinea -pigs inoculated either subcutaneously or int ra.pe ritoneally,each with 1 c.c.of a turbid su spen sion of what must have been a relatively enormous dose of virulent tubercle bacilli, the two earliest deaths occurred 21 day s after inoculation. In th ese cases the abdominal organs were diseased, but no macroscopic tuber cles could be found in the lungs. Similar negative pulmonary findings occurred in t wo animals dying, one afte r 24 and the ot her after 4U da ys, th ough in both these case s the tracheobronch ial nodes were notably involv ed, B ut in most deaths in th is series occurring after 28 days and up to the last decease, 115 days after inoculation, pulmonary tuberculosis Was plain enough . E ven so, in five cases, in guinea-pigs dy ing 59,67, 76, 76, and 115 days after inoculation, while very general tuberculosis prevailed , the lungs were noted as being involved in non-caseous bronchopneumon ia, but no macroscopic tubercles were discerned. It may be mentioned th at in this series, accordin g to macr oscopi c evid ence, implantation of tubercles in the kidneys, so rare und er ordinary dosage, was not un common . We may now proceed in lim ited detail to con sider the relations of rebreathed air to development of ex periment al tuberculosis as indi cated by the very meagre material at our dis posal. Tn fi gs. 3 and 4 are represented photogr aphs of the lungs of gu inea -p ig I of jar], and of gu inea-pig () of J ~r (i. 'I'h e former animal may, as regards any affection by rebreat he<1 all', be con sidered as one of th e controls. This guinea-pig had occupied the jar, more or less, fOI" foul' days antedating its inoculation. It seems probalile that its death was much hastened by the relatively high temperature, usually in excess of 2 ;')0 C., of its jar. It died after residence, for about on e-half the time, of 1:1!) da ys in the jar, supplied with fresh air, and l B5 da ys after inoculation. 'l'h ou gh its lungs were involved with a cons iderable ext en t of tubercul ous conso lidat ion , they collaps ed fairl y well on opening the chest, and th e pict ures sh ow , dec isively, how mu ch more ~xtens i ve was the patholog ical cha nge in the lungl-! of the guinea-pig in Jar (j, dying a fter only II Ii day s in th e jnr, and 11 ~ da ys after inoculation . 'l'he lungs of this ind ividual d id not perc eptibl y collapse on opening the chest. Our estimate of the relative extent of tuberculous involvement in lungs and abd ominal viscera of both groups of animals is indicated by the plus signs in the t able.

[June, ] 9:24

'lTBERCLE

~o pulmonary infection was found in guinea-pig :2, dying after Ci:2 days of confinement in jar 5. 'I'his animal's death was probably greatly a.cce lcrated by jar life, for it succumbed (it{ days after inoculation. As has already been mentioned, a control guinea-pig died li7 days after inoculation, but it WlLS a physically inferior subject: it showed no pulmonary lesions. Possibly a more reliable comparison of the effect of conditions of life within the jars and within the animal cages open to the air should be expected from examining the lungs after killing the still living animals. This was done on .Iuly (j in the case of the five rerua.i n i ng jar guinea-pigs, 212 to :2:20 days after their inoculation, and on ,J uly 7, in the case of the three remaining controls, 21il days after inoculation. As will be seen later, this method of observation was pursued in our second series of experiments.

FIG. 3.

FIG. 4.

FIG. 3.--Lnng" of guinea-pig from Jar 1, subjected to f resh The animal died 135 days after i nocu lat ion and 13:J day" of life in FIG. 4.- Lung" of guinea-pig from Jar G, subjected to foul The animal died 112 days after inoculation and IIG days of life in

air. the jar. air. the jar.

I n general, the pathological condition of the five guinea-pigs remaining in the jars on July 6, when all were killed by illuminating gas, was proportional to their length of residence in the jars. 'I'he records of the autopsies are given in the table. Possibly an adequate idea of the macroscopic evidence of the extent of infection can be given by denoting the degree of infection, from very slight to extreme, by the characters + to + + + + +. Only two guinea-pigs, occupants of ars ;1 and 4, continued to live in the jars under the conditions noted, from the beginning to the end of the experiment. Both had long presented signs of profound intoxication. The autopsies showed, especially in the animal from jar 3, relatively slight abdominal infection, but the lungs of

June, 19241

EFFECTS OF REBREATHED AIR

435

neither collapsed on opening the chest, and were nearly solid WIth tuberculous deposits. The guinea-pig in jar 3 deserves special mention. From near the beginning of the experiment this animal was peculiarly sonmolent, sitting flat with hair ruffled, and many times it was thought that it would succumb to disease, though its behaviour was probably the result of somnolence, a symptom due to a certain low percentage of C02, and especially mentioned elsewhere [:2]. An inordinate appetite characterised this animal and probably preserved its life. 'I'he conditions of guinea-pig ~ of jar 1 and of guinea-pig 4 of jar 5 Were noteworthy as showing how long, '21'2 days, the lungs can escape severe involvement in a general infection. 'I'he former was not exposed to rebreathed ail', and the latter had probably been confined too short a time, ~D days, for the development of pulmonary deposits depending on environment. The number of animals sacrificed in the foregoing experiments was, of course, far too few to establish any definite conclusions; but the evidence thus far gained indicates positively that guinea-pigs inoculated with a Small dosage of tubercle bacilli and subjected for a sufficient length of time to an environment of rebreathed air, so fouled as to contain from 0'5 to 2 per cent. of CO 2 , tend to an excessive development of pulmonary and a mediocre degree of abdominal tuberculosis, as compared with control animals similarly inoculated. The period of life, also, of guinea-pigs respiring used air is notably less than that of the controls. SECOND SEHlES OF THE EXPERIMENTS.

A group of guinea-pigs was inoculated precisely according to the technique and material previously used, except that the size of the dosage of tubercle bacilli WILS multiplied 1,000 times. That is, each guinea-pig received a subcutaneous injection estimated to contain one oue-huudrcd-thousamlth «roooon rnilligl'lLlll of virulent tubercle bacilli, human strn.in, On July 5, 1~J22, Dr. Corper prepILl'ed the water-clear suspensions and inoculated, as before, 14 young male guinea-pigs. Six of these were relegated to lifo in the jars and eight were reserved as controls. It was proposed to kill all these animals and compare their pathological conditions after allowing time enough for extensive development of disease but not enough to cause futa.litiea from tuberculosis. Within three weeks of their inoculation three of the controls died without apparent cause, until- it was found that, the attendant had heen washing the animal cage with a commercial disinfectant. ~Vhen this was discontinued the fatalities ceased. Six guinea-pigs of the series legan their lifo in tho jars on July 10, 1!J:J2. All were killed by illuminating gas o~ Septomber 2, after ILn interval of 54 days. During this period the six guineaPigS remained in the jars approximately 70 per cent. of the time. Re-examined On August 1, 23 dlLyS after inoculation, all the guinen-pigs showed distinct lymph node enlargement. with occasional ulceration, in the left groin (site of inoculation) ~nd slightly palpable nodes in some in the right groin. All IIlLd gained slightly In .weight except guinea-pig Ii of jar G, which had lost about !J per cent. Of its height: ){e-e~amined again on August :Jl, ~;l days after illocuhLt.ion, all animals ad glLlI10d weight, hut the controls hall gamed most and were m much better Ph,Ysical condition than the jar guinea-pigs, though tho wholo series showed eVidences of intoxicution. The jar animuls were gassed and autopsied on September 2, and tho controls On September 4. It was often difficult by inspection to be sure of the nature and

436

TUBERCLE

[June, 1924

extent of apparently pathological lesions in the lungs. In these, as in all other cases, the lungs were first studied in a state of collapse, and then when distended with 10 per cent. formalin injected by the trachea, after which they were preserved in the same medium. The pathological findings in the two sets of guineapigs were of very distinct lymph node enlargement, more especially marked in the JILl' animals, and of deposits in the spleen and sometimes in the liver, also more marked in the jar animals. The lungs of the guinea-pigs subjected to rehreathed air showed more evidence of tuberculous deposits than did those of the controls. Numerous microscopic preparations were made of the lungs of the animals in each series; for this work we are greatly indebted to Mr. Max Goldberg and D. M. B. Lurie. Hyperplasia of the lymphoid tissue WILS more in evidence in animals from jars 2 to 6 than in the controls. Nevertheless, we must admit that our estimates of the respective extent of pulmonary infection in the two groups of guinea-pigs of this series are founded on impressions rather than on measurements.

With this qualification, fatal to crucial evidence, the results of Series 2 of these experiments may be summarised in the statement that the pathological condition of the animals which had been subjected to rebreathed air, and especially the pulmonary infection, was more profound and extensive than that of the animals that had not respired such air and that there was more or less direct proportion between the deterioration in the air breathed and the pathological effects observed. THIRD SERIES OF EXPERIMENTS.

Series 3 of these experiments had the following design : In a number'of guinea-pigs, inoculated with a small dose of tubercle bacilli, allow time for the development of clinical tuberculosis and then subject six of the animals to confinement within the jars, and after a chosen period, submit the whole group to autopsy and pathological comparison. The procedure used was similan .to that previously outlined. Sixteen male guinea-pigs were inoculated subcutaneously by Dr. Cor per, each with one millionth (0'000001) milligram of virulent tubercle bacilli on July 8, 1922. Examined on August 1, after 26 days, a variable degree of lymph node enlargement was found in the left groin of each animal. On September 5, 59 days after inoculation, six of the guinea-pigs were placed in the jars. Unfortunately, a high mortality affected the animals of this series. Eight of the 16 guinea-pigs died during the course of the experiment, including two that had been assigned to the jars. These deaths were possibly due to poison in a disinfectant referred to above.

Autopsies on the control guinea-prga dying 2, (i, 18 and 25 days after inoculation showed no pulmonary tuberculosis. Autopsies on the guinea-pigs dying in jar 2, 74 days after inoculation and after 15 days of jar treatment, showed extensive lymph node, and abdominal tuberculosis, but the lungs were negative. The guinea-pigs from jar 4, dying on the same date, showed It few apparent tubercles in the lungs in addition to extensive abdominal affection. All the remaining guinea-pigs were gassed September 20,80 days after inoculation and 21 days of treatment of the jar guinea-pigs. In most cases we were not able to decide, from macroscopic appearances, whether or not the lungs were tuberculous. '1'0 this statement exception must be

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EFFECTS OF RKUREATHED AIR

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made in the case of the animal from jar (i, whose lungs showed several well-marked tubercles. On e of th e con t rol pigs also presented an apparentl y ch eesy area in one lung. This animal was in very bad clinical condition when sacrificed . For some reason the clinical deterioration of th e animals of th is series was far greater than seemed accountable for by the extent of th eir tuberculosis, and we venture to draw no definite conclusions from the experiment. Possibly the guinea-pigs of this group were the forerunners of an ep ide m ic disease that carried otI a num bel' of our animals during the autumn. S L"J.DIARY AXIl C ONl" LC S IO NS.

The authors of thi s research venture to put it on record because it seems to strengthen the foundation for a promising working hypothesis rather than with the presumption of furnishing crucial evidence for a theorem. It may he reasonably maintained that guinea-pigs, inoculate'd with 0'000001 mgui. virulent tubercle bacilli, when confined for long periods in well-cleaned, communicating jars, ventilated by an air current which allows the accumulation of 1 ';", to ~ per cent. CO 2 of respiration in the last jar of the se r ies, are subject to a higher mortality and somewhat more rapid and profound extension of disease than are control animals maintained in ope n cages. These results do not prove that fouling of the ventilating current by rehrel1thed air is the onl y deleterious agent in the case. Mere confinement of the aniuial at a relatively high temperature, hut in pure air, as witnessed by the first occupant of jar 1, of the first series, is probably inimical to its health. Nevertheless, there is distinct evidence that the animals resid ent in jars ;j to ti, inclusive, suffered fr om more extensive tuberculous invasion than the controls. In other w ords, the deleterious effect of confinement ill the jars was roughly inverse to the purity of the ventilati'ng cur re n t as est im at ed hy its con t en t of C O~ . Finally, the eviden ce collected ind icates that in rebreathed air the tuberculous process is som ewhat prone to select ive invasion of the lungs, with possibly complementary escape fr om infection of the abdominal Ol· gans. In view of the evidence presented elsewhere [2J for a specific sensitising action of the products of respiraton, we are encouraged to associate the fOUling of rebrcathed air with the deposition of tubercle in the lungs.

RKFBmmCES. M. .r., and AMOSS, n. L. JOIITlt. Mcd. Res ., 1911,35. H., and GUTSTEIN, H. AmcT. Journ , lIyg., 1923,300. KHAlT8J<:, A. K ..Imcr. J:CI'. ru«, HI:2:2, 1. [41 Mu.i.au, W. S. Ibid., 1\11\1, 6'-'!). 15J 81<:\\1,\1,1" 11., Ih:uxEH, C. F., and Powxr,r., C. iu«, 1917,220.

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