PUBLIC HEALTH REPORTS VOL. 40 APRIL 24, 1925 No. 17 VIABILITY OF B. TYPHOSUS IN STORED SHELL OYSTERS By CONRAD KINYOuN, Assistant Bacteriologist, hlygienic Laboratory, United Stztes Ptiblic Ilealti Serviee The object of this work was to determine whether oysters con- taminated with B. typhosuis and then stored unider uisual market conditions woul(l remain potentially infectious over a length of time sufficient to allow them to reach the consumer. Conflicting opinions are now current as to the length of time the causative agent of typhoid fever can remain viable in the oyster, and even as to whether the oyster can harbor the organisms at all. Obviouisly an oyster which harbors typhoidl organismns for as short a time as 24 hours becomes a potential infecting, agent for thlat time. Practi- cally it is of interest to know whether the time elapsing between the remov-al of the oyster from the bed and( actual consumption after passing through customary commercial channels is sufficient for oysters to rid themselves of possible infection. As early as 1603, oysters were incriminate(d in intestinal disor(lers, when suspicion was directed toward them by an illness of Henry IV of France (7). It was not uIntil the close of the nineteenth century, however, that oysters and shellfislh as agents of (lisease transmission receive(d particular attention. In October, 1894, Conn focused attention on the oyster by his investigation of the now famous Wesleyan outbreak, an(d thoughl only thlree outbreaks of typhoid fever were definitely traced to the oyster before 19,25, these stimulated wide interest and consequent study, with atten(lant epidemiological and bacteriological investigations. It is agreed that the medium of infection of oysters with B. ty- phosuis is water; but there is a wide (livergence of opinion uipon tlhe viability of the organisms in water. De Giaxa (12) fouind( numerous typhoid bacilli nine dtays after inoculation in ordinary sea water an(d 25 days after inoculation in steiilized sea water. I-le ma(le no ex- aminations later than thlese. Cassedebot, quoted by Frankland (1), foun(d the typlhoi(d organiisms (lestroyed in sterilize(d sea water within 48 houirs. Frankland (1) found 1 per cent and 3 per cent salt water prejui(licial to ty)hoid. Foote (2) conieludes that even in extremely cold weather, typlhoid bacilli will live in unsterilized brackish water (0.06 to 0.15 per cent salt) at least eight days, wlile in warnm(er 36275--25t-1 (819) April'24, 1925 8.120 water they rapi(dly (limiinish in numbers and can not be (letected after thlree weeks. Iler(linan and Boyce (3) conclude that typhoid bacilli w-ill not flourish in cleani salt water. Klein, quotedl by Reille (7), states that according to his experiments sea water is favorable to the survival of typhoid, an(l Reille (7) corroborates this finding. Burdoni et al., quoted by Conn (10), "proved the typhoid bacillus would live in sea wN-ater for 14 days." Dc Freytag, (11) found that the typhoid bacillus would live in concentrate(d salt anid gelatin solution for five months, buit not six months, at room temperature. To check the viability of the cultures used in tile present experi- nent, flasks containing 500 c. c. of 3.5 per (lent andl 30 per cent salt water were sterilized, then inoculated with a half slant eaclh of the Rawling and the Hopkinis strains of B. typhosus and kept at room temnperature. From day to (lay 0.1 c. c. from each flask was trans- ferred to Endo )lates and glucose brotlh fermentation tubes; and wlhen growth resulted, it was proved to be B. typhosus. This test slhowed that in 3.5 per cent sterilized salt water the organisms remained viable during the course of tile experiment-15 days- while in the 30 per cent salt B. typhosus was not proved after 24 hours. As to viability in the oyster itself, Harrington (13) cites the conclusion of Polak thlat during transportation the life processes of tlhe oyster have an inimical influence upon bacteria, diminishing their numiiber and, in certain cases, destroying them entirely; but Harrington states tiat this conclusion is opposed to that of others who liad found tilat the typlioid fever organisms live longer in the tissues and juice of the oyster than in the sea water. Field (6) cites experiments slhowing thlat mnany oysters are damaged and destroye(d during storage and trainsportation, thlus affording a favor- able mediunm for the multiplication of typhoid organisms. Previous laboratory work gives a range of infection lasting from eight days to six weeks. Foote (2) stated that, with oysters kept at a teulperatuire ranging from 500 to 65° F. (100 to 18.30 C.), the typhloid orgranismn lives longer in the stomiach and in the juice of the oyster thain it does in the water in whichi the oyster grows, and that if therc is an increase in organisms this increase takes place within the first two weeks, after whicil tilere is a decrease, but that the typhoid bacillus may be found even 30 (la-s after the date of in- fection. Boyce and lfcrdxnan (4) recovered typhoi(l organisms 14 days after infection; Fieldl (5, 6) 9 (lays after inf'ection, an(l from 4 to 6 weeks if oysters were cooled; Reille (7) froml 9 to 28 days; Klein fromn 7 to 11 (lays; Stiles (8) from 7 to 21 days. Park (14) states that infection is transitory and that oysters usuallv cleanse themnselves in lrom 6 to 8 davs. Gorham (9) gave an opinion, based on the seasonal counts, that oysters Iiiberniate. 81) April 24, 1:)25 Following this suggestion as to oyster hibernation, the New York City health departinent slhowed that infection (lid not occur in hibernating oysters whlen the surrounding wateI had 13O(M'() typhoid bacilli per c. c. Pease, *luoted by Patirk; (14), states that at 2.2° C. oysters close their slhells so tightly as to be im1pervious to particles of dyes in aqueous solution. The oysters use(l in the tests lhere reporte(l were of a large salt- water variety, tong(e(l fromi Tangier Sound(I in the Chesapeake Bay and broulgilt to Washington by power b)oat. By the tine they had reached Washington they had been out of water eihlt (lays. Two buslhels were purchiase(d oIn February 4, 1925, brought to the Ily- gienic Laboratory, and stored outdoor-s for the night. The followitng morning they were brought insi(le and cleaned roughly by scrubbing with brush an(l cold tap water. They were then placed in 6 large glass aquarium jars, each containing about 16 liters of sterilized tap water to whiclh 3.5 per cent sodijum chlloride C. P. had been ad(le(l. The amount of water was sufficient to cover the oysters generously. Air was passe(l througli this watet in a slow stream, allowingr ample aeration. After two lhours, when the temn- perature in tile jars ranged from 240 to 280 C., they were eaci inoculated withi two-thir(ds of a liter of 24-hour bouillon culture. The inoculum consisted of 2 liters each of Rawlings and(i Hopkins strains of B. typhossus. Previous to inoculation it was notice(d that the oysters had opened their shells. Six in each jar were tested for viability. On slight stimulation all closed their shells. During the night, air was passed through the water every two hours for 10 minutes. Cultural checks were made of the cultures used for inoculation. After 24 hours' feeding, the oysters were taken fromn thc aquariums, drained, and placed in galvanize&l-iron buckets. One portion was placed in a laboratory cold roomn, which is maintained at 100 C., and the other portion, covered with ice, was placed outtdoors. The, latter was re-ice(Idaily. During, thie test the mnean (laily outside temperature range(l from 27° F. to 580 F. (-2.80 C. to 14.40 C.), with an average of 430 F. (6.10 C.). From day to day individuals from each lot NweLre opened and cultured. Opening wtls preceded hy flaming the slhell thoroughly with a Bunsen buiner, pryina the shell apart witlh a flame(d oyster knife, and cutting the attachmneiit miiuscle of the right slhell. Cultures were obtained from the slhell liquidl with a pipette, anid from the stomnach by searing, dissecting, out anid opening the stomachl, and culturing with a loop, all un(ler aseptic conditions. The cultures were made on Endo plates, from which colonies were fislhed to Russell's double sugar me(liun, and to glucose brothl fer- mentation tubes. These presuimiptive tests were followed by testing April 24, 1925 8'22 motility, agglutinating power and indol production, action upon gela- tin, and fermentation reactions upon levulose, sorbitol, dulcitol, lactose, sailicin, iiositol, anI( dcxtrine. Unless an organism reacted typically to all these criteria, it was not considered to be B. typhos ts. Motility wtas observed from 24-hiour broth cultures, which were also used for aggcrlutinating tests. The latter were performed on microscopic slides by a(ldingr a (drop of culture to a dilution of monovalent seruni. Indol pro(luction was tested by moistening tile plugs of control tubes on eachl culture with a concentrated solution of oxalic acidl. Enlows' lne(liuln was use(d for the fermentation reactions. Adequate controls on a1llmedia and reactions were run. No atteinpt was made to estimate the nuimber of typhoid colonies otuantitatively on the plates from day to day. It was noticed, how- ever, that -from a very larg,e number at the first, there was no appre- ciable diminution until after thc eighthl day.