Shigella strains isolated by the writer near Oran, Algeria, in September, 1943,

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1 AN ADDITIONAL SHIGELLA PARADYSENTERIAE SEROTYPE' WILLIAM H. EWING2 Received for publication December 1, 1945 In recent years new Shigella strains have been reported by several investigators. Boyd (1940) described a number of Shigella paradysenteriae serotypes from India, many of which were found to have wide distribution. Working with microorganisms isolated in India and the Middle East, Sachs (1943) found Shigella cultures belonging to the group which do not attack mannitol. The occurrence in India of non-mannitol-fermenting microorganisms which differed antigenically from both Shigella ambigua and Shigella dysenteriae was mentioned also by Boyd. Two such strains of Shigella were studied in North Africa (vicinity of Casablanca and Bizerte) by Christensen and Gowen (1944). Type A and type B of Christensen and Gowen were found to be identical with two non-mannitol-fermenting Shigella strains isolated by the writer near Oran, Algeria, in September, 1943, and identical with Shigella sp., Sachs Q771 and Q1167, respectively. Maclennan (1944) and others also studied members of this group in the Mediterranean area. Shigella strains which were similar to those. described by Sachs were reported from the United States by Gober et al. (1944). In the course of our work in the Mediterranean area during the past 2 years a number of microorganisms were encountered which differed serologicaily from known Shigella strains. The purpose of this communication is to present the results of an investigation of one of these-a Shigella paradysenteriae by virtue of its biochemical reactions but serologically distinct from recorded types. In the following pages this serotype will be referred to as Shigella paradysenteriae, Lavington I. SOURCES OF CULTURES Since the history of the isolation of this microorganism is somewhat involved, it is presented in detail so that possible confusion in the literature may be avoided. Apparently the Lavington I serotype first was isolated in Casablanca during May and June, 1943, by Major A. H. Stock of the 2nd Medical Laboratory (Stock, 1945). The microorganism occurred in epidemic proportion in a United States Army installation and was isolated from the feces in 214 cases of bacillary dysentery. Although the cultures presented biochemical characteristics of Shigella'paradysenteriae, they did not agglutinate in any of the available antisera. It was concluded by Ernst and Stock (1943) that the microorganism was probably an additional serotype. In January, 1945, Stock submitted his type T culture to this laboratory for comparison with our unclassified cultures. A small quan- 'This investigation was made in the Bacteriology Section of the 15th Medical General Laboratory, United States Army, located at Naples, Italy. 2Major, Sn. C., Res. With the technical assistance of Sergeant James L. Gravatti. Present address: New York State Veterinary College, Cornell University. 433

2 434 WiLLIAM H. EWING TABLE 1 Sources of cultures CUI.TU NUMBER SO8JKCZ OcAz.ny ISOLATUD BYOX ZCKIVED 2-24 (Type T)*. Lavington It. Lavington I*t * * * t t Patient, Italian Carrier, Italian Food handler, German Casablanca England England France France France N. Italy N. Italy N. Italy N. Italy N. Italy Stock, 2nd Med. Lab. Francis, Emergency Vaccine Lab. Heller, 1st Med. Gen. Lab. Francis Heller * Cultures were used for antiserum preparation. t Both Lavington I cultures originated in Dr. Joan Taylor's laboratory. t Two cultures were isolated from the same individual. Heller 262nd Station Hosp. 262nd Station Hoop. 262nd Station Hosp. 262nd Station Hosp. 2nd Med. Lab. 2nd Med. Lab. 2nd Med. Lab. 2nd Med. Lab. 2nd Med. Lab. U. S. Hospital Ship 300th General Hosp.

3 SHIGELLA PARADYSENTERIAE. BEROTYPE tity of antiserum made with the type T culture in 1943 was also supplied. Stock's type T is in our series. During early March, 1945, an antiserum (labeled Lavington I) prepared with a new Shigella paradysenteriae type was received from Lieutenant-Colonel A. E. Francis, R.A.M.C., of the Emergency Vaccine Laboratories, East Everleigh, England. Francis received cultures of the new type from Doctor Joan Taylor, of Oxford, who isolated it in England (20 cases) and from Captain George Heller, of the 1st Medical General Laboratory, who recovered a number of cultures from cases in France. Francis (1945) found that Taylor's Lavington I and Heller's type culture (6900) were identical, as shown by reciprocal adsorption tests. The Lavington I and strain 6900 cultures were received from Francis during May, 1945, as were cultures 8944 and 8945 from Heller. Cultures , 607, 608, 613, and were isolated from cases in northern Italy in the vicinity of Florence and Modena by Stock and personnel of the Second Medical Laboratory during the latter part of 1944 and the spring of The remaining cultures were isolated by various United States Army hospital laboratory workers and sent to this laboratory for identification (7 cultures), or were originally isolated here (16 cultures). The sources of individual cultures are listed in table 1. BIOCHEMICAL STUDIES The cultures isolated here were recovered on SS agar (Difco), but Stock's were picked from desoxycholate citrate agar (D.C., Baltimore Biological Laboratory). The colony form on these media, on eosin methylene blue, and on plain agar plates conforms to the descriptions recorded for other dysentery bacilli. Variation of the S to R type was noted in most of the cultures, although no completely rough forms were seen. Small colony variants appeared in some cultures, but these were not studied in detail. Carbohydrate media were prepared by adding Seitz-filtered solutions of the various test substances to an autoclaved basal medium made up of beef extract (0.3 per cent), peptone (1.0 per cent, bacto), sodium chloride (0.5 per cent), and Andrade's indicator. The final ph of this medium was about 7.2. Glucose, lactose, sucrose, and glycerol were each employed in 1.0 per cent concentration. The other carbohydrates were used in a concentration of 0.5 per cent. Simmon's citrate agar (Difco) was employed in tests for citrate utilization. The Voges- Proskauer and methyl red tests were done with 24- and 48-hour cultures, respectively, grown in MR-VP medium (Difco). A 40 to 50 per cent (weight per volume) potassium hydroxide solution containing 0.3 per cent creatine was employed in the tests for acetylmethylcarbinol production. Indole tests were made by two methods. The first was the Gnezda test in which filter paper strips were impregnated with a saturated solution of oxalic acid, dried, and suspended over 2.0 per cent peptone water. The alternate method for indole was the standard 1.0 per cent tryptone water culture tested after 24 hours' incubation with Kovac's reagent. Tests for the production of hydrogen sulfide were made with the aid of strips of filter paper soaked in a saturated solution of neutral 435

4 AS WILLIAM H. EWING lead acetate, dried, and suspended over the 2.0 per cent peptone water cultures. Many Shigella strains produce blackening of such papers. Motility tests were made with two media, the first of which is a modification of the Jordan et al. medium used by Salmonella investigators both to determine motility and to separate phase variants (Edwards and Bruner, 1942). The alternate motility medium was prepared by adding 0.25 per cent agar to 1.0 per cent tryptone water. Both motility media were inoculated with a loop at the top of the medium column. Tests for urease production were made with a medium devised by Christense (1944). Since the formula for this medium has not to our knowledge been published, it will be included here: distilled water 1 liter, glucose 1.0 g, peptone (Difco) 1.0 g, sodium chloride 5.0 g, monobasic potassium phosphate (KH2PO4) 2.0 g, phenol red g, and agar 20 g. The ph is adjusted to 6.8 to 6.9, and the medium is sterilized in the autoclave. When the basal medium has cooled to 50 to 55 C, 80 ml of a Seitz-filtered 20 per cent urea solution are added, and the medium is tubed in approximately 3-ml quantities in Loeffler tubes and allowed to solidify so as to have a deep butt and a short slant. A heavy inoculum is made on the slant without stabbing. Although some Aerobacter cultures may yield a positive or doubtful reaction within- 18 to 24 hours at 37 C with this medium, as compared to a delayed trace or entirely negative reaction with the highly buffered medium of Rustigian and Stuart (1941), no Shigella or Salmonella species thus far tested has given even a trace reaction after prolonged incubation. The Christensen medium is an exceptionally valuable one in routine work for the early recognition of Proteus and paracolon Aerobacter cultures. All cultures which yield an acid butt and q. alkaline slant on Kligler's medium (modified with added sucrose), regardless of gas or blackening from hydrogen sulfide, are inoculated onto the urea medium. Cultures which are positive at a preliminary reading made after 3 to 4 hours' incubation at 37 C or which show trace or doubtful reactions following overnight incubation are eliminated from subsequent serological and biochemical tests. Used in this manner the urea medium has aided materially in the examination of thousands of feces specimens. For the differentiation of Proteus cultures, however, the medium of Rustigian and Stuart is essential. Tests for the reduction of trimethylamine oxide were made by the method of Wood and Baird as outlined by Weil and Black (1944). The tests were performed with 24-hour cultures on two separate occasions with two lots of medium. Appropriate positive and negative contrbls were included on each occasion: Shigella sonnei and Shigella dispar cultures were positive, and microorganisms of the S. paradysenteriae Flexner group (I through VI) were negative, as were several cultures of S. dysenteriae. All biochemical tests were incubated at 37 C with the exception of motility tests, which were incubated at about 28 C on top of the incubator (7 days). Negative carbohydrate media were held for periods varying between 28 and 38 days. Gelatin and citrate tubes were kept for at least 21 days, the gelatin being

5 SHIGELLA PARADYSENTERIAE SEROTYPE testd for liquefaction at intervals of 4 or 5 days. The final reading of the urea medium was made after 7 days' incubation. The biochemical and physiological reactions of the first 27 cultures listed in table 2 were studied in some detail. The remaining 7 cultures were tested with only those substrates customarily employed in the identification of dysentery bacilli. All cultures were nonmotile, methyl-red-positive, and Voges-Proskauer-negative. Sodium citrate was not utilized, gelatin not liquefied, nor urea hydrolyzed by any of the cultures. Indole was produced by all cultures, and tests were positive by both methods. Three cultures (24-605, Lavington I, received from Francis, and 6900) produced a slight yellow-brown discoloration of the lead acetate papers after 48 hours' incubation; the other cultures were entirely negative. Acid but not gas was produced within 24 hours from glucose, mannitol, arabinose, and xylose by all cultures. As indicated in table 2, maltose utilization was delayed. All cultures produced a small amount of acid in maltose broth on the first to the fourth day as evidenced by a pink coloration. Further incubation resulted in eventual strong acid production with a bright red medium in 3 to 35 days. Had a 21-day observation period been used, several of the cultures would have been recorded as negative or as doubtful. Sealing the maltose tubes with corks dipped in paraffin tended to hasten the reaction in most cases. Strong acid reactions were obtained after 2 to 12 days' incubation with 27 cultures so treated. There was one case in which acid was produced from maltose on the fifth day in both the sealed and unsealed tubes; also there was an instance in which a longer period was required in the sealed tube. The 27 cultures were retested in maltose broth, ph 7.4, with bromeresol purple as the indicator. The weak acid reactions were not noted with this indicator, but definite reactions were apparent after 3 to 12 days' incubation, the majority of cultures requiring 9 to 11 days. ' Six cultures (table 2) fermented sorbitol within 24 hours, sixteen required 48 hours, and the remaining cultures required 7 to 8 days before strong acid reactions occurred. Weak reactions were noted in sorbitol broth in 1 to 5 days; these became strongly acid upon further incubation. Four cultures (table 2) that were negative by the usual method of testing produced acid reactions in sealed tubes. All cultures failed to ferment lactose, sucrose, salicin, dulcitol, rhamnose, and adonitol after prolonged incubation periods of 28 to 38 days at 37 C. Acid without gas was produced within 24 hours from trehalose, galactose, and fructose by 27 cultures tested on these substrates. Likewise, acid reactions occurred in glycerol broth after 6 to 13 days. Slight acid production was noted in glycerol on the third to the sixth day of incubation. This same grou'p of 27 cultures failed to ferment inositol and raffinose. Cellobiose was not utilized in 38 days by any of 25 cultures tested on it. The group of 27 cultures reduced trimethylamine oxide. Two cultures (m 280 and 8-770) of S. paradysenterae, Boyd V (P.143), were tested at the same time as the group of 27 cultures of the 437

6 438 WILLAM H. EWING TABLE 2 Biochemical reactions of Shigella paradysenteriae, Lavington I, cultures CGLUCOSE, LACTOSE, ~wr AJWMOSE, ~~ MOTILITY CULTURE M NITOL, SUCROSE MALTOSE SORBITOL ADOMNTOL, AT GLATI XYLOSE, SALICIN DULCrTOL X.. VP UREA ARABINOSE 2-24 (Type T) A (A) Lavington İ A - (A) (A) + Iavington İ A (A) (A) A - (A) (A) A - (A) (A) A - (A) (A) A (A) (A) A (A) (A) A (A) (A) A (A) (A) A (A) A A (A) (A) A.-.(A- A (A) A0- (A2-- A (A) A (A) (A) A (A) A A (A) (A) A - (A) (A) A (A) (A) A (A) A A - (A) A A (A) (A) A (A) A A - (A) (A) A (A) (A) A (A) (A) A - (A) (A) A (A) (A) S.A - (A) A A (A) (A) A (A) (A) AA - (A) A - (A) (A) A (A) (A) ( )indicates delayed reactions, 48 hours or more. Lavington I type. Except for the fact that neither culture fermented maltose (30 days) and one culture failed to produce acid from glycerol, the results were identical with those listed for the Lavington I type (table 2).

7 SHIGELLA PARADYSENTERLAE SEROTYPE 439 BEROLOGICAL STUDIES -. Methods. In addition to those received from Francis and Stock, antisera were prepared with the Lavington I, 8944, type T (2-2-4), , and cultures (table 1). Cultures selected for immunization were plated, and smooth colonies were picked to infusion agar slants in 16-by-160-mm culture tubes. After incubation for 20 to 24 hours the slants were tested for smoothness in 1:500 acriflavine, and in 1:10 Lavington I antiserum for agglutinability. The growth was removed with formalinized (0.3 per cent of 40 per cent formaldehyde) solution, 10 ml per slant. Vaccines so prepared were injected into rabbits at 3- to 4-day intervals in doses increasing from 0.5 ml to 5.0 ml. The first dose was administered subcutaneously and subsequent doses were given intravenously. The animals were test-bled a week following the last injection, and, if the titer was found to be satisfactory, the animals were bled from the femoral artery. Antisera were preserved with a mixture of equal parts of ethyl ether and phenol, ml per 1.0 ml of serum. Bacterial suspensions for agglutination were prepared in the manner employed by many investigators (e.g., Wheeler, 1944). Antisera were diluted serially, 1:20 to 1:5,120, in the usual manner, and all tests were read with the unaided eye following overnight incubation in a water bath at 50 to 52 C. Antisera to be adsorbed were diluted 1:10 and added to an adsorbtive dose calculated to be slightly in excess of that required. Adsorbing suspensions remained at 37 C for 20 to 22 hours; after this they were placed in the icebox for 24 hours. An adsorbing dose consisting of the growth from two 90-mm plates of infusion agar (20- to 22-hr cultures) effectively removed the homologous agglutinins from 1.0 ml of 1:10 antiserum. There were two exceptions to this, in which cases a larger adsorbing dose was necessary. In the experiments designed to determine the serological relationship of the Lavington I cultures to known Shigella strains, preliminary tests were made employing only three dilutions of antiserum, 1: 50,1:100, and 1:200, and a control. If positive or doubtful results were obtained with any antiserum, that antiserum was diluted serially and the suspension retested. Results. (1) It was noted that the Lavington I antiserum supplied by Francis as well as the type T antiserum from Stock agglutinated antigens 2-24 (Stock's type T), Lavington I, 8944, and our culture to titer. It was then established that adsorption of the Lavington I antiserum or of type T antiserum by any one of the cultures removed all agglutinins for the homologous culture. (2) Antisera were made with cultures isolated in Africa (2-2-4), England (Lavington I), France (8944), and Italy ( ). These four sera and their homologous cultures were used in another group of adsorptive tests. Master dilutions were made with each of the four unadsorbed antisera, and the dilutions were tested with the homologous and three heterologous cultures. Each antiserum agglutinated the four suspensions in dilutions of 1:2,560 to 1:5,120. Each of the four antisera was then adsorbed with four cultures, the homologous

8 AAA WILLIAM H. EWING and three heterologous. The 16 adsorbed sera thus obtained were then tested with the same four cultures. The results of these 64 tests were negative. (3) An antiserum prepared with culture (table 1) was employed in a third series of adsorptive tests to ascertain whether or not any differences could be noted between the various cultures isolated in Italy and elsewhere. Antiserum agglutinated all cultures in a dilution of 1:5,120, and a few cultures exibited weak reactions at 1:10,240. This antiserum was adsorbed with the first 24 cultures listed in table 1. Each of the 24 adsorbed sera thus obtained was tested with suspensions of the homologous ( ) culture and the adsorbing culture. In each case the adsorbing culture removed all agglutinins from the antiserum. (4) Studies designed to reveal the serological relation of the Lavington I cultures to other Shigella types were undertaken. Preliminary tests had indicated that the microorganisms bore little or no serological relationship to previously described types. Formalinized. suspensions were prepared in bulk with cultures , Lavington I, 2-24, and These suspensions were tested for agglutination in three dilutions (1:50, 1:100, 1:200, and control) with 41 unadsorbed Shigella antisera. Readings were made after ovemight incubation in a water bath at 50 to 52 C. No agglutination occurred with antisera of the Shigella paradysenteriae, Flexner group, types I through VI, in any dilution tested. Three Flexner II antisera were employed. Likewise, negative results were obtained in antisera prepared with members of the S. paradysenteriae, Boyd group: Boyd I (170), Boyd II (P.288), Boyd III (D.1), Boyd IV (P.274), Boyd V (P.143), and Boyd D.19 (Boyd VI). Two Boyd V antisera were employed. No agglutination occurred ini an antiserum induced by culture (S. paradysenteriae serotype with strong serological relation to members of the Boyd group, especially Boyd IV and V, and some Flexner group relationship-unpublished data; Francis, 1945; Wheeler, 1945). The Shigella alkalescens antiserum employed was one prepared with culture 1-2 which has antigens A, B, C, D (Stuart, 1944). No relationship to S. alkalescens was detected. The four suspensions were also tested in antisera made with five microorganisms which have the biochemical reactions of members of the S. paradysenteriae group but which differ serologically from established types. The four Lavington I cultures were unagglutinated by these antisera in three dilution tests. No significant relationship between the Lavington I and the lactose-positive Shigella cultures was detected. The four suspensions were tested with 1:50, 1: 100, and 1: 200 dilutions of three Shigella sonnei antisera (two S and one R) and with six Shigella dispar antisera. The latter included types I, II, III, and IV (Carpenter, 1943, 1944), a Shigella madampensis antiserum supplied by the Army Medical School, and an antiserum made with culture , isolated in Italy. Culture bears some serological relationship to Carpenter's type II. The 1:50 dilution of S. madampensis and one of the S. sonnei (S, 5493) antisera were doubtful, but it was found that when these sera were diluted serially 1:20 to 1:1,280 a doubtful reaction occurred in the 1:20 but not in the higher dilutions.

9 SHIGELLA PARADYSENTERIAE SEROTYPE Antisera prepared with Shigella sp., Sachs Q771, Q1167, Q1030, Q454, Q902, and J.P.6 were tested with suspensions of the four Lavington I cultures. A doubtful result was obtained in the 1:50 dilution of the Q771 antiserum but not in the two higher dilutions. This antiserum was diluted serially and the suspensions retested. The repeated tests were doubtful at 1:20 but were negative in all higher dilutions. The Lavington I cultures were negative in the other five Shigella sp., Sachs, antisera in all dilutions of the three-tube test. Antisera received from Francis against the two motile Sachs types (B.81 and B.105) also failed to agglutinate the Lavington I cultures. Lastly, the Lavington I suspensions were tested with a S. dysenteriae antiserum furnished by the Army Medical School and with a S. ambigua serum. Agglutination was not apparent with these antisera in the dilutions employed. (5) The next procedure investigated was the converse of the tests just described. Formalinized suspensions of various Shigella cultures were prepared and tested with native antiserum (table 1) in dilutions of 1: 20 through 1: 640. Suspensions of the following cultures were tested: S. paradysenteriae, Flexner I, II (two cultures), III, IV, V, and VI; S. parady8enteriae, Boyd I, II, III, IV, V, and D.19; culture 2-193, S. sonnei (two cultures), S. alkalescens, S. madampensis (Army Medical School, 43-M4), and culture ; Shigella sp., Sachs Q771, Q1167, Q1030, Q902, A.12, and J.P.6; S. ambigua, S. dysenteriae, and a number of unclassified cultures. With four exceptions, these suspensions were unaffected by antiserum Culture 2-193, , and S. sonnei (Kasauli) agglutinated weakly (1+) in the 1:20 dilution. One S. sonnei culture ( ) was agglutinated in the 1:80 dilution. Antiserum was absorbed with the S. sonnet culture. The factor causing weak agglutination was removed, but the titer of the antiserum for the homologous culture was not altered. (6) Although S. ailkalescens and S. paradysenteriae, Boyd V (P.143), cultures did not agglutinate in antiserum and cultures of the Lavington I type were not agglutinated by S. alkalescens or Boyd V antisera, the possibility of the presence of "hidden" antigens in these cultures which might be related was investigated. S. alkalescens antiserum (1-2) adsorbed with culture and tested with the homologous S. alkalescens (1-2) and cultures was found to be unaltered by this treatment. Likewise, adsorption of antiserum with a culture of S. alkalescens did not affect the titer for the homologous culture. Antiserum was treated with a Boyd V (P.143) culture (8-770). No reduction of titer was noted when the adsorbed antiserum was tested with culture Nor did adsorption of Boyd V antiserum with culture result in any alteration of its titer for the Boyd V culture. (7) Slide agglutinative tests were made with heated (100 C, 30 minutes), unheated, and alcohol-treated suspensions of cultures , 8944, 2-24, and Lavington I. Unadsorbed antisera were used in dilutions of 1: 5 or 1: 10. The 41 antisera employed in the three dilution tube-agglutinative tests, together with Shigella sp., Sachs A.12, two polyvalent Flexner antisera, and five Lavington I antisera (table 1) were used in the slide tests. 441

10 WILLIAM H. EWING The unheated, heated, and alcohol-treated suspensions of the four Lavington I type cultures were rapidly and completely agglutinated by all antisera of the Lavington I type at 1:10 dilution. Doubtful, delayed results were obtained in S. sonnei (S) and antisera, dilution 1:5. The alcohol-treated suspension of culture 8944 and the heated suspension of the Lavington I culture reacted slightly with S. sonnei (R) serum at 1: 5. Tests with all the other antisera were uniformly negative with the three kinds of suspension. Formalinized suspensions of cultures , , and , and heated suspensions of the last two, were tested in 19 "Shigella diagnostic serums" (Lederle) with negative results in all instances. (8) Serological studies on the possible relationship of the Lavington I serotype to Salmonella were made by Captain D. W. Bruner. Eighteen of the cultures listed in table 1 (including 8944, 6900, and Lavington I) were given to Bruner, who prepared alcohol-treated antigens from them in the manner in which Salmonella 0 antigens are prepared (Edwards and Bruner, 1942). The 18 antigens were tested in various antisera containing agglutinins for all the described 0 antigens of the Salmonella group. The antisera were employed in the dilutions used in diagnostic work. No relationship was detected between the antigens of the 18 cultures and the Salmonella somatic antigens. SUMMARY AND DISCUSSION Biochemical and serological studies were made with a group of 34 cultures recovered from cases of bacillary dysentery and from carriers in North Africa, England, France, and Italy. The purpose of the investigation was to determine whether or not the cultures isolated in the different localities were the same and to determine what, if any, relationship the cultures might have to known Shigella and Salmonella strains. The biochemical studies clearly indicate that the microorganisms belong to the genus Shigella. The cultures compose a biochemical type made up of nonmotile, gram-negative rods, which are anaerogenic and do not produce acetylmethylcarbinol, utilize citrate, or hydrolyze urea. Lactose is not fermented and hydrogen sulfide is not formed. The criteria for entrance into the genus as listed in Bergey's Manual (1939) are therefore fulfilled. Further, the more limiting definition of the genus Shigella given by Borman, Stuart, and Wheeler (1944) would also include them except that the question of growth and of acid production at 45 C is unanswered, and the cultures have not been tested on bismuth sulfite agar. The present group of cultures is remarkably uniform in carbohydrate utilization, the only irregularities being the delayed fermentation of maltose and the variable results with sorbitol. Fermentation of mannitol and lack of acid production from lactose establishes the type as a Shigella paradysenteriae. The biochemical activities of the type are identical with those of S. paradysenteriae, Boyd V (P.143), as listed by Wheeler (1944). Two stock cultures of the latter type which were available to us differed, however, in that they failed to ferment maltose. These two cultures of Boyd V (P.143) reduced trimethylamine oxide.

11 SHIGELLA PARADYSENTERIAB SEROTYPE Failure to produce acid from dulcitol and rhamnose differentiates the type from typical S. alkalescens. The results of the serological studies indicate that the microorganism isolated by Stock in Casablanca is identical with those recovered by Taylor in England, by Heller in France, and by the writer in Italy. Four antisera prepared with cultures isolated in these localities were adsorbed in all possible combinations. The resulting 64 tests with 16 adsorbed antiserum samples were negative, indicating that agglutinins were removed in all cases. This finding confirms and extends the work of Francis (1945), who found that cultures isolated in England were identical to those recovered in France, as shown by reciprocal adsorptive tests. The antigenic pattern of the various cultures studied is similar, as is indicated by the fact that each one of 24 cultures is capable of removing the homologous agglutinins from as many aliquots of an antiserum. Cultures isolated since these tests were made were tested with several of these adsorbed (heterologous) antisera by slide tests, and the results were negative in all instances. No significant relationship between the Lavington I serotype and the other Shigella types was revealed when suspensions of the former were tested for agglutination in some 41 antisera prepared against Shigella types. Nor did any important relationship become apparent when cultures of various Shigella types were tested in an antiserum made with a culture of the Lavington I serotype. One S. sonnei culture agglutinated slightly in the 1:80 dilution of antiserum. This is not interpreted as being significant, however, because the titer of this antiserum was not reduced when it was adsorbed with the S. sonnei culture. The possibility of a relationship of deeper antigens of the cultures was investigated to the extent that "blind" reciprocal adsorptive tests were made with S. alkalescens, S. paradysenteriae, Boyd V (P.143), and cultures, and antisera were prepared against them. No relationship between the Lavington I serotype and the S. alkalescens or Boyd V was detected by this means. The possibility remains, however, that such a "hidden" relationship may exist between the Lavington I cultures and some of the other Shigella serotypes. The presence of a heat-labile factor which inhibits agglutination of certain Shigella cultures has been mentioned byseveral investigators (e. g., Stuart, 1944), especially in connection with S. alkalescens. Cultures of S. dispar, S. sonnei, S. paradysenteriae Boyd IV (P.274), Shigella sp., Sachs Q1030, and S. ambigua, as well as S. alkalescens, were encountered in Italy that agglutinated poorly or not at all until after the suspensions were heated. With the Lavington I serotype, however, no such factor was detected, which fact might be responsible for the lack of agglutination in one or another of the various Shigella antisera. Heated and alcohol-treated suspensions of several cultures were tested by slide tests in al of our Shigella antisera and in commercially prepared antisera with negative results. Such treatment of the suspensions did not affect their agglutinability in Lavington I antisera. No serological relation was found between the Lavington I serotype and the 443

12 AAA 4 WILLIAM H. EWING Salmonella. These test were made in connection with an investigation of SalmoneUa-Shigella antigenic relationships that is being carried on by Bruner and the writer. Thus, the serological studies on the present group of 34 cultures would indicate that they comprise a homogeneous serotype bearing no significant antigenic relation to the other ShigeUa types with which they were compared. Stock (1945) reports that the outbreak in Casablanca in 1943 in which he isolated 214 cultures of this S. parady8enteriae serotype was severe. The cases had temperatures of 103 to 104 F on the first day, and pus and gross blood were present in the stools on the first and second days of illness. By the third day the patients were improved, and after a period of recuperation they were returned to duty. The cultures isolated by Taylor in England were recovered from 20 cases of bacillary dysentery (Francis, 1945), and those from France occurred in "sporadic outbreaks" (Heller, 1945). Of the 28 cultures isolated in Italy (table 1), 13 were recovered from American soldiers who had bacillary dysentery with typical symptoms and temperatures ranging from 99 to 105 F. The average period of acute illness was 3 days, after which the patients became asymptomatic. One of these cases is complicated by the fact that trophozoites of Entamoeba histolytica were reported present in the feces. Two of our cultures were isolated from the same Italian civilian. This individual was ill with dysentery at the time the first culture was isolpted. The second was recovered about 3 weeks later, at which time the man was asymptomatic. A blood specimen obtained at the time of the second culture agglutinated the microorganism isolated from the same individual in a dilution of 1:80. Twelve of the cultures were isolated from Italian civilians in the course of routine food handler examinations. In two of these a history was obtained of their having had "diarrhea" during the previous 6 months. We were unable to learn whether or not the other civilians had had dysentery recently. One culture was isolated from a German prisoner of war serving as a food handler; no further information was obtainable in this instance. It was not possible to obtain enough information about the present group of cultures to furnish all the criteria required to establish the serotype as a pathogen. A similar situation has existed with most of the other Shigella strains when they were first reported. The writer feels certain that in time the Lavington I serotype will be accepted, for there is no doubt in his mind as to the pathogenic propensities of the microorganisms. In regard to the nomenclature of the type, we would suggest that the designation used by the English investigators be retained and the microorganism referred to as Shigella paradysenteriae, Lavington I, until such time as taxonomists assign it a number and place it among the other S. paradysenteriae serotypes. Heller (1945) has suggested the specific name Shigella etousa, but we are of the opinion that a specific name is not warranted or advisable. CONCLUSIONS Biochemical and serological studies are described which show that the group of 34 cultures reported upon compose a Shigella paradysenteriae type which is

13 SHIGELLA PARADY5ENTERIAE SEROTYPE serologically homogeneous and serologically unrelated in any significant way to any other Shigella type with which it is compared. The type is very similar to S. paradysenteriae, Boyd V (P.143), in its biochemical activities. Trimethylamine oxide is reduced. The work indicates that microorganisms of this type isolated in North Africa, England, France, and Italy by several investigators are biochemically and serologically identical. The serotype is referred to ass. paradysenteriae, Lavington I, and it is suggested that this designation be retained until such time as it can be given an appropriate number. ACKNOWLEDGMENTS We are indebted to Lieutenant Colonel A. E. Francis and Major A. H. Stock for antisera and cultures, to Captain George Heller for cultures, to Doctor Philip Carpenter for antisera, and to Doctor A. J. Weil for a supply of trimethylamine oxide and a set of the Lederle "Shigella diagnostic serums." REFERENCES BERGEY, D. H., BREED, R. S., MURRAY, E. G. D., AND HITCHENS, A. P Bergey's manual of determinative bacteriology. 5th ed. Williams and Wilkins Co., Baltimore, Md. BORMAN, E. K., STUART, C. A., AND WHEELER, K. M Taxonomy of the family Enterobacteriaceae. J. Bact., 48, BOYD, J. S. K Laboratory diagnosis of bacillary dysentery. Trans. Roy. Soc. Trop. Med. Hyg., 33, CARPENTER, P. L Antigenic relationships of the species Shigella dispar. Proc. Soc. Exptl. Biol. Med., 53, CARPENTER, P. L Personal communication. CHRISTENSEN, W. B Personal communiction. CHRISTENSEN, W. B., AND GOWEN, G. H An arabinose-fermenting bacterium of the lactose-negative, mannitol-negative Shigella group. J. Bact., 47, EDWARDS, P. R., AND BRUNER, D. W Serological identification of Salmonella cultures. Ky. Agr. Expt. Sta., Circ. 54. ERNST, K. F., AND STOCK, A. H Bacillary dysentery in the Atlantic Base Section, French Morocco, to June 12, Report to the Surgeon, Atlantic Base Section. FRANCIS, A. E Personal communications. GOBER, M., STACY, V., AND WOODROW, M A probably new type non-mannitol fermenting Shigella. Am. J. Hyg., 40, HELLER, G Personal communication. MACLENNAN, J Personal communication. RUSTIGIAN, R., AND STUART, C. A Decomposition of urea by Proteus. Proc. Soc. Exptl. Biol. Med., 47, SACHS, A A report of an investigation into the characteristics of new types of nonmannitol-fermenting bacilli isolated from cases of bacillary dysentery in India and Egypt. J. Roy. Army Med. Corps, 80, STOCK, A. H Personal communications. STUART, C. A Personal communication. WEIL, A. J., AND BLACK, J Species differentiation within the genus Shigella by test for the reduction of trimethylamine oxide. J. Bact., 47, WHEELER, K. M Antigenic relationships of Shigella paradysenteriae. J. Immunol., 48, WHEELER, K. M Personal communication. 445