MW ix llll‘lilll WIHIHHWIJ I \ \ ! II 144 552 THS_ BLGCMNG ANTEGENS 0E $£€iGELLA ALKEKLESCEMS AND! ITS ‘a'Akifiif‘éTS 3713525 fur the Emma «f M. $2. Ann-u {‘0‘ u- q “("91" lieui‘U'fik h All. Rab-“Lulu”; r"; : 3;.321'. ‘a 1‘ "9 f ‘ :52?.2: E? .-. ‘3‘ - .. . Nit"! é we“: aflLzzg. TH E513 This is to certify that the thesis entitled fl Blocking Antigens of ‘22 Shigella Alkaleacens F ' And Its Variants JQ‘ " presented by r» 7.; Margaret J. Carlson I , 1‘; l. ' '2 _' .2 has been accepted towards fulfillment 2 of the requirements for 7",ZA‘J' f' .3 .. M. 8. degree in Bacteriglogy 'l "‘: //VW filajor profes‘r m ...-~ g Date_12e£amhar_18,_1342.. M495 € 2' 4, ‘ \ ‘ flj —f l 7* fl ‘ ,- ——— q_ ‘ F J '. r‘. w I. . .‘. r' J .2 ' ,3. l . ,- , J . J . f - s . .J *' - ‘ ‘ . {'lr’ I l .« 4 ’ I u" r 4]" f3 1. .4 [I BIDCKING ANTIENS 0F SHIGEEILA ALKAISESCEIB AND ITS VARIANTS Margaret J’. gsrleon AW Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the mquiremente for the degree or ‘ MAS‘IEROI'SCMGK Department of Bacteriology 1947 THESIS Q.‘ I. II. V. VII. m'arcommrs Introduction Literature Hat erials Used , mperimental Methods A. Production of Antisera B. Preparation of Absorbed Antiserum Experimental Results A. Reactions of Absorbed Antiserum with Stock Cultures B. Reactions of Absorbed Antiserum with Representative Iroshly Isolated Cultures c. Inactivation of Heat-lab ile Substance 1). Biochemical Reactions of Cultures 3. Examination or Inagglutinsble Strains F. Abeo rption Experiments Discussion Summary Bibliography Acknowledgments I} "' .7, ‘ 19(1153 '5 INTRODUCTION The recent trend in the laboratory diagnosis of bacillary enteric infections is toward the use of the slide agglutination ‘test. This method.makes possible identification of intestinal pathogens within ferty-eight hours of receipt of specimens, an interval which can be further reduced when epidemic conditions prevail. The reliability or the slide agglutination test depends primarily on the production of‘highly specific, high-titer anti- sera and the existence of the organisms in an antigenically "smooth" state at the time of test. In most instances, cultures of Shigella of recent isolation are antigenically smooth and readily agglutinated by specific antisera. There are, however, certain freshly isolated strains of Shigella which can be ag- glutinated in specific antiserum only after heat treatment. These occur with sufficient frequency to be annoying to the diagnostic bacteriologist and a puzzle to the immunologist. It is the purpose of this work to investigate the in- agglutinable state of one species of Shigella. I. LITERATURE 'ihe inagglutinability or certain members of the genus Shigella in homologous antisera when tested in the live state has long been known. It is also known that such strains can be ren- dered agglutinable by boiling. This characteristic was apparently first noticed in Shigella dysenteriae (M), (2, 14). Brauh and mm: (3. I.) reported a heat-labile antigen in strains of Shigella paradisenteriae, Flaxner, which inhibited O-agglutinat ion. They also reported a method of treating serum by "immunizing" it against heated organism (1.). Similar reports of destruction of insensi- tivity to agglutination have been made by Schutze (13), Wail, Black and Farsetta (20) and Olitzki et al (12). That Shigella alkalescens possesses a heat-labile anti- gen 01' a similar type has been repeatedly mentioned. In discuss- ing the antigenic relationships of Shigella alkalescens, Stuart et al (16) state that heat treatment or certain strains was necessary to obtain agglutination. Iheeler (18) nations the necessity of treating Shiglla alkalescens antigens for the slide test to tem- peratures or 60 to 70 degrees Centigrade for one hour. Fulton (8) also found "some strains (or g. alkalescens) require heating to render them agglut inable." {the explanation or the phenomnon or inagglutinability or "blocking” was first sought in the cellular structure of the -1- -. . .— .- -. v bacilli exhibiting this characteristic rather than in the prOper- ties of their specific antisera. Stuart and Rustigian (16) felt the behavior of the blocking antigen was suggestive of the pre- sence of capsular substance in certain strains of Shigella alkalescens. Wail (l9) believed the phenomenon to be connected with some structural element of the cell, although examination under the light microscOpe and also by electron microscope re- vealed no difference in the morphology of agglutinable and inag- glutinable cells. More recently a number of studies upon the properties of normal and hyperimmune human ears have disclosed (12) the pre- sence of non-agglutinating antibody in human ears to Shigella % and Salmonella 3m. The development of prozones occur- ring with certain human sera and somatic suspensions of Salmoy £13 m were found (10) to be determined solely by the treat- ment of the bacterial suspension with heat. Some investigators have attempted other methods of solving the problem presented by the presence of "blocking” factors in agglutination tests. ‘Ihe sensitization of antigens by treatment with dye particles (ll), the develoynent of pre- cipitin tests (5) and the separation and concentration of thermolabile precipitinogens for use as antigen in the produc- tion of antisera against heat-labile substances (15) are some of the methods undertaken. Thomen and Frobisher (l7) and -2- .Archer (l) have shown that it is possible to produce specific bacteriophage against the dysentery organisms. These investi- gators mentioned that bacterioPhages were effective with cul- tures rendered inagglutinable by other procedures due to rough- ness as well as with smooth cultures. The use of 'phage typing as a diagnostic tool awaits refinement of the procedures and greater knowledge of the interrelationships existing between the various dysentery 'phages. It has been the experience in the Bureau of Laboratories, Michigan Department of Health (6) that the majority of strains of Shigella alkalescens are inagglutinable by slide test upon pri- mary isolation. The isolation of other species of Shigella with "blocking" substance has not been noted, perhaps because of the limited species and types encountered. 80 unique is this charb acteristic of Shigella alkalescens that it has proved to be a pre- sumptive identity test for the species. Ferguson et al (7) found that strains which appear to be Shigella from their reactions on triple sugar agar, and which are stable in physiological saline and unstable in acriflavine hydrochloride solution?, are subse- quently confirmed in identity as Shigella alkalescens. No explanation has been made for the instability of these strains in acriflavine solution . It is noteworthy that 1“Randi-lemme monohydrochloride, 1:500, in physiolOgical saline strains of Salmonella m containing Vi antigen are also un- stable in acriflavine solution, leading one to conjecture that the "blocking” substance of Shigella alkalescens, like the V1 of Salmonella 331%, may be a surface antigen. The present investigation is an attempt to determine the possible antigenicity of "blocking" substance in Shgella alkalescens and to learn if practical application can be made of the information. II. MATERIAIS USED A. Eggistgz Antiserum. The antiserum referred to herein as Registry antiserum was prepared by the immunization of rabbits with a strain of Shigella alkalescens obtained from Dr. Joseph Falsen, Dysentery Registry, The Bronx Hospital, New York City. This organism pos- sessed the biochemical characteristics of a classical strain of W W was citrate negative and inagglutinable in acriflavine solution. There was no evidence that this strain con- tained blocking substance. B. Acriflavine The acriflavine solution was a 1:500 concentration of acriflavine (trypaflavine) monohydrochloride in 0.85 percent sodium chloride. C. Sterilizigg Filter Pads Type ST (Bacterial Tests) filter pads manufactured by the Hercules Filter Corporation, Patterson, New Jersey, were used in Seitz filters for sterilization of serum. D. glutintion Slides Agglutination slides, Type A-1474x, obtainable from Clay-Adams Company, Incorporated, New York, were used. A slide of this type permits the simultaneous mixing of a number of dif- ferent antigens with portions of antiserum and holds at a minimum the amount of evaporation which occurs during the mixing and in- cubat ion period. III. MENTAL WTHODS A. Production 23 Antisera Two cultures, Nos. 2269 and 5628, were selected at ran- dom from a group of thirty-seven isolations of Shigella alkalescens which failed to agglutinate in the Registry antiserum and which were agglutinated in acriflavine hydrochloride solution. These isolations were made in the Diagnostic Division of the Michigan Department of Health, Lansing. Antisera against these strains were produced in rabbits by the following immunization procedure: Two intracutaneous in- Jections of 0.5 ml. each were made on alternate days with antigen killed by the addition of 0.4% formalin to saline suspensions of the organisms. After a two-day period of rest, intravenous injections of live antigens were begun. These were given on alter- nate days beginning with a 0.25 ml. volume and increasing the dos- age 0.25 ml. at each injection until a dosage of 1.0 ml. was reached. Inoculations were continued at this volume until a total of 5.0 ml. of live antigen had been given. All antigens, both killed and live, were adjusted to a turbidity equalling that of a No. 2 McFarland nephthelometer (approximately 600 million organ- isms per cc.). 2 Twenty-four hours after the final injection, the animals were bled out and the serum immediately separated from the cells. Sterilization was accomplished by passage through a Seitz filter, using a Hercules Type ST pad. No preservative was used. Strain 2269 produced an antiserum with such a low titer for its homologous organism that it was considered to be useless as a basic serum from which to prepare an absorbed serum for the slide agglutination test. are titer of the antiserum produced by Strain 5628, as determined by tube agglutination tests, was l:20,000 with the homologous antigen and l:80,000 with the Registry strain. B. Preparation 91: Absorbed Antiserum The steps in the preparation of an absorbed antiserum may be followed by reference to Table I. The raw serum, diluted rm 1. SHIGELLA moms mm, 15623 Slide Agglutination Reactions Unabsorbed Tollowing Following Following Serum First" Second" Final Living Antigens Absorption Absorption Absorption (Dil.1:10) (Dil.1:10) (Dil.1:10) (Dil.l:10) Flexner V, 3090 ~-l~+-I+ moderate 4» slow 1 slow - Flexner W, In) C +++ moderate + slow 3 slow - Flexner W, IIa R +1- slow + slow 1 slow .- Flexner X, 691. 4+» rapid + slow 1 slow - Homer 1, 1533 - - - - Flexner I, 1806 «MH- instant + mod. - - nexner X, 1807 ~++++ instant + mod. - - Flexner Y, R w++++ instant ++ mod. : slow - Flexner Y, 6094 v++++ rapid ++ mod. + slow - Plexner Y, [.12 «HH- instant ~4+++ rapid + slow - Flexner Z, R - - - - Manor 2, 21.50 - - - - Boyd 103, 1049 - - - - Boyd 170, C - - - - Boyd P—ll.3 - - - - Boyd P-ll9, II + moderate - - - Boyd 88 + slow - - - BOYd 13-288, NeMe " "' " ' Boyd F274, M H» moderate - - .. Boyd D-l, M - - - - Boyd D-19, M -++++ rapid + slow - - S. typhi, 703 «H-++ moderate + slow .. - Sh. whigua, NeYe '9‘ 31m - "' - Sh. ambigua, H 4+++ slow ++ slow 4» slow - Sh. alcaligenes + slow - - - Sh. alkalescens,R l++++ rapid ++++ rapid rH-H rapid +-H-+ rapid Sh. alkalescens rt-H-t- rapid ~++++ rapid++++ rapid ++++ rapid #5628 Sh. sonnei, 1055 - - - '- ‘Absorbing strains: I""‘Absorbing stra ins: +H+ Complete agglut ination ++ Agglutination, 50$ _-_+_ Doubtful agglutination Flemners X-1806, 15-1807, and M94 Ilexners Y-Reg. , and Y-Ll2 with physiolOgical saline 1:10, was tested by the slide agglutina- tion technique with live, eighteen-hour, smooth cultures of repre- sentative strains of Shigella which had been grown upon nutrient blood agar. The cross reactions are shown in column 1. The tech- nique of the slide test, the method of absorbing the serum, and the interpretation of the results are the same as previously de- scribed (6, 7). In this case, the strains selected for removal of antibodies causing cross reactions in the unabsorbed serum were: Sflella Erastenteriae, Flexner X, Strain 1806 Shigella paradysehteriae, Flexner X, Strain 1807 . Shigella paradysenteriae, Flestner X, Strain 691. Shigella paradysenteriae, Flexner Y, Registry Shigella paradysenteriae, Flexner Y, Strain 1.12 The result of the absorption of the serum with these organisms is shown in columns 2, 3 and I. of Table I. Following removal of the factors responsible for cross reactions, the titer of the absorbed serum, as determined by tube agglutination tests, was 1:10,000 with the homologous antigen and l:40,000 with the Registry strain; the slide test was shown to yield a ++++ rapid reaction with both the 5628 and Registry antigens. IV. WTAL RESULTS A. Reactions 3; Absorbed Antiserum 3.939. S3295 Cultures The absorbed antiserum was first tested in parallel with the antiserum produced with the Registry strain. The initial comparison was performed with a group of thirty-seven strains of -3... Shigella alkalescens which had been collected from the magma- tic Division over a period of eight months and stored in the 1y0philized state until the time of test. Members of this group of cultures were tested in the unheated state with the 5628 ab- sorbed antiserum and the absorbed Registry antiserum by means of the slide agglutination technique. The results of this experiment appear in Table II. All but three strains, Nos. 1.71.6, 6590 and 711.1,, ex- hibited an agglutination reaction of ++ or stronger with the ab- sorbed 568 antiserum. with the exception of Strain 5708, which also failed to react with acriflavine, all strains gave a negative or doubtful agglutination reaction with the absorbed Registry antiserum. 2 It will be noted that the three strains, Nos. 1.71.6, 6590 and 711.1,, reacted in acriflavine, providing presumptive evidence that blocking substance was present. ‘mis was substantiated by their failure to react in the absorbed Registry antiserum before heat-treatment and their agglutination in the same antiserum after heat-treatment. One possible explanation for this behavior may be the fact that Nos. 1.71.6 and 711.1, were shown to be citrate positive, motile variants of Shigella alkalescens. These two characteris- tics were often found in strains inagglutinable with absorbed 5628 antiserum. m II. COMPARATIVE M11016 OF STOCK cum Live Antigens Heat-treated culture In with #5628 1th Registry Antigen and Mar Acriflavine Antiserum Antiserum Rag. Serum (1311.1:10) (1:114:20) (1311.1:20) 726 ++++ 4+» rapid . l++++ moderate 2123 ++++ .++ rapid _+, »++.+ moderate , 211.5 ++++ 4+» rapid + ++++ moderate 2181. +0-H H—H» rapid 1 -H-H- moderate 2263 H» ++++ rapid - ++++ rapid 2269 ++4+ +-H-+ rapid - »++++ moderate 3173 -H-H- ++++ rapid + slow ++++ moderate 3787 ++++ 4+» rapid - -H-++ moderate 21.55 ++++ 4-H-+ rapid 1 +-H+ rapid 3855 -H++ -H~H- rapid - +-H-+ rapid 1.215 4+++ «H» mod. - -H-++ moderate ALZ) ++++ ++++ rapid - «4+ moderate 1.1.32 ++-|-+ +4++ mod. . ++++ rapid 1.727 ++++ ++++ rapid - “4+ rapid 1.71.6 HH - - ++H> rapid 51.61. +-H-+ 4+» mod. - ~++++ moderate 563 +H-+ -H-+-t- rapid - 4+++ rapid 5692 «+1» ++++ rapid «- -H-++ moderate 5708 - ++++ instth instant 5889 4+++ 4+++ mod. 4» slow ++++ moderate 6590 «H - - ++++ moderate 6731. +++t ++++ rapid - ++++ moderate 6743 HH 4+» rapid + 44-» moderate 71“ H-H- - - 4+» moderate 7015 ++-H» 4+: rapid - 4+++ moderate 7050 +I-++ +4 mod. - +r++ moderate 7060 ++++ H—H rapi - ++o~+ rapid 7078 ++++ ++++ rapid - ++++ rapid 7081 4+» «4+ instant - «H-H- moderate 711.6 ++++ 4-H» rapid - ++H~ moderate 7149 ++++ 4+ slow -_._ 4+» moderate 7156 +H+ +-H- slow - ++++ rapid 7218 ++++ ++e+ rapid - «+1- rapid 7276 ++++ +H-+ rapid g ++++ rapid 7286 ++++ ‘H-H- rapid 1 4+..- rapid 75M. +«H-+ H mod. _-|_-_ ++++ rapid 7539 -H~H- +-H-+ rapid ; ++++ rapid 7577 ++++ ++++ rapid 1 ++++ moderate st +H+ rapid )H-++ rapid +++o~, Complete agglutination ++, Agglutination, 50$ -10- After heating in a boiling water bath for thirty minutes, saline suspensions of all strains gave a HH- agglutination re- action with the absorbed Registry antiserum by the slide technique. B. Reactions 2?. Absorbed Antiserum with Freshly Isolated Cultures The same general procedure was followed for the paral- lel testing of the absorbed 5628 antiserum and the absorbed Re- gistry antiserum with freshly isolated cultures. 'Ihese cultures were characterized by (l) agglutinability in acriflavine hydro- chloride solution, (2) inagglutinsbility in physiological saline and (3) failure to react with the absorbed Registry antiserum in the slide test. A total of fifty-two fresh isolations were examined. Four- teen of these failed to agglutinate with the absorbed 5628 anti- serum. ‘Ihe balance were agglutinated to some degree in the ab- sorbed 5628 antiserum. The results of these typings are shown in Table III. Heat treatment of antigen, followed by test of agglu- tinability in absorbed Registry antiserum, was discontinued after the first few strains were examined. This test was performed in the Diagnostic Laboratory as a basis for identification of the cultures as strains of Shigella alkalescens and repetition of the test appeared superfluous. The inagglutinable strains were subjected to further test as will be reported later in this paper. all“ TABIE III. C(MPARATIV'E REACTIONS OF MESH 001mm , Live Antigens Heat-treated Culture In With #5628 with Registry ““3” an ‘1 Numbers Acriflavin Antiserum Antiserum “’3' 5°” (Di1.l:10) (Dil. 1:20) (Dil. 1:20) ”886 -H-++ ++-H- - +H+ *3451. ++++ - - ++++ 222 ++++ ++o - ++++ 223 ++++ +++ - ++++ 259 ++++ +++ slew - ++++*** 1.1.9 44+:- +++ slow .- 842 ++~H 4-H - 951. ++++ ++++ moderate - 101.0 ++++ ++++ moderate - 1095 ++++ +++ moderate - 1099 ++++ +++ - 3701. ++++ - - 3717 ++++ 44-H- moderate - 3917 ++++ ++ moderate +4- slow 1.31.0 +|>++ -- ++ slow 1.817 +-H-+ +++ rapid - 6757 -H~H~ -H-+ moderate «- 6881 4+++ ++ moderate - 7139 4+» 4+ moderate - 7745 ++++ +++ moderate - 4201 ++++ -H-+ moderate - 5186 ++++ +H» moderate - 5535 ++++ ++++ rapid - 261.9 ++«H- 4+ moderate - 2875 ++++ ++ moderate - 3105 41+: +++ moderate - 3143 ++++ -H-+ moderate - 3278 +++I~ +4- slow - 31.37 4+» - '- 3643 +H-l- ++ - 3694 ++H~ ++ - *Similar reactions obtained with cultures 31.87, 755, 1718, 1277, 1.192. L792. 5146. 7015. 1.527 and 4135- "Similar reactions obtained with cultures 2682, 728, 31,90, 3520, 96. 83435. 3m. 771. 774. 363. 1346. 6325 and 6757'. “Balance of cultures gave ++++ reactions; tested in Diagnostic Laboratory prior to inclusion in study. -12- C. Inactivation 33 Heat-labile Substance Experiments were conducted to determine the time and tanperature required to destroy or inactivate the heat-labile substance in the Shigella alkalescens strains under study. Sus- pensions of Strain 5628 in physiological saline were subjected to temperatures of 56 degrees Centigrade and 100 degrees Centi- grade for varying periods of time; the latter temperature was se- cured by immersing small tubes containing saline suspensions of the organism in a distilled water bath held at a rolling boil. These results are shown in Table IV. It was found that one hour and twenty-five minutes was required to inactivate the heat-labile substance at the 56-degree temperature. Inactivation was secured in the boiling water bath in 1.5 minutes. Agglutination in the absorbed Regis- try antiserum appeared within 1. 5 to 2.0 minutes after combina- tion with heat-treated antigens and rapidly progressed to the +H+ degree. The prolongation of the period in the boiling water bath failed to speed up the initial appearance of agglutination. A representative group of Shigella alkalescens con- taining the heat-labile substance yielded the same results upon being subjected to the boiling water bath. (See Table 17.). The one exception was Strain 2269. This was one of the strains which had been selected at random for the production of anti-blocking antiserum. However, it had produced serum with a titer so low as to be considered useless for the experiments under consider- -13.. ms Iv. INAC'I'IVATION or 9mm: 5628 Tile Temperatures, Degrees Centigrade Minutes 100 56 1 ++++ - 2 ++++ - 3 4+++ - 4 ++++ - 5 ++++ - 10 ++++ - 15 ++++ 4- 30 - 1.5 - 60 ++ 85 +4-4- Overnight ++++ Quantity of Test Material: 0.5 m1. saline suspensions TABIZ V. GGEPARATIVE AGGIBTINATION REACTIOI‘B 133m WING AND INAC‘I'IVA'JIED mums Culture Registry Antiserquil. 1:20) #56$ Antiserum Rushers USEeatei .Heated 1.5.min. unheated 1.215 1.71.6 7015 7081 71-44 711.6 7149 7156 751.4 41.32 7050 slow '5 E llllllllllIlllllllllllltllllllllIIII iiiiiiiiiiiiiififiiifi.fiiiiiiififii iiié‘éifiiiiiiiifiififiiii..+.+ - . . . . . . .. -15... ation. The behavior of this strain might be interpreted as in- dication that it contained a greater concentration of blocking substance which caused it to rail to be affected by the 1.5 minute heating period . D. Biochemical Reactions A compilation of the data obtained by a study or the biochemical reactions of these organisms disclosed the fact that nine (NOSo 3451.. 31.37. 7141.. 4746. 7149. 7081. 7015. 71-46. and 7156) or the eighty-seven cultures studied were capable oi’ utiliz- ing citrate in three to five days and were motile in semi-solid agar. The biochemical reactions or the remaining seventy-sight strains conformed to the classical reactions of Shigella alkales- _c_e_n_§_ in their ability to ferment dextrose, maltose, mannite, xylose, sorbitol, dulcitol and produce indole. By reference to Tables II and III, it will be noted that the organisms which uti- lized citrate failed to react with the absorbed 5628 antiserum altOgether or were agglutinated weakly and slowly by it. In Examination 21:. _I_n_a_&1utinable Strains- As previously mentioned, fourteen of the freshly iso- lated strains failed to be agglutinated by absorbed 5628 anti- serum. Eleven of these were selected for further investigation in comparison with thirteen cultures which had been shown to be agglutinated in varying degrees by the absorbed 5628 antiserum. Of this group of twenty-four cultures, all but three (Nee. 1277, 4192 and 4340) were agglutinated by the unabsorbed 5628 antiserum as recorded in Table VI. 0f the three inagglutinable strains, strain 1277 has been shown to be a paracolon; strains 4192 and ‘4340 were repeat isolations from.the same person and have been shown on further study not to be members of the Shigella alkalescens group. 1'. Absorption Experiments One portion of absorbed 5628 antiserum was further ab- sorbed with Shigella alkalescens organisms of the Registry strain which had been heated for fifteen.minutes in a boiling water bath. .A second portion of absorbed 5628 antiserum was further absorbed with unheated Registry organisms. After the initial absorptions, both portions of the absorbed 5628 antiserum.were capable of come pletely agglutinating suspensions of the Registry strain but showed only a + or ++ reaction with the suspensions of the 5628 strain. .A repetition of the absorption resulted in that portion of the anti-blocking antiserum.which had been absorbed with the unheated suspensions of the Registry strain giving a doubtful to negative agglutination with the Registry strain and no reaction with the 5628 strain. The portion absorbed math the heated suspension of the Registry strain continued to be capable of agglutinating the ‘Registry strain with a ++ to +++ reaction and the 5628 strain with - 17 - TABIE VI. COMPARATIVE REACTIONS IN UNABSORBED AND ABSORM 5628 ANTISERUM Culture Unabsorbed Absorbed Numbers Antiserum Antiserum 96 ++++ instant ++++ instant 222 ++++ instant +++ instant 259 4+++ rapid +++ slow 1.49 ++++ rapid +++ slow 755 +++ moderate - 842 ++++ moderate 4+++ rapid 868 ++++ instant «H-H rapid 951+ ++-H- rapid ++++ moderate 1040 ++++ rapid ++++ moderate 1099 ++++ rapid m 1277 _+_ - 1718 ++++ rapid - 3701. -H-++ rapid - 3717 -H-++ rapid 44+.» moderate 3917 ++++ rapid ++++ moderate 4135 ++++ rapid .. L192 - - - 4340 - - 1.527 ++++ instant - 4793 +++ moderate - 5146 ++++ instant - 6325 «MH- moderate 4+++ rapid 6757 +++ moderate -H-H- rapid 7015 4++ instant «- a +4- to +++ reaction after the repeated absorption. It would ap- pear'that the Registry strain possesses the blocking antigen in some degree but in quantities insufficient for manifestation by the slide technique. Unfortunately complete reciprocal absorptions employing Registry antiserum and 5628 organisms were not possible because (1) heating renders the 5628 organians agglutinable in Registry cam and (2) a test for completeness of absorption would be point- less due to the normal inagglutinability of 5628 strains in Re- gistry antiserum. v. DISCUSSION The blocking substances appear to be a constant character- istic of the strains of Shigella alkalescens exhibiting that on primary isolation. They are also capable of stimulating antibody production in the form of agglutinins. The agglutinins produced by these so—called "rough" strains include those possessed by the classical strains of Shgella alkalescens plus a slight, but de— finite, antibody against the "heat-labile“ antigen. An antiserum was built against a strain which had been carried upon artificial media and also lyophiled for some time. However, it agglutinated the majority of stock cultures and re- acted equally well with freshly isolated strains. Five of the 'nine organisns which failed to be agglu- tinated with the absorbed 5628 antiserum possessed the common -19.. characteristic of motility and ability to utilize citrate after approximately three to five days. It would appear that these variants differ antigenically as well as biochemically from both the classical strains and those containing the heat-labile block- ing antigen. The results obtained by testing the inagglutinable strains against the unabsorbed 5628 antiserum suggested that there.might have been a decrease in the titer of the unabsorbed serum due to storage. It appeared that the quantity of specific antibody had been depleted in the absorbed antiserum. Tube agglu- tination tests employing unabsorbed 5628 antiserum with both.ths Registry strain and strain 5628 failed to confirm this assumption. The absorption process apparently completely removed or decreased certain important antibodies present in the unabsorbed antiserum. It is interesting to note that unheated suspensions of Registry strain organisms were capable of removing all antibodies for both the Registry and 5628 strain from.the absorbed 5628 anti- serum. The ngistry strain, therefore, would appear to possess the "blocking" antigen as a recessive or masked characteristic. The only effect of heating appears to be in rendering the Registry strain a little less agglutinable in the absorbed 5628 antiserum; its reaction is more on a par with that of the 5628 organisms whereas fermerly it exceeded them. The saving in time between the hour previously recom- mended in literature and the one minute actually required to in- activate the strains included in this study has been applied successfully to cultures in routine diagnostic work. Since this particular part of the work was completed and was in use, Fulton and Curtis (8) have reported results of a similar nature. 1e 2. 3. 4. 5. VI. SUMMARY Using as antigen Shigella alkalescens organisms possessing heat-labile "blocking" factor, it is possible to produce an agglutinating antiserum in rabbits. This antiserum pos- sesses a titer, demonstrated by tube agglutination tests, of l:20,000 for the homologous strain and 1:80,000 for the Registry strain. An absorbed anti-blocking antiserum, suitable for use in testing by means of the slide technique, can be prepared from raw anti-blocking antiserum.by absorption.with the ap- propriate organisms. Such an absorbed antiserum agglutinated 73 (84%) of the eightybseven "rough" strains of Shigella alkalescens under test, regardless of the interval elapsing'between isolation and testing. The blocking antigen is heat-labile and can be inactivated by temperatures of approximately 100 degrees Centigrade in 1.5 minutes. Organisms failing to be agglutinated by the absorbed anti- blocking antiserum.were readily agglutinated by the unab- sorbed anti-blocking antiserum. It appears that removal of factors producing cross reactions with other members of the Shigella group depletes the anti-blocking antibodies sufficiently to prevent agglutination of certain strains. - 22.. 6. 7. Of nine citrate positive, motile variants possessing the major antigens of Shigella alkalescens, four gave evidence of containing some degree of blocking antigen; the remain— ing five failed to react with anti-blocking antiserum. .Ahsorption of absorbed antieblocking antiserum with unheated suspensions of the Registry strain removed all antibodies for both the Registry and 5628 strains. .Absorption of absorbed anti-blocking antiserum.with heated suspensions of the Registry strain fails to remove either the Registry or 5628 antibodies. The reactivity of Regis- tryhabsorbed anti-blocking antiserum with Registry antigen was lessened but no change was noted in the degree of reaction with 5628 antigen. -23- l. 2. 3. 1.. 5. 6. 8. 10. VII . BIBLIOGRAPHY Archer, G. T. 1..., _B_. dysenteriae, Flexner; group identifi- cation by bacteriophage and ”group" serum, J‘. Trop. lied. and Hyg., 48:78-85, August-September, 191.5. Bauch, R., Zentralbl. f. Bakt., I. 0rig., 81:228, 1918. Braun, H. and Unat, E. R., Uber thermolabile leibesantigene des Ruhr-bazillus Flexner, Istanbul Seririyati, 24:109-115, 191.2. Braun, H. and Unat, E. K., Treatment of serum inmunized against bacteria, Tip Dunyasi, 15:1-8, 191.2. Draper, R., Precipitin, agglutinin, indole and methyl red reactions of dysentery bacilli, M. J. Australia 2:84-90, July 22, 191.1. Perguson, VI. and Carlson, 11., Rapid identification of Shi ella by serologic methods, Paper presented at Micfiigan Branch of S. A. B., March 16, 191.5. Ferguson, R., Branston, M., McCallum, G. and Carlson, R., Rapid identification of Shigella in a public health Fulton, M. and Curtis, 8., Bacteriology of a collection of Shgella strains typed by Heil's method, J’. Infect. Dis. 7 :198, May-June, 191.6. Haves, W. , Prozone phenomenon in relation to the agglutina- tion 01' _S_e tthi, Brits Jo EIPOI'. Paths 28:98. April. 191.7- Jones, L. R., Serologic agglutination of antigen-coated dye particles, Proc. Soc. Exper. Biol. 8. Med. 58:124-28, .Tune, 191.5. Morgan, 11. T. J. and Schntze, H., Non-agglutinating anti- body in human antisera to Sh. shi a and S. t hi, Brit. J’. Exper. Path. 27:286. October, 191. . ' U . k I . . t r. C . . o ( . . p l I I O s D r . . . O s. ' z . F O . t . _ e a. e n . O r. ' . ’ s! O O I r r. . C 13. 11.. 15. 16. 17. Bibliography (Con't'd.) Olitzki, L., Shelubsky, M. and Koch, P. H., Thermolabile substance of Shigella dysenteriae Shig, J'. Hyg. 44:271-275. January, 1946 Schutze, H., Agglutinability of Bact. Shiga, 3’. Path. 8:. Bact. 56:250, 1944 Seligmann, R., Zentralbl. f. Bakt., I. 0118., 79:71, 1917. Shelubsky, M. and Olitzki, L., Separation and concentration of a thermolabile precipitinogen from a. dysenteriae , Shga. 3’- Hys- 45:123. May. 1917 Stuart, C. A., Rustigian, R., Zimmerman, A., and Corrigan, F. R., Pathogenicity, antigenic relationships and evolutionary trends of Shigella aLkalescens, J. Im- munol. 47:425-437, November, 1943 Thomen,1.. F. and Frobisher, H., Ir., Study of Shigella by means of bacteriOphage, Am. J. Hyg. 42:225-253, November, 1945 Ihseler, K. H., Serological identification of dysentery bacilli, Am. J. Pub. Health 34:621-629, June, 1944 Weil, A. .T., Dysentery. A progress report for the years 1942 to 1946, .T. Imrmrnol. 55:363. April, 1947 Weil, A. 1., Black, .T., and Farsetta, H., Serological types of Shigella paradysenteriae (Flexner); types with dual primary antigens - practice of typing -- discussion, I. Imunol. 49:341-351, December, 1944 -25.. To those members of the staff of the Bureau of laboratories, Michigan Department of Health, without whose cooperation and counsel this study would not have been possible, I wish to express my appreciation. I." C. REC-1'35 h f s m R A R m I.- Y P- s R E .v. N U E Ti Al lllllUllllHll