O _ , — # — — k WM \ 2&3 Mk 3ACT’ERSOFHA5-E TYMNG 05 STRAINS ‘9? SALMOE‘EELLA TY'FHi QCCURRENG EN Miiii’i’fifim‘i’ WW?! SPECIAL REHRENCE 1’0 ESGUKTEONS ERQM ‘Z‘YSJH'SED CARNERS “flask ‘32:: ‘54“ $an 31” M. S. Mi’filfifififi S?A?§ CCELLEGE 3342mm Q Hanémgon 195653 M-796 Date \ 1 .‘ ' l l I o t l '1'. \f ”AJ"; and” are ~ A. y¥$ 't .L «hf: , ‘f'wi; _ . . " f t . b "I ' .3119"; I " \ .O- 7. _ ,‘ 9'6,“ J {271‘ ' . ' " " f a H .‘Iv ' l'JIIn '2' . '?" LVN l: .‘ ." (”Pr 3." {At . ‘ 9“ w \ l‘T'J'. ‘ f l". l ll"- .y.l i ‘w' l“. ‘ ' €' ._, W . In ‘_ L t , ’1 ( \, ‘ '1“ . fk'l" . ‘ . t4 I,~ ‘an "I ‘ ,‘ f n _'. ' , ‘I 'k t ' l A“. .l‘ '1 l I \v' I’m I, If. ' 1' .~ '. H .l ’ | \I! X }l\ u' '1' \ I l" ‘ s - I“ ‘. I I ’ l I W. ~ . | I In. l , ‘ | v ’ . ‘ ~ 1 ' ' I . ’i a ' ' I ‘ll I' g '. f. ' l‘ l r .t l f I . '. I a ‘ - f , 1 i I ‘ . p ‘. 1 , ‘0‘ _ J .. ‘5' L: 0 . A I ‘3 ' 1w I " ‘ \ 5 t ' \ ‘ ‘ . . . z ,7 \ | ’ J I o ‘ I ' ‘ l X‘ l | ' l " ‘ a ‘ This is to certify that the thesis entitled Bacteriophage Typing of Strains of Salmonella Typhi 0ccurring in Michigan with Special Reference to Isolation from Typhoid Carriers. presented by Norman D. Henderson has been accepted towards fulfillment of the requirements for “$18.. “degree in_#§8:c.l’§§°l°gy Iajor professor May 24. 1948 ( t '1 \ l \ r‘. a - I. r a l 1'\ . ' \ .l v t. l ‘ lll.rllll\.{lflllll.lllllf1ll|l\ ll\lll BACTERIOPHAGE TYPITG OF STRAIKS OF SALKOTELLA TYPHI OCCURRIEG IN MICEIGAE WITH SPECIAL REF- BRBJCE T0 ISOLATICKS FROM TYPEOID CAREIBnS by Norman D. Henderson \ Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIEECE Department of Bacteriology 1948 THESIS é/‘Iffi’ 6/ m -—-s-v-~1 *‘Dd-‘d III‘TROD‘L CTICN . ~~1:-T\-np ELIE-V—1-\—’cfi .-.-".-'.'"' .: .I. is IZTITLDS ’ f‘x'lf- ‘3'? I'", ‘ 'f‘ 7"," C ' '1". ‘TT .-~‘/\.— -._J . DISCUSSICE O C OZTCLUS I CH IIBL IOGEAI‘EI 3 y‘— 2 I"..- o 'flyfiw -0— I'm". 'v-ncm r.U.L‘:..' v...4..3uJ\I. .14.“; COZ'CETT SUT ES H“ 2:3 :3 o: o q L- \J) H \ n .YI'I’10'DUCT I CIT In 1900 there were 5,122 cases of typhoid fever reported to the lfichiaan State Board of Health with 824 deaths (1901). During l9Q7, a total of 77 cases and five deaths were recorded by the Bureau.of Disease Control of the Mich igan Department of Health (1948). Figure I depicts the reported death rate due to typh oid fever from 1900 to 1947 (Michigan Department of Health 19L8). In spite of modern sanitation, public health education and the deveIOp— ment of better laooratory tech qlues, typhoid fever persists as a small residuum due for the most part to the existence of carriers of Salmonella typhi*. The majority of cases occurring in 1947 was of a sporadic nature. One outbreak, which 1 as caused by a previously ulcetected carrier, accounted for 17 cases and one death. Bradley (l9b3) re: ers to tnese sporadic cases as "c11in:s in the con lex armor of modern sanitation" and states that "it is only by counting hese sporadic cases which fail to initiate outbreaks that we have a true measure of the efficiency of our system." Presumably the chronic carrier is always son ewhere in the background, but his relationship to a series of cases may be remote. Bradley followed 23 sporadic cases in adjoining counties in England. Using epidemi- ological methods and the bacteriOphage typing technig1e, he was able to locate and identify the responsible carrier on a dairy farm *The nomenclature used in this paper is that of the Kaufinam White Schema. 100 miles from the locality of the cases. Bacteriophage typing supplies the epidemioloaist with a tool which enables him to trace an outbreak or a series of isolated cases to the source. Once it has been ascertained tnat a person is a typhoid carrier or case, it is now possible by a relatively simple laboratory procedure to identify not only the typhoid rganism itself but the actual type being excreted by the individual. This laboratory pro- cedure is the bacteriophage method of typing the typhoid bacilli. Deom Rate per IO0,000 Population 35 30 25 20 1 FIG. I TYPHOID FEVER DEATH RATE IN MICHIGAN I900 " I947 1 1 I L l l 1900 l905 1910 1915 I920 I925 1930 "335 194 4s HISTORICAL The question of phage specificity has been investi- gated since the earliest days of bacteriOphage study. d'Eerelle (1922) stated "It (the bacteriophage) attacks a certain number of species at the time of isolation, and possesses for ea h of tliese e a ainst Sniaella T o-.. , . -- A. J. ‘ "v.1" ‘ "I‘ ‘\_. —.fi-.o—..._— a variable virulence." Thus, a -vsenteri_§ may exhibit some lytic action on Salmonella. tLLIi. The 5L. 1 1. other condition also ezists in which a )ha 3e, showing activity a3a lIlSt one species, will demonstrate none when applied to a species of a different genus. Furthermore, a particular phage may be active against one strain of a bacterium, and not against a second strain of tie same speciw . Burnet (1927) demonstrated hat there was a close correl- ation between the action of a particular phage on a certain bacterial L fin .. sp ecies and the su M1 ea or somatic antiaens of that species. For example, a phage will show activity against S. tvnhi, Su1ionell 1 0 s Q enteritidis, and Salmons la pullorum, ea ch of w1c1cn nas in com.on the somatic antigen IX,XII as designated in TheK aufima nn -'i{hite Schema. The application of bacteriophage typing to the classi f1- cation of the genus Salmonella has not met with success, because organisms possessing the same antigen were lysed by one bacteriOphage. Felix and Pitt (193w? ) anrounced that virulent living strains of S. tvohi were resistant to agglutination by a pure "0" antiserum. This announcement was followed by a second report from the same au hers (l93wb) statin3 that virulence and res stance to a33lutination by "O" antiserum were correlated with the presence of an antigen which they called the Vi (viruler ce) anti3en. Kauffmann (1934) elaborated on the terminology of the Vi antigen by introduc- ing the terms V form and W form. A strain of S. tvr i that poss~ U) (3 (D U) the Vi antigen is said to be in the V form, while a strain not demon- strating this antir‘ By applying the terninolo:y of K2 finann to the work of Felix and Pitt, it is I!) nparent that a st1ain w.hi chc tains the V1 A. I4 antigen (V form of Kauffmann) will be agglutinated by a pure anti- Vi serum and will not be a33lutinated by a pure anti—O serum. Accordr in3ly, an or3anism devoid of the Vi anti3en (N form of Ka uffna nn) will be a33lutinated by a pure anti-O serum and will not be a33lutinated by a pure anti—Vi serum. Kauffmann made an extensive study of this phenomenon and found that an or3anism ma.y exist in the V, W, or V-W form; the latter being that condition in which a strain will exhibit partial agvlutination in both a pure anti-Vi and pure anti-O serum. “-1 The discovery of Felix and Pitt served as the starting point for a new line of research, and several authors, Sertic and Boulgalcov (193s), Scholtv—ns (1936,1937), Craigie and Brandon (1936) and Craigie and Yen (1938), worki 1n3 ind ependently, established the 0 fl 0 existence of bacteriOpha.3es s1ec11ic for the Vi antigen of S. 1 hi. The action of these phages was highly specific and lys did not occur with typhoid bacilli devoid of the Vi anti3en. Craigie and Yen (1937) exarii Md iffe rent strains of bacteriopnc~e which were active aga‘ inst the Vi antigen and found that the pha3es could be grouped into four types (see Table I). TABLE I CHAPAC'IERISTI CS OF THE VI BACTERIOPHAGFS Vi Phage Relative Thermal Neutralization Lytic activity ' Particle Death by antiphage for'V forms of Size Point sermm §b_typhi I II III IV Type I Large phage 67-700 C + - - a Lyses all V foams Type II Medium phage 69-720 C - + - - Develops a high selective lytic activity for the type Of.§; typhi on which it is propagated Type 111' Small phage 61-640 c - - + - Lyses majority of'V forms Type IV Medium Phage 59-620 C - - - + Lyses majority From the infornation contained in Table I, 'b e drawn: 1. 2. 3. of'V forms with exceptions of type F and D1 several conclusions may The four phages are specific for the V form of S. tynhi. Serolo;ically, the four types of phage are distinct. A variation occurs in the lytic action on V form strains of S. tvnhi, particularly in the case of Type II phage. Bacteriophage propagated on a strain of §; tvnhi deve10ps a high selective lytic activity or specificity for the substrate strain. The variation exhibited by phege II attracted the attention of Craisie and Yen (1938). They found that the affinities of this phage could be conditioned to a particular strain of §; tvphi on which it had been propagated. Using class1Mc 1 strains of S. tvn“, they determined that phage II, when grown on either the Rawlings or Watson strain, developed an eoual‘-y 11.i311 lytic activity for both strains, but lysed strain Ty2 only when applied in strong concenp trations. On the other hand, when grown on strain TyZ, phage II lost its ability to lyse the Watson and anlings strains and became hM hly specific for strain Ty2. By changing the prepagating strain, several preparations of ph age all \.ere ob tained that showeda selective affinity for a particular strain of.§3t rphi. The epidemiological aspects of this discovery were realized by Cra Mie and Yen, and the factors responsible for this phenomenon were investigated. The hypothesis, deveIOped by Crai 3ie and Yen to explain the variation of phage II, was that the pha 3e er ists as a series of mutants .hich differ from each other in their affinity for different strains of S. tvphi. This hypothesis, in turn, is predi- cated on the supposition that varieties of §; tvnhi occur which differ from each other only in their Ti antigen. The variations or diffe HT nces in Vi antigen are demonstrate d only by the action of "(1 ha e II. Thus, the increase in specificity of a phage preparation (“2 may be explained on the basis of selective propagation of the mutants specific for the substrate strain of §; typhi, with a corresponding decrease in the o ther mutant particles. By selective propagatio on of the mutants of phage II, a series of lytic preparati1ons was built up which demonstr ted a mi her relative affinity for the homolog gous strain than for the heterolOgous strain of S3 tvnhi. Such preparations, diluted so as to contain only an effective number of phage p rticles of the domin- ant homolOgous mutant, were employed to identify strains of S. tvnhi similar in type to that on which they were grown. The hypothesis of Craigie and Yen explains: (l), the ability of the phage to adapt itself to bacteria of heterologous type: (2), the high titer for strains of homologous type: (3), the lower titers for the v: rious heterologous bacte M1 , and (b), constant ra.tio between these titers. To standardize phage reactions so that the preparations could be used for typing strains of.§; tynhi, Crai gie and Yen decided on a "critical test concentration"*. This was defined as that dilution of he phag which; under the conditions of the test, just produced a sufficient number of plaques to give an area of confluent lysis on the homolOgous strain of S; tvnhi. The actual dilution emplo; ed rm s determined by two fa.ctors: l. The time and condition of incubation of the tests. 2. The diameter of plaques produced by the phage on the homologous strain of.§; tvnhi. Upon extending their procedure to a number of isolations of S; typhi, Craigie and Yen found that these strains were differ- entiated into well defined groups or types. New phages were built *Abbreviated as C.T.C. against representative s trains of each tVJG ani then checked against the original phage preparations. In this manner, it \Ias discovered +- U}; {D t simila .r tvpe strains vielded similar pha3es. This technicue was applied to strains isolated from a known carrier and from persons known to have been infected by the carrier. The results obtained indicated that all the strains related to a single carrier were identical in type. FLirther test inn; produced new t3'pes, in til at the time of their first comprehensive report, Craigie and Yen (1938) had studied 625 cultures representing ll different phage types. These were desi3nated A,“ -l, 32, C, 31, D2 ,3. F, G, E and L. The letter "I" was intentionally omitted in the designation of tvpes to avoid possible confusion with phage I. Other authors have added to the number of types originally reported by Craigie and Yen until there are now 25 o-:n types and subtypes. The rt actions of these pha ge es at their "critical test concentration" when tested against the corresponding series of Vi type str 21 ins of S. typhi are given in Table II. Experience has proved the stability of Vi phage II. It is now generally ccepted th at the phag ;e preparations should be taken as the standard and not the culture of type strains of.§& tvnhi. From the information available, it is apparent that preparations of Vi phaae II, once prOpa 3ated, tested and diluted to their critical test concentration, are, for all practical purposes, fixed in their specificity. The particular form of the Vi antigen of a given strain of S. tvphi is thought to ce a reasonably permanent characteristic. The epidemioloaic cal value of phage typing is based on this observation. .2 mom mom +++ I I +++ mom mom.mn I ST + + Ho Ho +|I I +|I A0 a++ +++ I do +I mom s+++ 8+9 A0 A0 mom Ho mmsdwam msOHmEdz I +++ mmswmaq pqosawnooIanm I mom .H mosden Sam I + 8+ ++ H. mom 5+ mom m mom ++ Om mmsvwam chan I a mnmsa pnosamqoo I go do do +++ I +++ Ho Ho +|I I +|| Ho I‘z -l I‘« 2 NH HA m c Nrm .En NR HE «G mqm mm Hg .O.B.o H1 mZOHBmuamdflm modzm Qfiifimdam HH mqmde 0 mm '4 Hagp.m Mo 0959 A few exceptions have been noted, but these are apper- ently rare in occurrence. Type A is sensitive to all Vi phage preparations. A recent report by Craigie and Felix (19%?) indicates that this type may result from dissociation. For example, a Type A culture was isolated from a Type T and a Type 0 carrier. The indi- cation is that this change may take place in carriers of long stand- ing. Type A has also been isolated from cultures of Types 31, C, D5, F1 and N. This point is of rest importance to the epidemiolOgist because the isolation of two types in an outbreak raises the question of a double source of infection. Helmer et al. (1930) have reported on the isolation of Types F1 and F2 from a single case of typhoid fever. A careful epidemiological study failed to disclose a double source of infection. Type O and Type C are peculiar in that they yield plages which are variable as to their plaque size. This condition seems to be a characteristic of the particular type of.§3 tvnhi involved because it has been found to exist in freshly isolated cultures. In addition to these variations within the types, there are other conditions which must be considered in the typing of strains of_§3 tzphi. The variation between the V form (Vi antigen) and the W form (0 antigen) has already been discussed. t will be recalled that the V forms are lysed by phages I, II, III and IV, and show considerable variability in the reactions with the type II phage. Generally speaking, the oraanisms sensitive to the type II phages can be further subdivided into three large groups. The alpha group is defined as being composed of those strains which are fully 10 sensitive to Type II phage. In addition, when used as the substrate strain for propagating pQEFB II, they produce a phage with a clear- cut lysis of that type. Thus, if wha-e II is transferred from one alpha type to another, it develops a distinct affinity for the new tvpe, shile retaining no residual lytic action for the preceding The beta group is made up of those types of S. tvnhi which 1 when used as a substrate strain produce a pha e that retains its affinity for the original strain even after several passages on the new type. The gamma group is made up of those strains which are resistant to the action of phage II. The three groups are arranged as follows: Alpha Group Beta Group, Gamma Group Typ A Type 32 Resistant to 31 BB phage II, but C Dl sensitive to D2 D3 phage I, III D4 and IV. El 32 F1 F2 G H J L1 L2 M The practicability of the method of Craigie and Yen has been well substantiated. Lazarus (1941) in the United States, Crossley (1942) in Canada, Buckle (1946) in Australia, Croclzer (1947) in South Q 1 Africa, and Cruichsnanx (19L?) in England have reported the results ll of phage typing in their countries. Two salient facts stand out in these verious reports: 1. The world-wide distribution of types appeers to be fairly uniform and the phage tvpes 01 §; trnhi are, for all practical purposes, steble. 2. Typhoid fever persists due to a small number of cases which occur in Spite of our existing methods of soni- tery control. epideniClOgist. The stctility of tries provides him with a guide wnerebr ne may trace a perticu;cr outtraak back to its source. The persistence of cases presents a constent challenge to his efforts to control and eradicate this disease. Creigie (l9hl) has suggested that on occasions the task of the epidemiOIOgist might be facilitated if records were aveileble regerding the type excreted by every known carrier in the area. Therefore, typings from carrier isolations have been included as a separete section of this study. ‘- 'ZL.I.US Tie t\.'erty—three standard Vi-phrge II preparations used in this study were suoplied, in concentrated form, byT lie Connausht Medica.l Re rch La uoratories, Univers city of Toronto, Toronto, Canada. The corresponding Vi-type strains of §; tvphi were also supplied. Instruction included with the pha ge kit directed that the pha 'ge preparations should be diluted to the C.T.C. and then tested against each of the Vi-type strains. To facilitate the typing of cultures, he 23 ph age s were combined by us into seven pools. Dilutions were computed so that each phage was present at its C.T.C. (Table III) Late in the study, Vi-o1:*ec I, II I and IV were sup: lied by Mr. Arnold Juenker of the Salmonella T;.m in ng Sta ion, Michigan Department of Health Laboratories. These preparations had been furnished originally by J. M. Desranleau of the Quebec Provincial Health La Moor .ories. The media necessa y for typing were prepared originally according to the instructions supplied byi ‘ eConnaught Laboratories. These were as follows: Agar Medium 'Bacto' nutrient broth (dehvdrated) 20.09 gms. Sodium chloride 7.59 Ems. 'Bactc' agar 20.09 gms. Distilled water to ' 1000.00 ml. 13 TABLE III COL-ZPOSITI ON AND PRICPAPATION OF POOLED PHAGES Individual Amount of Total . Total Final Pool Phages each phage Phage Broth Vol. Dilution I A,0,H 0.05 ml. .15 ml. 4.85 ml. 5 ml. 1-100 II E2,G,J 0.05 ml. .15 ml. 4.85 ml. 5 ml. 1-100 III F2,B2,B3 0.05 ml. .15 ml. 4.85 ml. 5 ml. l-lOO Iv K,D2,D4,F,D5 0.05 ml. .20 ml. 4.80 n . 5 ml. 1-100 V L2,M 0.05 ml. .10 ml. 4.90 ml. 5 ml. l-lOO VI Bl,Dl,El,Fl 0.05 ml. .20 ml. 4.80 ml. 5 ml. 1-100 VII n4n,Dé,L1,N 0.05 ml. .20 ml. 4.80 ml. 5 ml. 1-100 14 Phage dilution broth 'BCCto' nutrient broth (dehydrated) 8.09 gms. Distilled water to 1000.00 ml. Phage culture broth Eacto' nutrient broth (dehydrated) 15.09 ans. Sodium chloride . 7.09 gms. Distilled water to 1000.00 ml. n: these media. However, *1. Original tests were made us Craigie and Felin (1947) have stated that any good meat digest age 1 may be used as an alternative providing that the standarl reactions are reproduceable. Therefore, in the interast of economy and for convenience, we have su stituted the followinr media in place of .I. cto' nutrient broth agar: ’h the 'Eacto‘ nutrient broth and '3 .4 r2 B:oth Veal infusion 1000.0 ml. 'Eacto' pepton 10.0 g. Sodium chloride 5.09 gas. Adjust to p H 7.6 and filter #2 Broth Agar 'Bacto' agar 15.0 gms. #2 Broth to 1000.0 ml. Craigie had recommended that the cultures of the Vi-type strains should be maintained on Dorset Egg medium (3 parts whole egg, 1 part saline, slanted and inspissated). Medium of this formula was made up and dispensed in small screw—cap glass vials. In this form, 15 n the Dorset L Medium wes used for mainteininr the stock strains. :‘JIJ’ C CJ 3 All 23 stock strains were transferred to fresh slents at six month intervals following passage on nutrient agar and tests for specificity. Slents of the egg medium were also supplied to all laboretories in the State that would submit strn'ns for typing. Cultures thet proved refractory to Vi phage II were tested ser0105ically for O and Vi antigens in addition to being tested with pheges I, III and IV. For this purpose, two antisera were used: ‘1 1. A pure "0" antiserum containing antiwodies against the IX, V I XII antigen. Selmonelle_gellizarum was used for immuniz- etion. 2. An anti—Vi serum prepared by us. Salmonelle.hellerun was used for ’mmunizetion. 16 IETHCDS Our procedure for typing strains of §. tvphi by the bacteriophage method has been essentially tha of Craigie and Yen (1938) and Craigie (l9bl). Because we have followed Craigie's procedure with close attention to detail, the following description is quoted directly from Craigie (l9b1). "Two standard platinum loops, 2.75 mm. in internal diameter, No. 20 B & S gauge wire, are used alternately to apply the culture and phage to the plate. While one loop is actually in use, the other is coolizg after steril- ization. In transferring a loopful of broth ulture to the plate, the loop is used merely to direct the natural spread- ing of the broth and should not be rubbed over the agar. The shank of the 100p is angled so that the loop will lie parallel with the agar surface during inoculation, the loopholder being grasped between the thumb and first two fingers like a pen. A full loopful of broth culture is obtained by sharply moving the culture tube so that the loop ‘uickly breaks the surface of the fluid. Then with & the holder held in the may described and with the forearm and side of the hand resting on the bench, the loop 15 lowered until the drop held in it touches the surface of the agar. At this point, the loop is given a slight horiz— ontal circular movement by moving the fingers, guiding tne spreading of the drop over an area approximately 15 mm. in 17 q diameter. A number of areas, corresponcliz a to the number of J'C.) ‘\ -ha. *d ‘e prepa.rati ons us ed in the test, are inoculated eith each \ 4 ( strain. When the aiar has absorbed the fluid vhich has been applied (optimum 5 to 10 minutes), the pha Se is applied from the sta nc ard 100p to the center of th e inocm ated area. In this case, the loopholder is be at held horizontally between the thumb and fingers, these pointing downward with the side of the arm resting on the edge of the bench. The loopful of phage is lowered until it al:nost touclies the surface of tlze agar, when slight rotation of the loopholder fill move the loop into a vertical plane, thus allowing its contents to flow onto the center of the inoculated area. It is to be noted that the drop of phage must be allowed to spread naturally on the plate, and the actual contact of the loop and inoculated area is to be avoided as for as possible. "When the fluid wh’ch has been applied had dried sufficiently to permit of th e plates being safely moved, they are incubated for 2 hours at 37° C. and transferred to the cold room overr1i§ 1t. They are returned to the incubator and are ready for readina in u to 6 hours. It should be noted that the plates must not be stacked, since at tempera- tures significantly below 370 C., E; tvphosus will grow with- out exhibiting lysis with Vi phage. The method of interrupted incubation has two advantages. It permits the phage reactions to be observed at their optimam and before they are obscured.by late growth in t11e case of sor e stra ins. Epither, the intermediate 18 period in the cold room permits diffusion of the phage into the surrounding normal culture, thus enhancing the clerity of the reactions. The ToRCthflS are best otserved by oblique artificial illumination and e ainst a dark background." The results obtained when the diluted phages and stock strains were tested on %2 broth 25? are reproduced in Table II.* To aid in the preliminary screening of test strains, pooled phages were used. Stock strains were tested egainst the pooled phages and yielded the results reproduced in Table IV. *The form used in Table II is that of Craigie and Felix (1947). l9 moddem machoEdz I +++ moswwam Sam I + mowdwam owan I A moswwam pmomHHHOOIHEmm I mom mosdem OHOHE I S mamma pqodamnoo I Ho .Ho mom fl mom I I I z I I mom I I I I 9 up ..... .8 I .n .n I d + + ++ Ho 5+ 8+ I M u. n I... I N am. I h + + + I + + mom m H. I I SH. H 1.5 H. w I I I I Ho I I Nb I do I I x I I am I I I I I mom. I m I .HD I I I +++ I Hm .5 I I I I I I 8 mom mom I 40 I I I mm mm I I Ao I I I cmmmqm no I I mom I I I mqa I I I do I I I Na ,6. Hp II. mom M II. I a 9+ 4+ A+ 8+ a+ 4+ go o mom +++ +++ .afl Ao +++ +++ mm +++ E+++ +++ +++ . no +++ +++ mm I A0 nmfl I “an Hmfl I m 90 Ho A0 no Ho Ho Ho ¢ ZJWIB «RISE Hwfimrfilam 2 «NA mmfiqndmmdm mm «mmdmm madam mdna fiflpm HHP HP _> bH HHH HH H Hoom owwsm W73 Hefimaawm figmm Qanoom DH mflmdfi RQUTIIZ TYFITG IEUC Jfih Strains of S. tvphi submitted for typing Ierc QS‘“11" were streeked out on nutrient agar plates and six V form colonies picked and transrerred to an e7; slen . A swell amount of culture from the egg slant was inoc“let ed into 2 ml. of nutrient broth and the tube incubated at 37.5 C for E-h hours. At the end of h incubation period, ei 1-t inoculation sites were made on a nutrient agar plate as described above. After the broth had dried, one standard loopful of each of the seven pooled ph mg s was applied to a seperate inoculation site, the e1 ghth s>ot bein g left for control purposes. After the plm ges had dried, the plate wes inverted and inculeted for tvo hours and placed in tne ic ebox overnight. In the morning the pletes were returr ed to tie incu ator for four hours of 1 additional incubation. The )flE .ction resulting from the variou 0:; (D [0 pools was recorded and compared with the standard reactions as given in Ta ble IV. If confluent lysis was demonstrated in each spot, he culture was records; as type A and no further tests were performed. :hage action was apparent in one or more spots, he culture was retesteda -ainst tle ejecific phe;es nIkin: up each pool involved. p- ‘1 1 Incubation was repeated as before aid the results read the follow- ing morning. As an example. let us e.amine a.crl ture wnich, when tested with the pooled ph.:es, producedc nflu e11t l"sis in Pool I FIGURE II Reaction with pooled phages Reaction with specific phages 22 with only a few isolated plaquesa pfarent in the other inoculated areas (Iig :ure II). T'his culture 1% s ted with t? e indivitual phages making up Pool I, i.e., phages A,C, and H. The results, read the next morning, showed confluent lysis with phage C and onlv f a ew isolated plagues on the other txo in Ioculation sites. By com- paring these results with Table II, it can be seen that these reactions were comparable only with those of type C and that the culture was therefore type C. If no oh ge ction In 5 oh served I.ith any of the pooled phages, two addition 1 tests were performed. The strain was inocup lated into 2 ml. of broth and tested with phages I, III and IV using the procedure outlined above. If the culture demonstrated lysis with the three phages, it possessed the Vi antigen and was resistant to phage II for or e of two rcasons: 1. T1 1e culture w:s in tiie gamma .form and. was therefore untypeable. 2. The culture was of a new type not included in the present scliema and a phage would haVe to be propagated on it, tested against the stock strains and standardized. A culture refractory to the action of phage II was also tested serOIOgically as a check on the action of plL° gas I, III and IV. This test was performed on a glass slide using a IX, XII anti- serum, a Vi antiserum and a saline control. The strain reacting strongly in the IX,XII serum and weakly or not at all in the Vi serum wa.s in the h' or degraded V form. If the culture reacted 23 strongly in the Vi antiserun and weaxly or not at all in the IX,TII serum, it was in the V form. If this cultur3 yielded lysis with p1 ia-ge s I, III and IV th en it was afparently a gamma culture. (See I. bl v) . 2:15:31; v REACTICHS OF THE VLRIO"S FCRXS OF.§; TYPhI Sensitivity to Vi Phages Agglutination Form I II III IV Vi Serum "0" Serum V + + + + ++++ a V “ ‘ - - - ++++ Gamma + - + + ++++ - + - lysis ++++ - complete rapid agglutination ne differences between the V, W and Gamma forms are antar- ent from the reactions obtained with the Vi pha'es. These same differ- ences are confirmed by the use of Vi and "O" antisera. However, no combination of these rca gents 1ill disti « sh betxme n a true gamma form culture and one hat is of a new Vi type and therefore not included in the tz'pe II phages. To sep Mr te these cultures, we have resorted to what we will call tne 1:0molo: u Sphar C) e technique. A loopful of a concentrated phage corr U03Cl“? to one of the alpha tm pes was ap“lied to an inoculation site of t11e test strain. If no lytic action was observed, the culture was in the gamma form. However, if a few isolate d plagu es Isre observed, several were cut out so that the pla‘ues, a small amount of culture and the underlying agar were removed. This section wes plsced in a tube containing 2 ml. of nutrient broth. The tube was incubated until lysis was observed, or for four hours. The tube was then placed in a 56 C water bath for 45 minutes. This destroyed the bacteria but did not effect the phase. Serial tenfold dilutions of the phages were k-a made and tested egeinst the culture in the usual manner. If the phage action appeared strong, the new phase wes tested against the stock strains and the results compared with the standard reactions. If the results were comparable with any of those in the standard chart, then the culture was merely a vrrisnt of the existing type. However, if the reactions differed from those presented in the standard chart, we were dealing with a new pnnee tvne, end the culture would heve to be tested exhaustively with the stock strain to determine its own standard reactions. 'inel r. '2. U After ell tests with the Ph3§LS were completed, the ori broth suspension wes used to inoculate media containing certein bio- Chemicals to confirm the identity as.§; tvnhi. The following media were inoculated: Liquid single sugars, dextrose, lactose, sucrose, maltose, ennite, sorbitol, Iylose end dulcitol: other media, Urea medium, Peptone water for Indol end H28 production, Voges—Proskeuer medium, Simmon's citrate agar slants end 0.3% eger for motility. Three cultures submitted for typing proved not to be S. trnhi. All others produced typical biochemical reactions with some variation in the fermentation of xylose and dulcitol, the production of H25 and the degree of motility. There was no annerent correletion between these verietions end the phage types identified. T‘“L“ VI $1.51.: VI PHAGE TYPES OF SALMOIELLA TYPHI ISOLATED IN MICHIGAN SEPTEKEER l9h7 T0 MARCH 1948 ISCLATICIS FROM ISOLATIOIS FROM CLIIICAL 035:2 Geno IO CARRIERS Total Total Case Carrier TOTAL Pheg Isola- Isola— Isola- Tyne Original Re eat tions Original Retest tions tions A 6 o (6) 17 12 (29) 35 s1 0 o (o) 1 2 ( 3) 3 22 o o (o) u 3 ( 7) 7 33 o o (o) 3 3 ( 6) 6 c 3 5 (8) 15 11 (26) 34 31 5 1 (6) u 2 ( 6) 12 DA 2 o (2) 5 1 ( 6) 8 D6 0 o (o) 2 o ( 2) 2 El 33 51 (8a) 18 19 (37) 121 2 ( 5) 20 lo -\1 '0 k»: I... 2, lo E I... 56 65 (121) 73 55 (128) 249 w Form 2 o (2) 18 8 (26) 28 Gamma Form .2 11 '30 .3 1.6. 129) .52 FIIAL TOTAL 67 76 (1L3) 105 79 (18A) 327 26 rIs2'*2~2:2" 7:? .2" av»... V‘n v Ta le VI conte ins a Jn2ry of all typin between July, 19t7, an hay, l9h8. The die nostic end carrier cul oure s submitted for typing were about evenlv divided. The division between orixinsl and rene p. .l U L '3 st e)eci.ens is also about egua . The submission of du7p licate Specimens nrovide d us with the opportunity to m2ke 2 thorough checn on the stability of types. In over 300 typings, we hcve found only one C2se er1isitin possible discrenan v. During the period covered by this study, we have bed one opgortunity to st°HCr the st2bility of types in 2 limited C2rrier- borne eniuenic. An outbreak of tyghoid fever, 2.7))2. rcntlv veter- (3’ orne, involving 17 persons occurred in one of I Family 8., livin5 on tne outskirts of the Ci y, sunglied about a dozen families, as well 2s thcflSClVQS, with water from a sh2llow well. During the letter p2 rt of July, the f2nily discontinued the flush toilet required a septic t2nh which wvs ins sta lled near the well. Meanwhile, the nei5h1bors continued to use water from the well. Durin5 late July and.August, a total of 15 cases of typhoid fever develOped in the vicinity of family 5. Upon investigation, it was discovered that the well water was dan erou sly :olluted. The indicated source of pollution wes the new septic tank end the well was condemned as soon as its mensce was discovered. A total of 30 strains of §; tvnhi from 17 individuals 27 involved were submitted for typing. All were phage type El. Among these streins were severel from two members of Fenily S. who were proved to be carriers. The originel source in the femily was the mother who hed hed typhoid fever 15 to ?O yeers before. A grand- cnild, presumably a contact carrier, was also found to be discherging é; tyihi, type 3 . Attemn s made to culture §; tynhi from the well water proved unsuccessful. Up to the tine of this report, 60 cultures isolated from this outbreaz have been received for typing. All were identified as type El end none proved to be untypeeble. The cultures identified as Db were 211 of the sub—type HP55. 5. These cultures represent the newest of the phere types. Creigie (l9fl6) identified this tvpe among cultures submitted for typing from an out- 0. '3 I break of typhoid fever in one of Michigan's larger cities in 19b6. The disease was contracted by 22 people at a wedding breakfast. Epi— demi0105ical investigetion indiceted contaminated food. A previously nknown cerrier wes discovered among the people who served and prepared the food. Creigie made an extensive study of the 17 cultures submitted 3 and reported Tvoe DhEPEé as beine isolated from the carrier end eight be type Dl. Arrengenents are being made to study the organisms being excreted by the cerri r. All cultures of type DhEPEé identified during this study were isolated from persons known to have been iniected 2s a result of the outbreoh described ebove. The two cultures identified as Type D6 were isolated from two chronic carriers. Reactions originally obtained with the pooled pheges were indefinite, portiel lysis being observed only in Pool VII. he homologous phage techni us was employed and the phage produced ’30 Avg was identified :2 being similar to the stenCnrd phage I’. V Q 0 9 0 o / Lntypeeble cultures in tnlS series represent 23.85p of o a a o A AA/ 0 - all isolations as compared YltL 41.7op reported by Eucnle in his survey of the litersture. As inciccted in i- f «3’ 1.1 (T) q H 0 d n D '1) un t:.'17:€ C)": l e C cultures were divided into the h and rrnne form. The W form cultures ‘! " ” o o w A]. o ,- made up 8.;6p of the total strains, while 13.22; were in the gamma N e I I / . a 1' '1‘ \ form. Anong the carrier str2ins, l¢.llp were in the t iorn and / A ‘ -8 . ‘ r-r “R\c lo.30p we-e 1n the firm 9 form. Included smong the 23 N form on tures are seven cultures from a carrier 2rd 3 single contact which were proved to be small 1 colony verieties of S. tvpni. The cultural chrrecteristic— were C similar to those described b orris (19kl, 19L3). however, we were not 2ble to reproduce the satellite uhen menon: neither were we able to secure a successful ty.ing on 21y one of the strains submitted. r J \O SUKHA:CI A study of the Vi Uhcge tyfies of S. tvtLi is presented. A detailed review of the literature was mede to develop the lines q 0 ‘L ~ .A . . A» i‘ - r .— "‘3 I: .. -' .. UeCnnlCUU Lit as an ali t0 tee e11ceuiolo 1st. I! vr- ,w‘q— A ”’3 v a. we- 1- . I“ ~ “‘1' 0‘ ~\ e huVC c- emihed ;;7 cultuies of S. t :41 Ly tie gauge -_- 0.. .1. " t. -"l 1.- 0. r- '3, , . 'y~-\""' tgleF Letiod. St. ins in *ded 1n tLis stud; mere Slbulvbed ,_J C'- fron t11e l: torrtories of the Lic.‘ igcu DeUrr 1 of LBS-1th end other leboretories in the state. T;e Ciltures received were dizided roughly into tvo groujs: (l), isoletions from cl'ui Cl crses end (3}, isolep tions from chronic carriers. T1ere i.ere lb} strains (£3.73?) submitted frou ir WEi id1rls clinicallv diagnosed as Levine tvgnoid fev=r. This I total was made up or 67 original and 76 reuest streins. He were able to secure positive typing on 121 cultures (3h.6lfi) waile 22 cultures were untyUeeble. The typedble strains rerressuted seven types, i.e., A, C, D1. De, D6, 31 and Fl. 31 was the most prevalent tyUe, making fl/ 7 t o o q o o o o a up 69.44p of tee pos1t1ve tvping SGCITBQ 1.1ta clinical isolations. )\ \ There were 18$ strains (55.~op) suonitted from carriers (chronic or contact) for tv1ing. Type i, not represented in the ilentificetions from clinicel cases, was identified in a culture from a chronic carrier. Success ul tryings were secured on lb str miis thich proved to be sub-tyres of type 3. This type, as vell rs D5, was not —‘ *v- ‘ . ‘-. ~~ .‘ r- : - - ,- ' P H * regreSeuted in tie cultures out fined from CllfllCcl C9918. 30 Lt se of more than academic interest trcruse it could be con- jectured tLet tLer is s verirti or in tLe gcthogenicity of the various chge types. If true, this mi Lt account for the yreeony derence oi certain range tvUes. The V io.m could Lot be recovered . p.‘ ,./ iron 56 carrier st:21:s (J3.9;}). Positive typings ve re com“leted on 2L9 (70.1ep) or tLe teri received for typing. Furtner a Jplice -tions b) I \) -0 (0 cf ’3' m H. S 'I‘ O H lm N f the honolorous Uhp"e technicue snould name it possicle for us 0 to reduce the numcer of untywesble strains. We also have received three new type strains not r=rreserte1 by nneges in our typing kit. Plrges propugeted a einst these and tested ag-inst tLe so- elled untypeable strains sLould furtler reduce the percentage of unsuccess— ful t;rpings. 2. 31 COZCLCSIOIS FA. H 3 The types of S. tvULi wLicL heve been ident ied during the course of this study are limited in~numher end.are readily distinguisLeble. Typing results indicste that the phege type is a reletively stable characteristic of.§; tvnhi. The epideniological value of phsge typin; is based on the fact that the form \ en, and therefore the phege type, is steble — of the Vi anti followin' pessege from one person to another. In C.) u‘ The information ostained, as a result of phage typing, is of epid niolOgical significance, especially es concerns the typing of strains isolated from known typhoid carriers. 9. 10. 13. 31 13“! "nv I (~13 Erelley, L. H. 1943 An enick lioloviccl stu_d" of Erctcriuh tvphosun type 34. Pritish‘ Me edicel Journs l 1:L38. Bu 72:18, G. 19146 Le t3: 1: 1 o rcteriun tvfhcsum. Medical \_. ‘ I n/ Journel of Austzelie, 2:,05. Burnet. F. M. 1927 elstion,911s oetiecn heet—steole e“ lutin- s o:ens end sen itivitv to becter flirge in tlze Selnonelle Proun. Eritish Journal of Experime ntel P? tholog: 8:1 M1 Creigie, J. 19L6 Personsl communication. Creigie, J. 19Ll Tvping of typhoid hrcilli with type II Vi phege. Tlie Cvclo::e-dia of Medicine, Surgery and Specialities, Service volume:5. Creigie, J., end.3rrndon, K. F. 1936 The lehorst r3 identifi— crtion of the V form of Brcterium tgjhosus. Canndien Public Health Journal, 27:105. Creigie, J., e.nd Pelix,.&. 1947 Typing of tveltoid crcilli with Vi b? cteriophnge. The Lancet, June lit. p. 87?. J., and Yen, C. H. 1937 V b cte ium tvrr‘o