SOVIET VIABLE PASTEURELLA TULARENSIS VACCINES · from Pasteurella tularensis strains 155 and 15 'recovered" (also translated as "renewed" or "restored"). These instructions bear an

SOVIET VIABLE PASTEURELLA TULARENSIS VACCINES

A REVIEW OF SELECTED ARTICLES

W. D. TIGERTT

The United States Army Medical Unit, Walter Reed Army Medical Center, Fort Detrick,Frederick, Maryland

I. Preface...................................................................... 354II. Introduction............................................................................. 354

III. Tularemia and Pasteurella tularensis in the U. S. S. R.................................... 355IV. Development of Vaccination against Tularemia in the U. S. S. R., 1934 to 1956............ 357V. Laboratory Studies on Vaccine Strains of Pasteurella tularensis ........................... 361

VI. Production Techniques .................................................................. 363VII. Vaccination, Skin Testing, and Revaccination............................................ 364VIII. Current Status and Future Programs..................................................... 366IX. Literature Cited...................................................................... 368

I. PREFACE

Efforts to develop methods of vaccine pro-phylaxis for prevention of tularemia began some30 years ago. Various nonliving preparationshave been used in animals and man. Because ofthe apparent inadequacy of killed vaccines,Soviet investigators turned to a viable prepara-tion, first used in man in 1942. Today viablevaccines form a major portion of those Sovietpublic health measures intended for the control oftularemia.

It is the purpose of this report to describe thedevelopment of these viable vaccines. Since suchstudies in man had not been conducted elsewhere,the list of literature cited obviously is limitedalmost entirely to articles and monographs bySoviet authors. The sources were determined, inpart, by their availability in translation, and itmust be appreciated that the coverage is notcomplete.

Gratitude is expressed to the numerous indi-viduals and agencies through whose effortssource materials were assembled.

II. INTRODUCTION

Ampoules of "viable" tularemia vaccine werebrought to the United States from Russia fromthe Institute of Epidemiology and Microbiologyimeni N. F. Gamaleia (Gamaleia Institute) byShope in 1956 (88). The label indicates seriesno. 672, control no. 3207, date of expirationJanuary 25, 1958, and the name "cutaneoustularemia live vaccine." Accompanying was a

set of provisional instructions (9) for the controland use of the dry live cutaneous vaccine madefrom Pasteurella tularensis strains 155 and 15'recovered" (also translated as "renewed" or"restored"). These instructions bear an originat-ing date of August 9, 1955. It is specified thereinthat the expiration date, if the vaccine is held at4 C, is 2 years from the date of manufacture.The two strains (155 and 15 "restored")

mentioned in the title of the provisional instruc-tions are described by Olsuf'ev et al. (69-71).Either may be used and either strain, dependenton the method of culture, will dissociate.

Cultures from these ampoules have been shownin several American laboratories to contain twocolony types. One of these, termed "white" inSoviet publications (or "blue" when colonies areviewed with oblique light), is immunogenic forguinea pigs. The other colony type, termed''gray," does not appear to be immunogenic.

In the 1955 control procedures, the "immuno-genic" type must constitute 20 to 30% of thetotal number of organisms present in the vaccine.Discrimination is on the basis of colonial mor-phology after incubation for 4 to 5 days, followedby refrigeration for 24 hr.The 1955 provisional instructions specify that

guinea pigs should tolerate, without death for 1month, the subcutaneous inoculation of 1,000,000total cells of the vaccine, while it is permissibleto lose up to half of the total of 25 test whitemice receiving a subcutaneous inoculation of1,000 cells of the vaccine. When the surviving

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mice are challenged with a dose of 1,000 teststrain cells, 90% must survive for a period of 15days.

Olsuf'ev (68) states that the test strain used inthe above described challenge is called 503. Itwas isolated in June 1949 from Dermacentorpictus Herm. in the Moscow area (37, 38) andhas had 405 guinea pig passages. In the 1955provisional instructions (9) it is indicated that 1cell is capable of producing death in mice in 5to 7 days. The test strain is grown for 2 days oncoagulated egg medium. Olsuf'ev and Emel'-ianova (67) characterize this strain by statingthat the lethal dose of subcutaneous administra-tion is 1 microorganism for white mice andguinea pigs, while 108 or 109 organisms arerequired to kill white rats. Apparently somerabbits are capable of surviving an intravenousadministration of 1,000 organisms, and thesubcutaneous LDh is 109. (Olsuf'ev will be referredto extensively in this review. He is Director ofthe Tularemia Laboratories, Department ofNatural Focal Infections, in the GamaleiaInstitute of the Academy of Medical Sciences ofthe U. S. S. R., Moscow. Gamaleia Institute isthe largest unit of its kind in the Soviet Union(12, 13). According to Sil'chenko (92, 93), thistularemia laboratory was inaugurated in 1929under the direction of Khatenever. Olsuf'ev'searly work was on the vectors of tularemia inAlma-Ata, beginning in 1934. He was appointedto his present position in 1948 upon the death ofKhatenever. He is a Corresponding Member ofthe Academy of Medical Sciences of the U. S. S.R. (69, 70). Emel'ianova, associated with Olsuf'evin various publications, will also be quoted.She, too, is a member of the staff of GamaleiaInstitute and her publications on tularemia andP. tularensis began as early as 1942 (37, 38).)

III. TULAREMIA AND PASTEURELLATULARENSIS IN THE U. S. S. R.

Tularemia first was identified in the U. S. S. R.in 1926, occurring in epidemic form along theVolga in the Astrakan District (92, 93). Therewas initial recognition in, or perhaps extensionof the disease to, areas east and west of thisdistrict during the period 1929 to 1938. During1940, all of the foci in the south of Russia becameactive, and the disease appeared to spread to thewest as far as the Dnieper River (45). Sil'chenko(92, 93) relates these outbreaks to an increase in

voles and house mice, associated with the dis-ruption of war (also, see Tsareva (97)). In thewinter of 1941 and 1942, Jusatz states that nearly30,000 cases were reported in the lowlands nearthe Caucasus and some 37,000 cases occurred inthe area around Rostov. In some villages, theentire population was involved. Epizootics andepidemics are stated by Borodin et al. (16) tohave occurred in the Stalingrad area in 1934,1937, 1940 to 1942, and 1945 to 1946.

In the volume on Epidemiology in PracticalExperience of Soviet Medicine in World War II,there is a considerable section on the "greatexperience in preventing and combating tula-remia" acquired by the service (62). Sil'chenko(92, 93) described investigations done underdifficult front-line conditions and control of thedisease in the trenches and in the civilian popula-tion behind the lines. Mikat and Kuhlmann (61)described the clinical picture in 900 Germansoldiers who developed tularemia as a result ofparticipation in the "War in the East." (For adescription in English of a preantibiotic andprevaccination epidemic in deportees in Siberia,see Glass (41)).

Another example of the magnitude of theproblem is given by Shmuter (87), who describestularemia as it occurred in sugar factory workersin 1948 and 1949. At various factories the over-all attack rate ranged from 10 to 20%, and incertain sections up to 75% of the workers becameill. He cites figures of 1,207 workers on the sicklist because of tularemia infection with a totalloss of 24,353 working days, and specifies thatreporting of cases was incomplete.The public health significance of this disease is

likewise attested to by the large amount ofmedical talent which has been devoted to itscontrol through large-scale vaccination, treat-ment, and control of hosts and vectors.The geographic distribution of tularemia in the

U. S. S. R. can be appreciated readily by a glanceat the map in Fig. 1. Laboratories, tularemiacontrol centers, or reported endemic or epidemicfoci are underlined thereon. Except as they maybear incidentally on the main subject of thisreview, i.e., viable vaccines, no effort will bemade to discuss the several different types ofendemic foci or the programs intended to controlor eradicate hosts and vectors. It is generallyagreed that case fatality rates of tularemia inRussia are lower than in the U. S. A. U. S. S. R.

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over-all rates are about 1.0% (72). Yet Sil'-chenko (92, 93) specified that Stradowskiisuccumbed to the disease while investigatingwater-borne outbreaks in 1933, and also saysthat the study of the disease in the Omsk arearesulted in the death of Krol' in the early 1930's.In a recent summary the death of two or fourmembers of a family is recorded, the infectionhaving resulted from ingestion of an infected hare(82). However, Elkin (32, 33) seems to summarizecurrent opinion with this statement: "It is truethat when tularemia was first studied it seemedvery dangerous, but at present scarcely anyoneconsiders it so."

Several authors have advanced the belief thata part of the difference between Old-World andNew-World tularemia was due to a difference invirulence of the respective strains. RecentlyOlsuf'ev et al. (72) have compared the U. S. S. R.strain 503 (described above) and the U. S. A.reference strain Schu. They state that Schu iscapable of killing all rabbits in very low doseswhile 503 kills only with doses of 109 cells. Schualso produced more deaths in rats than did 503at comparable dose levels. For various wildrodents the lethal doses were very similar, but thetime to death was shorter with Schu. Russianstrains usually did not ferment glycerol, whileSchu did. Immunologically no differences weredetected.

IV. DEVELOPMENT OF VACCINATIONAGAINST TuLAREMIA IN THE U. S. S. R.,

1934 to 1956

Sil'chenko (91) states that the development oftularemia vaccines was begun in the U. S. S. R.by Khatenever. El'bert (26, 27) notes that he,Gaiskii, and Kudo first demonstrated in 1934that experimental animals inoculated with aweakly virulent tularemia culture were protectedwhen subsequently inoculated with a virulentculture and suggested the possibility of such aprocedure in man. According to Sil'chenko (91),Gaiskii and El'bert began work aimed towardsuch a vaccine in 1935, and by 1942 at the IrkutskAnti-Plague Institute had developed a strain,"Moscow," which had a weakened virulence andhigh immunogenic characteristics.

Elsewhere (50) it is stated that Gaiskii usedtwo methods of attenuation: "(a) by the actionon Bact. tularense of immune serum; and (b) bydrying the strains at thermostat temperature."This strain was tested in volunteers and proved

to be completely harmless. Six months later sixof these inoculated persons were subjected to anexperimental infection with a virulent tularemiaculture. The review article states that the testfully confirmed the experimental data obtainedearlier on animals and proved the harmlessness ofthe vaccine as well as its high protective effec-eiveness. After use on several thousand individualsthis strain apparently was lost.By 1944 Gaiskii had succeeded in obtaining

new strains of P. tularensis "according to aspecial method he developed," namely, attenua-tion on artificial culture media (see Emel'ianova(37, 38)).One of the attenuated strains which was

nonvirulent to guinea pigs and relatively virulentto white mice was called by Gaiskii "Bowillonstrain 15." (This is probably a typographicalerror; "bouillon" seems more likely.) A secondstrain of this type was named "Ondatra (musk-rat) IV." In a report by Faibich and Tamarina(39), it is indicated that Khatenever claimedthat he provided the already attenuated strain15 to Gaiskii. As originally prepared, this producthad to be used within a week after preparation.To overcome this limitation, Gaiskii then de-veloped a "dry" vaccine.

Clinical tests with this vaccine were reportedby Kosmachevsky (55). Strains 15 and OndatraIV were inoculated subcutaneously; the latterstrain was dry. When these vaccine strains wereemployed separately, it was not possible todistinguish the reaction of one from the other.Kosmachevsky concluded that vaccination withattenuated strains was harmless.

El'bert et al. (25, 30) developed the method ofinoculation of tularemia vaccine by the dermalroute (comparable to vaccination for smallpox).In 1945 this investigator, with various workersat the Rostov Anti-Plague Institute, prepared a"liquid yolk" vaccine (ZhTV). The originalpublication of this "liquid yolk" media byDroshevkina (19) has not been reviewed. In laterpapers, reference is made to the use of an eggyolk suspension in normal saline for vaccineproduction, as well as the preparation of avaccine from a 2-day-old tularemia culture on the"solid egg medium of McCoy," suspending thepreparation in equal volumes of the fluid yolkmedium and normal saline (20, 21). (Instructionsfor preparation of vaccine issued in 1957 (18)specify that after the egg yolk and saline havebeen mixed the material should be coagulated in

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a water-jacketed hot air incubator for 1 hr. Thetemperature of the water is 80 C.)

After this liquid vaccine had been used in a

variety of animal studies it was applied in man.

Initial work was with a 50% glycerol-virusvaccine with a titer of 2 X 109 per ml (26, 27),and the statement is made (25) that vaccinationcould be accomplished with as few as 3 to 4organisms. The vaccine, which had a very shortshelf life, was administered by the cutaneousroute in 24 tularemia-affected localities. Localreactions occurred in most instances; e.g., in one

series of 950 persons the rate was 94%. Systemicresponses were mild, if noted at all, and in no

instance was there time lost from work. Thevaccination programs were initiated after theoutbreaks of illness had reached their peak.Prior to vaccination 11.1% of the population hadcontracted tularemia; afterward 4.3% of thosenot vaccinated became ill, in contrast to a rate of0.36% among the vaccinated. The cases in thevaccinated persons occurred only during thefirst 12 days after administration. It was thusconcluded that vaccination during the incubationperiod was harmless and apparently resulted inshorter and less severe forms of the disease or

even in symptomless forms. In 1946 Gaiskii andEl'bert were given a Stalin prize for their jointwork (91).

Faibich and Tamarina (39) of the ScientificResearch Institute for Epidemiology and Hy-giene (NIIEG) describe in detail the laboratorycharacteristics of strain 15 and Ondatra IV.They state, "thus the experiment on white miceand guinea pigs has shown that the strains fromwhich the dry living tularemia vaccine of NIIEGhas been and is being prepared are not completelyavirulent for small laboratory animals; the strainOndatra IV is virtually avirulent for guinea pigsand weakly virulent for white mice, while thestrain 15 is, conversely, more virulent for guineapigs and less virulent for white mice. Thus bothstrains possess in one degree or another a 'resi-dual' virulence." These authors conclude thatstrain 15 is generally much better than OndatraIV, but then hasten to point out, "at the same

time it must be kept in mind that inasmuch as

the vaccinal strains were not identical in anti-genic and immunogenic respects, the preparationof a polyvalent dry vaccine from several vaccinalstrains of tularemia is expedient." They indicatedthat a spontaneously attenuated strain 10

"Bulukhta" obtained in 1944 from the SaratovInstitute was available for study, and that whileit was less reactive for guinea pigs and white micethan strain 15 it possessed high immunogenicproperties. An intent to study strain 10 in man

was stated.The subcutaneous inoculation of 12.5 to 25

million organisms of strain 15 produced agglutina-tion and allergic responses in man at 1 month inall individuals studied, while lower percentageswere obtained with strain Ondatra IV. More than30,000 men were vaccinated. Strain 15 could beadministered to recovered human patients indosages of 25,000,000 organisms subcutaneouslywithout producing serious side effects.For measuring the "allergic response" to the

vaccines, Faibich and Tamarina used intracuta-neous inoculation of "tularin," a suspension ofkilled P. tularensis. Khatenever (50) stated thathe first used this for diagnostic purposes in 1931and 1932. His preparation was heat-killed at 65C for 30 minutes and, following intradermalinoculation, promptly produced a reaction char-acterized by hyperemia and edema. El'bert andGaiskii are credited with the validation of a

similar procedure for evaluating the response tovaccination. Khatenever specified a concentra-tion of 108 organisms per ml. Kalitina (47) statesthat the "usual" diagnostic dose of tularin is 108killed organisms in 0.1 ml. A report in 1950 (23)specifies an identical concentration and indicatesthat a positive response will be manifest in 6 to 8hr. More commonly the test was read at 24 to 48hr (64, 65).A second report in 1946 by Faibich and Tama-

rina (39) is concerned with the methods initiallyused for the preparation of a dry vaccine. Sincecurrent methods are a direct outgrowth of thiswork, certain of their statements are quoted."Our own numerous investigations enabled us topropose the following media for mass-productionof tularemia bacteria preparations: (i) Semiliquidagar consisting of pancreatic hydrolysates of liverand gelatin, sucrose, white gelatin, starch, agarand water. (ii) A thick agar medium in two vari-ants, the first of which contains pancreatic hy-drolysates of liver, blood and gelatin, milk, serum,cysteine, starch, table salt, agar and water, andthe second, pancreatic hydrolysates of blood,autolysates of yeasts, milk, serum, cysteine,starch, table salt, glucose, agar and water....The hydrolysates of liver and blood, contrary to

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the generally accepted method, were preparedwithout preliminary thermal treatment, i.e.,from raw material ... [and] were treated withsulfuric acid aluminum. In the growth media a

slightly alkaline reaction (pH = 7.1 to 7.2) was

established and in the drying media, a neutralone. The proposed media are sterilized in an auto-clave at a temperature of 120 C.," The sucrose

content was 4% and the gelatin 1.25 to 1.5%.These gave a yield of 1.5 to 3.0 billion organismsper ml of media, the higher figure coming fromthe thick agar. Prior to lyophilization, additionalsucrose up to 10% was added to the semisolidmedium and the material was dispensed in 1-mlamounts. If the thick agar medium was used theorganisms were "washed" with the drying me-

dium (i.e., sucrose-agar-gelatin) and then dilutedwith the same material to a titer of 2 to 5 billionorganisms per ml. After being placed in ampoulesthe vaccine was prefrozen, then dried for 14 to 18hr without added heat, and sealed under vacuum.

The authors believed that the sucrose (or maltoseand lactose) "fixes the amount of water" and"safeguards the microbes against excessive dehy-dration." "Agar facilitates their longer preserva-

tion in the dried state," while "gelatin merelyimproved drying conditions" and permits thedrying of small numbers of organisms. In any

event, absence of any of these components was

found to produce an inferior product, as was

storage without vacuum sealing. Such a vaccinewas immunogenic for man after storage at 2-4 Cfor 1.5 years, at 18 C for 270 days, and at 26 Cfor 75 days. Using NIIEG vaccine prepared in1951, which had been stored at temperaturesranging from a winter low of -2 C to a summer

high of 18 C, Martinevski (59) noted that withaging the local skin lesion and the incidence oflymphadenitis declined markedly. No correlationwith viable count was recorded.

Control procedures specified by Faibich andTamarina included testing for purity, lack ofharmful effects in guinea pigs, autogenicity inmice, and the titer. The measure of the last men-tioned property was the limiting dilution of thevaccine at which there was perceptible growth oftularemia organisms in tubes containing yolkagar (52).

According to Vachkov and Pronina (98), mass

prophylaxis vaccination was begun in 1946. Anearly example is given by Borodin (15), who de-scribes a vaccination program with the liquid

product in the Volga-Akhtubinsk River valley.Because of an outbreak of tularemia, 11,622persons were inoculated during the summer of1946. Of those inoculated, 0.17% became ill,while in the noninoculated the incidence of diseasewas 3.4%. Most of the cases in the inoculatedoccurred within 2 weeks after vaccination, andthe disease was of a mild form. In subsequentyears additional vaccinations were given, and thecumulative number of individuals inoculated by1954 was 81,739. Another early program is notedby Panaiotti (75), who states that between 1947and 1951 21,000 persons were vaccinated inYartsevskaya. According to Vachkov and Pro-nina (98), during these early years mass vaccina-tion programs were conducted in numerous areas.The results obtained were "excellent," and in1951 the practice of vaccination was changed tothe interepizootic period rather than waitinguntil an epidemic seemed likely. U. S. S. R. publichealth forms dated 1955 include printed entriesfor the reporting of cases of tularemia and for"injections against tularemia" (94).

In a monograph edited by Olsuf'ev (64), it isclearly indicated that most of the work done inRussia before 1951 had employed the liquid eggyolk vaccine (ZhTV). The Gaiskii vaccine strain15 was used in most instances, and the Faibichstrain 10 was added (sic) to the NIIEG vaccine(i.e., the dried vaccine).

(In some recent translations (69-71, 79), refer-ence is made to liquid yolk-sac (sic) tularemiavaccine in use from 1948 to 1952. These aretranslation errors, and "liquid yolk" is the properterm. Although the response of embryonatedhens' eggs to the vaccine strain has been reported(7), there is no suggestion that this medium everhas been employed for vaccine production.)

In the 1953 monograph (64) there is a longreview article by Olsuf'ev. He examines the vari-ables that must enter into an evaluation of fieldresults in a live vaccine program, such as methodof storage, method of use, source of vaccine,immunological characteristics of the population,method of selection, accuracy of observation, andtype and amount of skin test antigen used. Hethen notes that although the number of observa-tions with the dry NIIEG product were limited itappeared that the number of clinical and immu-nological responses were somewhat higher whenthe dried product was used. He postulated thatthis difference is due not only to the method of

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preparation of these vaccines, "but also to thedifferences of the strains [sic] included in them."

Olsuf'ev then considers the relative merits ofthe intracutaneous and the subcutaneous routesof inoculation and concludes:

"As compared with the subcutaneous injection,the skin method of vaccination is considerablymore simple in its application and produces goodimmunological results. The skin method isconvenient, because the procedure of a skinvaccination reaction makes it possible to countreadily the results of the vaccination, conducted.The subcutaneous method of vaccination makessuch a count impossible. In the event of a suddencontamination (at the time of vaccination) ofthe vaccination material, the skin method ismore safe. This permits extensive enlistment ofintermediate medical personnel for the implemen-tation of skin vaccinations at any center. Thus,the entirely satisfactory results of mass vaccina-tions, and the considerable simplicity of theapplication technique justify considering theskin method of vaccination in tularemia incidenceas the more promising method, especially if thedry skin vaccine of the NIIEG type is to beused."

Olsuf'ev continues with a statement that theproblem of revaccination has not been adequatelystudied, and with a consideration of the "Epide-miological (Prophylactic) Effectiveness of Vac-cination," intermingling observations on ZhTVand NIIEG vaccines. Nearly all reports show amarkedly lower incidence of disease in those vac-cinated, and there is one example of a rate amongthe vaccinated of 0.0049% (sic). Although thenumber vaccinated usually is not included, hemakes the point that, on an oblast scale, vaccina-tion to be effective should include upward of 90%of the "threaten contingents." (An oblast is thefirst order division of an individual soviet republicand may vary markedly in size and population.)The following paragraph is also of interest.

"In conclusion, it must be noted that, thanksto the vaccinations, cases of intralaboratorytularemia infection ceased; yet prior to the injec-tion of the vaccine it has been practically im-possible to prevent them, regardless of the strict-est possible personal prophylaxis."

Olsuf'ev notes that the vaccine organisms arenonviable after 1 month and that "individualsvaccinated with ZhTV or NIIEG are protectedfrom tularemia infection through the skin,

respiratory organs, alimentary tract, etc." Earlierin the paper the following pertinent sentenceoccurs. "The question as to the quality of thevaccinations conducted can be settled only on thebasis of spot checking of the vaccinated contin-gents by means of a tularin test; the first checkupshould be conducted a year after vaccination."

Also in the Olsuf'ev monograph (64) is anarticle by Shmuter (86), which specifies thelaboratory control procedures of that period andindicates that vaccines prepared by two agenciescould vary in potency. According to Shmuter,"Tularemia vaccine strains used in productionmust possess the following properties. Uponinoculation of white mice with doses up to 1 mil-lion microbe cells, 30% of the mice perish. A doseof 1 billion microbe cells fails to kill guinea pigs.An immunizing dose for white mice consists of1,000 microbe cells, for guinea pigs, of 10,000microbe cells."Shmuter conducted studies with production

lots of vaccine obtained from two institutes, bothderived from strain 15 originally. He believedthat better results were obtained in man with theproduct having high mouse virulence.

According to Sil'chenko by 1955 Olsuf'ev hadaccumulated a "great number of observations"with a dry vaccine, in which he had found re-sponses in about 97 %. It is stated that until 1956,vaccines prepared at the Gamaleia Institute usedvaccine strain 15 of Gaiskii, prepared by theaeration method (52).

Detailed instructions for use of the dried vac-cine were issued from Gamaleia in 1953 (8), andan example of the field program is given byOvasapyan (74), who states that 121,204 personsin the Leninakanskiy Basin (Armenian S. S. R.)were inoculated by scarification with dry livetularemia vaccine from October 1952 to the end of1954. The vaccine response reported was 97.5%.Karakulov, Mertsalov, and Zhokin (48) indicatethat during 1955 and 1956 about 700,000 personsin Kazakhstan were inoculated. Kucherov et al.(57), apparently describing a part of the sameprogram, specified that the dried vaccine was ob-tained from Gamaleia. Their figures for fourregions in West Kazakhstan are 114,000, orpractically the entire population. Sil'chenko (91)makes the following additional statement. "Thechange to the production of dry antitularemiavaccine at the Institutes [sic] must be regarded asjustified." The date coincides with the issuance

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of the provisional instructions for the use of drylive cutaneous tularemia vaccine noted in theinitial paragraph of this summary. It must be,then, that El'bert (29), writing of the immuniza-tion of 60 million individuals, is referring to avariety of types of immunization employed overa period of 12 to 14 years.

V. LABORATORY STUDIES ON VACCINE STRAINSOF PASTE URELLA TULARENSIS

The identification of strains ranging from fullyvirulent, partially virulent but immunogenic,and avirulent, particularly as measured in themouse, resulted in a variety of laboratory studiesintended to characterize these various organisms.

Olsuf'ev and Emel'ianova (67) state thatEl'bert and Gaiskii in 1941 described a differencein the agglutination of virulent and avirulentcultures, the avirulent cultures producing a finegranular agglutination, in contrast to the clumpsseen when virulent cultures were similarly exam-ined. On the basis of these 1941 studies, El'bertand Gaiskii proposed that the virulent cells con-tained two antigens, one of which was graduallylost in the process of attenuation, and was com-pletely missing in the avirulent strains.

Aleshina and Pucheva (5) described a correla-tion between virulence and fibrinolytic activity.Lytic activity was most marked when testedagainst fibrin from animals fully susceptible totularemia infection.

In 1947 Karpusidi (49) described the char-acteristics of certain atypical and "little virulent"strains of P. tularenesis. Some of these werecapable of growth on ordinary laboratory media.Biochemical reactions were variable. All strainscould be agglutinated by specific antisera, and,at least in some instances, passage through ani-mals resulted in restoration of virulence.

Tinker and Bibikova (95) report on the capsu-lar fraction of P. tularensis, indicating that thisis present only in virulent tularemia organismsand is often absent in the case of avirulentgrowths. This is thought to be a thermostablepolysaccharide which is not a complete antigen.However, the capsular fraction of the virulent andof the vaccinal strains would result in the produc-tion of a precipitin reaction against tularemiaimmune serum. Both the virulent and vaccinalstrains had allergenic components, while corre-sponding tests in sensitized guinea pigs made withcapsular fraction of the avirulent strain did not

cause allergenic reactions. Later there is a reportfrom Tinker and Elfineva (96) on three strains ofP. tularensis, namely, an avirulent strain (no. 11),an attenuated strain (no. 38) used for vaccinemanufacture [sic], and the highly virulent strain47. (It is not known whether the Russian strain38 and the American strain of the same numberare identical.) In vitro the leukocytes of normalguinea pigs were more active against the avirulentstrain and lowest against the virulent strain.When inoculated into guinea pigs the virulentstrain produced a maximal increase in the phago-cytic index, a less intense reaction was observedwith the attenuated vaccinal strain, and some ofthe avirulent strains showed no change at all.Similar findings were observed on the phagocyticactivity of normal human blood with respect to alow response, or a low index, in terms of the viru-lent strain and a higher index in regard to thevaccinal and the avirulent strain. In recoveredcases the phagocytic index was high for the viru-lent and the vaccinal strains, and there was someincrease in the indices in those individuals vacci-nated against tularemia.

Studies on virulence, attenuation, and colonialmorphology were described by Emel'ianova (34).She states that by repeated (many) passages inlaboratory animals any virulent strain can bechanged to an avirulent strain. This change to"moderate virulence" was associated with theappearance of two colony types: (i) a small whiteopaque raised colony and (ii) a large transparentflat grayish colony. Killed cultures of both wereallergenic in guinea pigs. However, type 2 (largeand transparent) did not kill mice in doses of108, while the small colony type sometimes waslethal for mice.The medium used by Emel'ianova was fish-

cysteine agar and blood as formulated by Kolya-ditskaya and Shmurygina (52). Such a medium,with or without aeration, was used for vaccineproduction at Gamaleia until 1956 and wasknown to produce marked dissociation. It ap-pears also that these two types could be inducedby prolonged incubation. Primary growth of thevirulent opaque colonies stopped by the 4th or5th day. Then on the 9th to 11th days transparentdaughter colonies were observed about isolatedcolonies. Puchkeva (80) also notes that on artifi-cial culture media at 37 C, "the virulence of thevaccinal strain for mice gradually increases andagain diminishes in relation to the length of

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culture." Maximum lethality for white mice wasnoted with a 24- to 48-hr culture. Three- to 15-day-old cultures showed a material decrease inlethality for this animal. The 24- to 48-hr culturesshowed maximum immunogenicity (measured bysubsequent challenge) in guinea pigs, while the10- to 15-day cultures had very poor immuno-genicity. Emel'ianova and Shipitsina (35) reportdetailed chemical studies on virulent strain 21(S form) and an R variant derived from 21 as aresult of 250 passages on agar. In a 1957 paper(67), Olsuf'ev and Emel'ianova describe theirconcepts of the "Antigenic Structure of Pasteu-rella tularensis." They use conventional terminol-ogy for colonial types of the organism, referringto S and R, and indicating that S is virulent,while R is avirulent. They visualize the fullyvirulent tularemia organisms in the terms em-ployed for the description of typhoid bacillus.Thus, there is a somatic complex symbolized 0,and an envelope antigen symbolized Vi. Theavirulent strain has lost the envelope or Vi an-tigen. Tests are described indicating that an-tisera against the R antigen will not aggluti-nate S organisms. On the other hand, S antiseraagglutinate R organisms poorly, if at all. Absorp-tion studies are described to support this conceptfurther. "The virulence and immunogenic prop-erties of tularemia bacteria are due to the presenceof the Vi antigen. If this antigen is lost thebacteria become avirulent and nonimmunogenic.The Vi antigen may be destroyed by heating orby the action of alkaline solutions. Both the Viantibody and the 0 antibody are thermolabile."There is a description of the "living culture of a'renewed' Gaiskii 15 vaccine strain." This strainwas agglutinated by S and R sera at a dilution of1:1,280. The reaction with the S serum produceda stable agglutinate and, with R serum, an un-stable agglutinate. The authors assume thatwhile an envelope antigen is present, it is presentin such a small amount that agglutination byboth S and R sera is possible. The vaccine strainis therefore described by them as being SR andin terms of antigens to 0 (Vi). In continuation,the S strains of P. tularensis are denoted 0 cul-tures.

Biochemically, the capsular antigen containsprotein, polysaccharide, and lipids. Verenikova,Kontorina, and Bakhrakh (100) also describe theseparation of polysaccharides from virulent andvaccine strains of P. tularensis by heat and acetic

acid treatment. They state that the "vaccinestrain 15" on a weight basis provided morepolysaccharide than did the virulent strains.This polysaccharide is allergenic and can be usedfor skin testing but was not antigenic and did notprotect rabbits.

In an article submitted on May 29, 1957,Emel'ianova (36) describes "Characteristics ofTularemia Vaccine Strains According to Labora-tory Indices." It is stated that the work reportedtherein "served as the basis for the presentoperating specification in production, control anduse of cutaneous, dry live tularemia vaccine."The complete paper should be consulted by any-one specifically concerned with this vaccine. Sheexamines the problem of dissociation, identifiesthe immunogenic colony type, and specifiesmethods of examination to insure that an ade-quate number of immunogenic cells is includedin any preparation.

She then turns to an examination of the re-quirement for "retention of residual virulence"in vaccine strains, a concept originally stated byEl'bert and Gaiskii. By this is meant the reten-tion of the capability to kill a proportion of testmice while failing to kill guinea pigs. She statesthat if graded doses (102 to 101 cells) of an appro-priate product are given subcutaneously to equal-sized groups of white mice, the number of animalsthat die is proportional to the size of the dose andthat 30 to 50% of all animals so inoculated shoulddie. At least 90% of the survivors should with-stand the subsequent inoculation of 1,000 cellsof strain 503. Guinea pigs should tolerate 1 billioncells. At least 80% of guinea pigs vaccinated sub-cutaneously with 10,000 cells should withstandthe subsequent inoculation of 1,000 cells of strain503. Usable vaccines must also elicit a cutaneouslesion when inoculated by scarification.

Emel'ianova then notes that the originalGaiskii strain 15 apparently had become soattenuated that it no longer met these criteria."After passage through animals highly sensitiveto tularemia the vaccinating effectiveness of thestrain increased significantly." This variant wasnamed "no. 15 restored" ("regenerated" or "re-newed") and meets the laboratory indices noted.Finally, mention is made of "refinements inproduction technology" resulting in productswith a high percentage of immunogenic cells.Kolyaditskaia, Kuchina, and Shmurygina (53)state that as early as 1954 phenomena suggestive

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of phage infection were noted in the Gaiskii15 strain. Similar findings were also observed in1957 in the "restored" strain. Apparently thewhite (immunogenic) colonies are the typeusually involved; the changes vary from an "al-veolar colony morphology" to complete lysis.)

Coincident with the efforts to restore theGaiskii strain 15, a search was made for newvaccine strains (69). "By means of attenuation onartificial culture media and selection of coloniesa new strain 155 was obtained which when testedon laboratory animals showed properties an-swering the requirements." In the title of thepaper this is called the Emel'ianova no. 155strain.

Vaccines were prepared at the Gamaleia fromstrains 15 and 155. Cultures were grown by thedeep method with aeration (see below for detailsof methodology). At the time these were preparedthe vaccine production methods had not beenchanged, so that the total counts averaged 2 X109 per ml with an average of 20 to 40% immuno-genic cells. The vaccines were used 4 to 6 monthsafter drying (69). Seven laboratories participatedin the field program, which involved 7,324 sub-jects, mostly adults. Vaccines were employed innormal dosages, at 1:2 dilution, and at 1 :10 dilu-tion. No significant difference in reactivity asmeasured by the skin tests was found. With un-diluted vaccines the average conversion rate was97%, and at 1 :10 dilution the rates were 65.8%for strain 15 and 69.1% for strain 155. Systemicreactions were rarely encountered with eitherproduct. Agglutinin titers at 30 days postvaccina-tion were slightly lower, on the average, in thosewho had received strain 155. The paper concludeswith a statement that "at present, strains 15(restored) and 155 have been introduced into theproduction of tularemia vaccine." This paper wasreceived for publication in July 1957. Officialinstructions for the production of cutaneous, dry,live anti-tularemia vaccine published by theMinistry of Health of the U. S. S. R. in 1957 (18)include methods of calculation of dosage forthese two strains as well as Gaiskii 15 (old).

VI. PRODUCTION TECHNIQUES

Data concerning the "refinement in productiontechnology" (36) and the "changes in vaccineproduction methods" (69-71) were described byKolyaditskaya and Shmurygina in 1957 (but sub-mitted for publication, July 10, 1956) (52). Prior

to 1956 production of tularemia vaccine atGamaleia was carried out by "growing a culturefor 9 days, by a series of successive subcultures ina variety of media (two generations in yolkmedium, then inoculating onto semisolid agar,solid agar, and semisolid or liquid medium foraeration)." Using fish-cysteine agar with blood asa check medium for demonstration of dissocia-tion, these investigators found that originalgrowth on coagulated yolk gave the greatestpercentage of "antigenic" colonies (white andopaque). Coagulated yolk gave 69 to 89% of suchcolonies, while the "standard vaccine growthmedium" gave approximately 3% antigeniccolonies. Consequently a change was made sothat seed cultures were grown on trays of fish-cysteine agar overlaid with a thin layer ofliquid yolk media (3 to 5 mm) and heated to 80 Cfor 70 min. With cultures grown on this as seed,production batches of vaccine were prepared,and in these the percentage of antigenic organismsrather regularly exceeded 50% of the total. It isnoted that this method materially decreases thenumber of eggs required.The 1957 instructions previously cited indicate

that seed lots so prepared may be dried in amenstruum essentially comparable to that ofFaibich and Tamarina (39).For vaccine production 48-hr cultures are

washed from the trays of fish-cysteine yolk agarwith normal saline containing 0.2% agar. Thismaterial is used for the inoculation of liquid orsemiliquid media.

These final media contain 20 to .30% hydrol-yzates of fresh fish, liver, meat or fish-mushroom,10% gelatin hydrolyzate, 1 to 5% yeast hy-drolyzate, 1.5% gelatin, 0.5% sodium chloride,1.0% glucose,. and 0.1% cysteine with a pH of7;2 to 7.3. For the, liquid medium the gelatin isomitted. These media are dispensed so as to halffill 5-liter flasks equipped to permit aeration withpreheated air at the rate of 1 liter of air per literof medium per minute.

Such flasks are inoculated with sufficientorganisms to provide an initial concentration of5 X 108 to 109 cells per ml. They are then incu-bated for 12 to 18 hr at 36-37 C. Incubationterminates when the density reaches 2.5 to 3.0 X109 organisms per ml. (All concentrations aredetermined optically according to standardsbased on typhoid bacteria.) At this time the cul-tures and medium are added to "media for

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drying," which serves to increase the amount ofsucrose and, if necessary, gelatin. This final ma-terial may be held for 10 days at a temperatureof 4-6 C.For drying, the material is dispensed into am-

poules so as to provide a film less than 1 mm inthickness. After freezing at temperatures of-30 or -40 C, drying is accomplished on eithermanifold or chamber lyophilization equipment ata pressure of approximately 100 ti. The maximumpermissible ampoule temperature at any time is-30 C. A final moisture content of less than 4% isattained. Final sealing is under a vacuum pressurenot exceeding 100 ,u. Shishkin (85), in a reviewarticle concerning the activities of the RostovAnti-Plague Research Institute, states thatworkers there use a "rational 18-hr drying sched-ule" for tularemia vaccine.At various stages in the preparation, tests for

purity are employed. Tests for potency have beendescribed in the previously noted article byEmel'ianova (36). Samples from each lot are sentfor checking to the Moskovskaya Gosundarstven-naya Kontrol'naya Labaratoriya. The 1955provisional instructions specified that eachInstitute preparing vaccine should examine threeto four series each year in humans not immune totularemia. Such vaccines must have producedpositive responses in 23 of 25 persons. This re-quirement does not appear in the 1956 instruc-tions (10), apparently having been replaced by asimilar type of test on the vaccinal seed strains.Such strains, when checked in man, should notproduce general reactions and should result in alocalreaction in 24 of 25 individuals, as well as ina positive allergic test and an agglutinin responsein 30 days.The vaccine is stored at a temperature no

higher than 4-6 C and has, under these condi-tions, an expiration date of 1 year from the timeof drying. Freezing is permissible. It may be re-controlled at this time, and, if potency is retained,the date can be extended for an additional 6months.

VII. VACCINATION, SKIN TESTING, ANDREVACCINATION

For vaccine administration the dried product isrehydrated with distilled water and used within4 hr. It is applied by the cutaneous route only(10). The shoulder of the individual to be vacci-nated is cleaned with alcohol. After this dries, 2drops of vaccine are applied 3 to 4 cm apart.

Through each drop two parallel scratches 1 cmin length are made in such a manner as to resultin the appearance of only tiny drops of blood.The vaccine is then rubbed into these scratchesfor half a minute. A special quill is used.

In a successful vaccination small vesicles ap-pear near the end of the first week and hyperemiaand some infiltration persist to the end of thesecond week. The area then develops a crustedappearance and all symptoms subside within atotal elapsed period of 3 to 4 weeks.There are numerous articles evaluating the

extent of these various reactions. The resultsseem to vary with the source of the vaccine, andthere is correlation as well with the size of theinoculum. In the 1958 report by Olsuf'ev (68),general reactions were noted in 4.5% of thoseinoculated and enlargement of lymph nodes wasreported in 3.0%. All nodes returned to normalsize. In earlier reports figures up to 10-fold theseare sometimes given.Vaccination of individuals recovered from

tularemia produces an increased percentage ofgeneral reactions. El'bert et al. in 1954 (28) re-corded a 3-fold greater incidence in recoveredcases than that seen in those without a history oftularemia. Thus, when vaccine is administered tolarge population groups, those individuals knownto have had tularemia are not revaccinated.Those obviously ill from any cause are excluded

from vaccination, as are known asthmatics andwomen during the last half of pregnancy or duringthe period of breast feeding. (With minor varia-tions these latter general exceptions to vaccina-tion are applied to all viable bacterial vaccinesused in the U. S. S. R. (18)). Ovasapyan (73)describes a case of tularemia in a woman at term.The agglutination titer in the infant was 1:2,000a few days after birth. By the third month thetiter was negative, as was the response to tularin.Kalitina (47) states that in vaccinated guineapigs pregnancy causes a lowering of the responseto intradermal tularin.

In the large program described by Kucherovet al. (57), children less than 4 years of age wereexcluded. The 1956 instructions specify that theamount of vaccine used for children of pre-school age is half that used for adults. Success-ful vaccination of children down to 1 year ofage is reported (81). Olsuf'ev (68) notes thatthere is "lesser stability of the immunologicreorganization of the organism in children from7 to 14 years of age immunized against tulare-

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mia." Piotrovskaia (79) describes the vaccinationof 642 children (6 to 12 years old) and adolescentsin 1948 and 1949. Six years after vaccination witha liquid product, 84% had positive reactions totularin and 90% had agglutinins.The 1956 regulations also specify that other

vaccinations should not be carried out coincidentwith that for tularemia (but see below).

In a study conducted in a mental hospitalMatveets et al. (60) report that in individualswith long-standing catatonic stupor "the vaccina-tion process and the resulting immunologicalreaction developed in the majority of cases to alesser extent than in healthy individuals." In ad-vanced parkinsonism the reaction was also lessthan normal, while in imbeciles and morons theresponse was identical with that of the healthycontrols. (Bisikalova, Predtechenskii, and Zhda-novskii (14) report modification of the immuneresponse in the rabbit under narcosis.)An interesting observation is recorded by

Klepko (51). In a military unit an outbreak ofinfluenza was observed 4 to 13 days after thegroup had received tularemia inoculations.Thirty-eight of 92 men developed influenza, andin these the incidence of extensive lymphadenitis(from the tularemia vaccine) was much greaterthan in those not developing influenza. The agglu-tinin response at 2 months was not modified.The influence of irradiation on the outcome of

administration of the P. tularensis vaccine strainto white mice is described by Shevelev (84). Adose of 374 r was given at varying intervals afterinoculation of 50,000 organisms (vaccine was ob-tained from Gamaleia). Maximum enhancementof fatality occurred when irradiation was given2 to 24 hr after infection. Shevelev considersthat this results in a coincidence of maximumorganism multiplication with the climactic stageof irradiation sickness. Irradiation at a later datewas without influence.The duration of the period of immunity fol-

lowing vaccination has been examined by nu-merous investigators. According to Sil'chenko(90), the first Conference to Study the Effective-ness of Tularemia Vaccine, sponsored by theMinistry of Public Health, was held in 1g49. Atthis meeting annual revaccination was recom-mended. At the 1951 Conference there was agree-ment that the period between vaccinations couldbe extended to 4 years. This represents an averagefigure, since Olsuf'ev (65) records "rare" cases oftularemia among vaccinated individuals in whom

an initial skin reaction to vaccination had beennoted. These people had received liquid vaccinea year prior to their illness.The 1956 instructions state that the period of

immunity conferred is 3 to 6 years (18). Aneditorial footnote to an article published in May1958 (79) states that "at the present time theperiod of revaccination for children and adultsliving in natural foci of infection is officially setat five years." Olsuf'ev in December of 1958 (68)makes an identical statement.

Persistence of the immune response has usuallybeen on the basis of a continued allergic responseto tularin. As early as 1953 Sil'chenko indicated(90) that a considerable portion of vaccinatedpeople retained this reaction for as long as 8 years.(There is one report (17) of a laboratory worker,known to have had a sharply positive skin test,who developed tularemia following a laboratoryaccident. She had been vaccinated 5 years pre-viously, apparently with one of the first vaccinesproduced.)

If previously vaccinated persons who havemaintained an allergic response are reimmunized(superimmunized), the entire response proceedsat a more rapid rate than on initial vaccination,and the percentage of general reactions may beincreased (28, 68). In those who have lost theirallergy the revaccination proceeds in the sameidentical manner as in a primary inoculation.Mention has been made of the intradermal use

of tularin to measure the existence of an immuneresponse. Sil'chenko (92, 93) and Olsuf'ev et al.(69-71) note that such a procedure frequentlyhas undesirable side effects. Both authors citework reported in 1953 by Maiskii and Shipitsina(58), who administered tularin intradermally to agroup 9 months after vaccination. They observedtissue necrosis at the site of injection in 10%,chills in 3%, malaise and headache in 14%, andsome lymph node enlargement in 29%. Kalitina(47) suggests that these reactions may be de-creased by a 1,000-fold dilution of tularin withoutsignificantly decreasing the number of positivelocal reactions noted. In individuals who hadrecovered from tularemia, even more dilutetularin gave a positive test. However, in 1958(69-71) full strength tularin (107 cells per injec-tion) intracutaneously was in general use.(Dunaeva (22) notes that intradermal tularininoculation should not be used in experimentalanimals prior to the conduct of studies in tulare-

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mia since it may alter the immunological reac-tivity.)Because of these adverse reactions efforts

were made in 1954 to evaluate the dermal ad-ministration of tularin. This route of inoculationfor diagnostic purposes had been used as early as1934. This method had also been used for researchstudies in man but had not been accepted for usein the field as a general rule. In evaluating thisprocedure, Olsuf'ev et al. (69-71) showed thattularin could be prepared either from a vaccinestrain or from a fully virulent strain.

In 1956, Olsuf'ev et al. (66) administeredtularin from a vaccine strain percutaneously.They noted that, in 3,867 previously vaccinatedindividuals, a positive reaction was obtained inabout 90%, while side reactions occurred in only1 to 3%. No necrosis was seen. Side reactionswere more common in 202 individuals tested whohad recovered from tularemia. Sil'chenko re-ported similar data in 1957 (92, 93) and notedgood correlation between the responses to thedermal and intradermal routes. He recommendedthat dermal tularin should be put into widespreadpractical use for measuring the effectiveness ofvaccination procedures. According to Muromtsev(63), during 1958 a directive for the preparation,control, and use of dermal tularin from a vaccinestrain was prepared. Olsuf'ev et al. (69-71)specify that the technique of administration ofthe tularin by the epidermal route is identicalwith the procedure used for vaccination, exceptthat a single drop of tularin with two parallelincisions is employed. Reading is done at 48 hrand evaluation is on the intensity of the localhyperemia and edema. The test material shouldbe stored at 4 C (64, 65).

In 1952 Maiskii and Shipitsina (58) demon-strated that a polysaccharide component of P.tularensis obtained by the Boivin type of extrac-tion could be employed for skin testing intra-dermally with a very prompt response. Similarstudies were reported in 1956 by Verenikova etal. (100), who used both the intradermal and thedermal methods of inoculation. Although con-sidered relatively specific, this method apparentlyhas not been used on any large scale.

VIII. CURRENT STATUS AND FUTURE PROGRAMS

According to numerous reports, the value ofviable tularemia vaccine in reducing incidence of

disease and controlling epidemics is amply con-firmed. Elkin (32, 33) states that it is equal ineffectiveness to smallpox vaccine. Olsuf'ev creditsmass inoculations, in combination with othermeasures, with the reduction of the animal inci-dence of tularemia in the U. S. S. R. by 19-foldwhen the period 1945 to 1950 is compared with1951 to 1957. Furthermore, according to Olsuf'ev,the effectiveness of the vaccine has been con-firmed in foci of every type and in the presenceof diverse routes of transmission. Zhdanov (102),speaking at the 13th World Health Assembly,gave to the viable tularemia vaccine primarycredit for the reduction of the disease in theU. S. S. R. He stated that vaccination had beenso extensively practiced that the number of casesof tularemia, "which in the middle forties usedto be as high as 100,000 per year, has now beenreduced to a few hundred cases annually."Yet the problem of control of tularemia still

continues. In a 1958 conference (56), it wasstated that the number of tularemia outbreaks inthe R. S. F. S. R. increased in 1957 over 1956. Inseveral oblasts (mainly in the Moscow area),85.3% of all illnesses were said to be due totularemia. These outbreaks were attributed tocomplacency of public health organs, curtailmentof prophylaxis, inadequate training for medicalpersonnel, and insufficient attention to vaccina-tion. Vachkov and Pronina (98) specified that aninadequate program was being conducted in theseoblasts. Panaiotti (75) indicated in 1959 that inYartsevskaya (Smolensk Oblast) no vaccinationshad been conducted since 1953.

El'bert, one of the original investigators (31),also comments on the proved effectiveness of theviable vaccine but goes on to say, "The next taskis to obtain new vaccine strains capable ofestablishing a defense against cultures of P.tularensmi with maximum virulence." Muromtsev(63), in discussing the future program of theGamaleia Institute, specifies a requirement forthe development of "protective antigens suitablefor prevention of certain infections against whichpreviously immunity could be conferred only bylive vaccines." Elkin (32, 33) states the need towork on "combined vaccines and simpler methodsof inoculation (dermal, by aerosol, etc.) sincewithout these there are insurmountable difficultiesin the way of mass vaccination against a largenumber of diseases."

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In the next few paragraphs some of the worknow in progress to improve or simplify this vacci-nation procedure is noted. For example, Faibich(40) considers the possible additive value of thesubcutaneous inoculation of strain 10 in "adju-vants." He concludes that agar, gelatin, gumarabic, Malva decoction, and glycerine wereeffective. For mice the minimum immunizing dosewith agar or gelatin was 10 organisms, whereas inphysiological saline the dose was 1,000 organisms.Faibich credits Zlatovskii with showing a similareffect in man.The possibility of combining viable tularemia

and smallpox vaccines and administering themsimultaneously by the epidermal route wasexamined in guinea pigs by El'bert et al. (25).The addition of other immunizing agents (againsttetanus, cholera, typhoid, parathyroid A and B,and bacillary dysentery) was recommended asbeing effective by Akimenko (1).

Pilipenko, Polyakova, and Shchekina (76-78)described animal studies on the simultaneousinoculation of live vaccines for tularemia andbrucellosis and showed that animals so vaccinatedwere resistant to challenge with virulent strainsof either Brucella or P. tularensis. Amanzhulovand Rementsova (6) in 1955 administered vac-cines for tularemia and brucellosis to humanssimultaneously but at separate sites. The proce-dure was well tolerated. At 1 month 89% reactedto tularin; at 5 months the figure was 97%, andat 1 year it was 81%. A similar trend was ob-served with brucellin. Antibody titers generallyparalleled the allergic reaction rate.

Studies on the simultaneous inoculation of livevaccines for tularemia and brucellosis wereinitiated at the Gamaleia Institute in 1953 (42,43). It was established that the procedure pro-duced a satisfactory response in guinea pigs toboth antigens. Shulygina (89) records a continua-tion of this program in animals with the no. 15strain (enhanced) of P. tularensis and the Brucellaabortus BA strain, the two being mixed beforedrying. After drying and rehydration, 1.0 mlcontained 10 P. tularensis cells and 22.5 X 109 B.abortus. Comparable vaccines were then tested inmen by the dermal route (44). A positive inocula-tion reaction was obtained in 183 out of 185, andmild lymphadenopathy was observed in only6.0%. On the basis of a report by Gubina, theResearch Council of Gamaleia Institute accepteda resolution in 1957 "to recommend the use of

combined brucellosis and tularemia vaccine inman (subject to approval of the Serum and Vac-cine Committee)" (12). It would appear that"official instructions" have been issued for theproduction of a dried brucellosis-tularemia vac-cine (13).-

According to Vakker (99), who apparently wasdescribing an immunization program in theKazakh S. S. R., 42,300 people were given asimultaneous vaccination by the skin methodagainst brucellosis and tularemia. Serologicpositivity was noted in 95.1% for the former andin 86.3% for tularemia.

Studies on combining viable vaccines againstplague, tularemia, brucellosis, and anthrax inguinea pigs have been reported (101). In thecombination the response to anthrax was unsatis-factory, while in the other three the protectionclaimed was said to be the equivalent of a mono-valent immunizing product. Kalacheva (46) de-scribed the immunization of mice with acombination of live plague and tularemiavaccines. Subsequently the animals were infectedintraperitoneally with Pasteurella pestis or P.tularensis. It is claimed that the combinationvaccination produced a more intense leukocyticreaction in the peritoneal fluid and a greaterreduction in the number of organisms obtained onblood culture than did either vaccine alone.

Considerable attention is being devoted to im-munization by inhalation. In 1954 El'bert et al.(28) described the comparative effectiveness ofnasal and cutaneous vaccination against tulare-mia in the guinea pig. They could demonstrate noimmunological difference between the two routes.The report also states that El'bert et al. in 1952showed the harmlessness and immunogenicity ofdye and liquid yolk nasal vaccination with strainOndatra. Aleksandrov and Gefen (2) summarizea series of animal studies involving vaccinationby inhalation of dried viable P. tularensis, strain15 (restored). Guinea pigs, after inhaling largeamounts, are reported to produce agglutinationand allergic responses comparable in all ways tothose noted following the subcutaneous inocula-tion of 20 million similar organisms. In a laterpaper, Aleksandrov et al. (3) indicate that thedosages ranged from "several hundred thousandto tens of millions of living microbes." Guineapigs so immunized survived in 67 to 80% of casesafter the subcutaneous or aerogenic infectionwith 100 to 1,000 times the minimal lethal dose

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of a virulent P. tularensis culture. Animals vac-cinated subcutaneously survived in 88 to 100%of cases, while all unvaccinated controls died onchallenge. Small laboratory animals are not con-sidered particularly satisfactory for such studiesbecause of the narrow cross section of theirrespiratory passages, the small minute volume,and their superficial type of respiration.

In 1957 studies in aerogenic vaccination ofman were initiated at the Kirov Military MedicalAcademy. Dry tularemia vaccine (strain 15, re-stored), in a computed dose of 750,000 organisms,was inhaled by 128 persons. Cubicles havingvolumes of 3.8 and 10 mi, as well as a tent of 12min, were used. The exposure time was 15 min."A general moderately severe reaction was notedin two individuals." This apparently developed8 to 12 hr after vaccination and was manifestedby fever, headache, and muscle and joint pains.One individual also had "an insignificant localreaction in the form of a rapidly progressivebronchitis. After three days these signs com-pletely disappeared." Immunological changesappeared as early as 1 week after vaccination. Atthe 30-day serologic examination the agglutinintiters were reported as higher than in the subjectsreceiving percutaneous vaccine.

Further data on reactions to aerogenic vaccina-tion with attenuated P. tularensis are given byKorostovtsev, Onikiyenko, and Khokhlov (54).They describe a brief febrile response in 1 of 16individuals inhaling 750,000 organisms and in 3of 8 persons inhaling 7,500,000 cells. In 1 (or 2)such individuals there was an associated decreasein the maximum pulmonary ventilation. Therewere no sequelae.

Aleksandrov et al. (4) describe studies onrespiratory immunization of man with variouscombinations of dried living agents. Brucellosis-anthrax-tularemia triple vaccine was given to 17persons with three general reactions. Brucellosis-anthrax-tularemia-plague tetravaccine was ad-ministered to 13 individuals with nine generalreactions. These reactions were of short durationand did not prevent the authors from recom-mending aerogenic immunization of people withthe combined vaccines. Subsequent skin testswith tularin showed positive reactions in mostindividuals participating.

Olsuf'ev (68) offers the following on respiratoryimmunization of man: "The nasal, aerogenic,and alimentary methods of vaccination do notdisplay advantages over the cutaneous method

and clearly yield to it on account of the non-availability of external observations for inocula-bility; they are not without danger, and it isimpossible to recommend them for practical use."However, it is evident from recent reports byAleksandrov et al. (4) that aerogenic immuniza-tion of man is under continuing study in accordwith a resolution of the Vaccine-Serum Commit-tee of the Minstry of Health.

Finally, in addition to summarizing thesecontinuing efforts to improve the usefulness ofbiologicals in the control of tularemia, this sectionrelative to current work would be incompletewithout noting that other methods of control ofthis disease are also receiving continuing studyand application. These include the ecology ofthe animal hosts, the methods of transmission,whether by vector or other routes, diagnostictools, and therapeutic agents.The recognized history of tularemia in the

Soviet Union began in the valley of the Volga-Akhtubinsk Rivers. Extensive vaccinationprograms were initiated in this area in 1946, 20years later. During the period from 1951 to 1957no cases of tularemia were reported (15). In addi-tion to vaccination of all personnel and revacci-nation as indicated, the plan for the control oftularemia in this area includes annual extensiveepizoological, zoological, and parasitological in-vestigations, rodent extermination, control ofwater rat activity, anti-ixodial tick measures,protection against blood sucking diptera, andcultivation of areas which remain as primary fociof tularemia (11).

Thus, Soviet investigators have provided acapability to deal with this disease in an accept-able manner. Major reliance is placed on the viablevaccine, supplemented, at least in critical areas,with a multidisciplinary control program. Thecomplexity of the ecology of tularemia, and theneed for simplified methods of its control, serveto generate problems that are receiving, andprobably will continue to receive, the attentionof the organized Soviet research effort.

IX. LITERATURE CITED'1. AKIMENKO, V. G. 1949. Associated vaccina-

tion against tularemia. Rostov Vol. 8:121-123.

'To avoid confusion, references include theabbreviation "Vol." when this is given, and"No." precedes the issue number. For the Zhurnalffikrobiologii, Epidemiologii i Immunobiologii the

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2. ALEKSANDROV, N. I., AND N. Y. GEFEN.1958. A method of aerogenic (inhalation)immunization and possibilities of improv-ing it. Voyenno-Meditsinskiy ZhurnalNo. 11.

3. ALEKSANDROV, N. I., N. Y. GEFEN, N. S.GARIN, K. G. GAPOCHKO, I. I. DAAL'BERG,AND V. M. SERGEYEV. 1958. Reactogenicityand effectiveness of aerogenic vaccinationagainst certain zoonoses. Voyenno-Medit-sinskiy Zhurnal No. 12.

4. ALEKSANDROV, N. I., N. Y. GEFEN, N. S.GARIN, K. G. GAPOCHKO, V. M. NIKITIN,A. A. ANTONOVA, AND V. V. MISHCHENKO.1960. Aerogenic immunization against somezoonoses with dry combined powderedvaccines. Voyenno-Meditsinskiy ZhurnalNo. 12.

5. ALESHINA, E. N., AND T. I. PUCHEvA. 1947.Fibrinolytic properties of the tularemiamicrobe. Rostov Vol. 6:96-102.

6. AMANZHULOV, R. S., AND M. M. REMENTSOVA.1958. Simultaneous vaccination of humansagainst tularemia and brucellosis. I.ZhMEI No. 2, 11-16 (Pergamon, Vol.29:177-181, 1958).

7. ANINA-RADCHENKO, N. D. 1953. Attempts atthe cultivation of the causative agent oftularemia on chicken embryos and infertileeggs. ZhMEI No. 10, 86.

8. ANONYMOUS. 1953. Instructions for theapplication of dermal anti-tularemia drylive vaccine. From Gamaleia Institute,7 March.

9. Anonymous. 1955. Provisional instructionsfor the control and use of the dry live,cutaneous vaccine from strains 155 and 15"recovered." U. S. S. R. Ministry ofHealth, 9 August.

10. Anonymous. 1956. Instructions for dry livetularemia vaceine. From Gamaleia Insti-tute, 21 August.

11. Anonymous 1958. Supplement: Combinationof tularemia control measures in zone of

abbreviation ZhMEI is used. The Trudy Rostov-skova-na-Donu gosudarstvennovo nauchno-issle-dovatel'skovo protivo chumnovo instituta (Pro-ceedings of the Rostov on the Don State ScientificResearch Anti-Plague Institute) is abbreviatedRostov. Other journal names are given in full.Where references appear in an abstract or inEnglish translation, this follows in parentheses.For the English edition of the ZhMEI, the shortterm Pergamon is used, and the pagination of thatpublication is given also. The State PublishingHouse of Medical literature is designatedMEDGIZ. English translations of titles are used.

construction of Stalingrad HydroelectricStation and districts adjacent to it, p.184-185. In N. G. Olsuf'ev, B. N.Pastukhov, and V. V. Kucheruk, [ed.],Problems of epidemiology and prophylaxisof tularemia. MEDGIZ, Moscow.

12. BARINSKII, F. G. 1958. Sessions of theresearch council and general scientificconferences held in the N. F. GamaleiaInstitute of Epidemiology and Micro-biology, Academy of Medical Sciences ofthe U. S. S. R. (1956 and three quarters of1957). ZhMEI No. 1, 155-157 (Pergamon,Vol. 29:165-168, 1958).

13. BARINSKII, F. G. 1958. The Jubilee Sessionof the Gamaleia Institute, etc. ZhMEINo. 6, 154-157 (Pergamon, Vol. 29:1010-1014, 1958).

14. BISIKALOVA, V. M., A. N. PREDTECHENSKII,AND V. I. ZHDANOVSKII. 1957. The effect -ofnarcosis on the course of the vaccinationprocess in rabbits immunized with livetularemia vaccine. ZhMEI No. 12, 98-101(Pergamon, Vol. 28:1777-1780, 1957).

15. BORODIN, V. P. 1958. Study of the effective-ness of anti-tularemia vaccination in zoneof construction of Stalingrad Hydro-electric Station, p. 152-155. In N. G.Olsuf'ev, B. N. Pastukhov, and V. V.Kucheruk, [ed.], Problems of epidemiologyand prophylaxis of tularemia. MEDGIZ,Moscow.

16. BORODIN, V. P., N. A. SPITSYN, A. P. SAM-SONOVA, A. P. KOROLEVA, AND V. P.CHUNIKHIN. 1959. Natural tularemia of theravine-steppe type. ZhMEI No. 3, 35-40(Pergamon, Vol. 30, No. 3, 43-50).

17. CHICHINSKAYA, 0. P., AND V. M. SHIPNITZ-KAYA. 1956. To the question of the per-sistance and degree of the post vaccinalimmunity against tularemia. Rostov Vol.8:205-207.

18. DIDENKO, S. I., AND N. G. KLYUYEVA. 1957.Collection of official instructions for manu-facture and control of bacterial prepara-tions and directions for their use: Livebacterial vaccines (Monograph). Ministryof Health, U. S. S. R., Moscow.

19. DROSHEVKINA, M. S. 1945. Fluid yolk mediumfor the cultivation of B. tularense. RostovVol. 4 (not reviewed).

20. DROSHEVKINA, M. S. 1947. Fluid yolk mediumfor the growth of tularemia cultures. II.Characteristics of the growth of the tu-laremia microbe in fluid yolk medium.Rostov Vol. 6:71-81.

21. DROSHEVKINA, M. S. 1947. Fluid yolk mediumfor the growth of tularemia cultures. III.

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Preservation of the vitality, virulence, andagglutinability of the tularemia culturekept in fluid yolk media. Rostov Vol.6:82-88.

22. DUNAEVA, T. N. 1959. Use of the intradermaltularin test in experimental infection ofanimals with tularemia. ZhMEI No. 3,17-22 (Pergamon, Vol. 30, No. 3, 19-25).

23. DYACHENKO, S. S., O. P. KHYZHYNS'KA, ANDS. HW. BUYALO. 1953. Allergy reactions inpersons inoculated cutaneously with liveegg-yolk. tularemia vaccine. Mikrobiol-ohichynyy Zhurnal Akademiyi NaukUkrayins'koyi R. S. R. Vol. 15, No. 1,27-32.

24. DZHANPOLADOVA, V. P. 1958. The problem ofimmunity to tularemia in man. ZhMEINo. 12, 106-107 (Pergamon, Vol. 29:1987-1988, 1958).

25. EL'BERT, B. I., I. S. TINKER, T. I. PUCHKEVA,M. S. DROZHEVKINA, Z. D. KHAKINA,M. E. KALK, V. P. ROMANOVA, AND K. F.KAVARZINA. 1946. Skin method for pro-phylaxis of tularemia. ZhMEI No. 11,3-10.

26.' EL'BERT, B. I., I. S. TINKER, AND T. I.PUCHKEVA. 1947. The cutaneous methodof the specific prophylaxis of tularemia.Rostov Vol. 6:7-20.

27. EL'BERT, B. I., I. S. TINKER, V. P.ROMANOVA, K. V. ZAVARZINA, AND T. I.PUCHKEVA. 1947. On the epidemiologicaleffectiveness of cutaneous vaccination withtularemia yolk vaccine against tularemia.Rostov Vol. 6:62-70.

28. EL'BERT, B. I., V. A. YUDENICH, M. M.KIRVEL, M. N. PRUDNIKOVA, G. S. KHANIN,AND A. L. MATSKEVICH. 1954. Comparativeeffectiveness of nasal and cutaneous vac-cination against tularemia. ZhMEI No.8, 71-72.

29. EL'BERT, B. I. 1956. Immunoprophylaxis oftularemia in theory and practice. Zdva-vookh-raneniye Belorussi No. 7, 7-12.(Abstracts of Soviet Medicine, ExcerptaMedica, Part B, 1957, item 1237.)

30. EL'BERT, B. I. 1957. Soviet immunology andthe problem of live vaccines. ZhMEINo. 11, 40-46 (Pergamon, Vol. 28:1558-1563, 1957).

31. EL'BERT, B. I. 1958. Live vaccines. Medit-sinskiy Rabotnik, 16 September 1958. p. 3.

32. ELKIN, I. I. 1957. Soviet successes in the fieldof epidemiology, and the next problemsfor scientific study. ZhMEI No. 11, 3-11(Pergamon, Vol. 28:1523-1530, 1957).

33. ELKIN, I. I. 1957. The Saratov Scientific

Conference. ZhMEI No. 6, 154-159 (Perga-mon, Vol. 28:920-922, 1957).

34. EMEL'IANOVA, 0. S. 1953. Morphology ofcolonies and the composition of the cul-tures of tularemia. ZhMEI No. 11, 37-43.

35. EMEL'IANOVA, 0. S., AND G. K. SHIPITSINA.1956. Biochemical composition of strainsof Pasteurella tularensis. DokladyAkademii Nauk S. S. S. R. Vol. 109:365-368.

36. EMEL'IANOVA, 0. S. 1957. The character oftularemia vaccine strains as disclosed bylaboratory tests. ZhMEI No. 8, 125-129(Pergamon, Vol. 28:1197-1200, 1957).

37. EMEL'IANOVA, 0. S. 1959. Variation of thetularemia organism under artificial condi-tions, p. 109-115. In V. D. Timakov, [ed.l,Microbial variation. Pergamon Presstranslation, New York.

38. EMEL'IANOVA, 0. S. 1959. Variations inPasteurella tularensis stored in differentways under laboratory conditions. ZhMEINo. 3, 22-26 (Pergamon, Vol. 30, No. 3,26-31).

39. FAIBICH, M. M., AND T. S. TAMARINA. 1946.Dry living vaccine of NIUIEG of the RedArmy (Naughno Issledovatel skii Insti-tut Epidemiologii I Giginny). Report I.ZhMEI Vol. 7:59-63. Report II, ZhMEIVol. 10:23-27.

40. FAIBICH, M. M. 1959. The problem of increas-ing the immunogenicity of live anti-tularemia vaccine. ZhMEI No. 10, 20-24.

41. GLASS, G. B. J. 1948. An epidemic of tula-remia transmitted by insects in settlementsof deportation, Aseno and Yaya, Siberia,Soviet Russia: Report of 121 cases. Am. J.Med. Sci. Vol. 216:411-424. (Also in Med.Clin. N. Am. Vol. 32:769-778, 1948.)

42. GUBINA, E. A. 1957. Combined immunizationagainst brucellosis and tularemia. ZhMEINo. 7, 19-23 (Pergamon, Vol. 28:942-947,1957).

43. GUBINA, E. A. 1957. Experimental study ofimmunization of guinea pigs with a com-bined brucellosis and tularemia vaccine.ZhMEI No. 11, 107-111 (Pergamon, Vol.28:1623-1627, 1957).

44. GUBINA, E. A., AND G. P. UGLOVOL. 1958.Study of reactions to vaccination of humansubjects with live combined brucellosisand tularemia vaccine. ZhMEI No.2, 8-11 (Pergamon, Vol. 29:173-176, 1958).

45. JUSATZ, H. J. 1952. Tularemia in Europe.In E. Rodenwaldt, [ed.], World atlas ofepidemic diseases, Part I. Falk-Verlag,Hamburg.

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46. KALACHEVA, N. V. 1959. The leukocyticreaction in mice immunized with livecombined plague and tularemia vaccine.ZhMEI No. 1, 43-44 (Pergamon, Vol.30, No. 1, 56-57).

47. KALITINA, T. A. 1958. A study of the skinallergic reactions as an indicator of thedegree of immunity in tularemia. ZhMEINo. 2, 16-21 (Pergamon, Vol. 29:182-186,1958).

48. KARAKULOV, I. K., E. M. MERTSALOV, ANDA. R. ZHOKIN. 1957. Some results of theprophylaxis of infectious diseases in theKazakstan S. S. R. ZhMEI No. 10, 11-14(Pergamon, Vol. 28:1384-1387, 1957).

49. KARPUSIDI, K. S. 1947. On the "non-typical"strains of the tularemia microbe. RostovVol. 6:107-118.

50. KHATENEVER, L. M. 1943. The allergicdiagnosis, specific prophylaxis and vacci-notherapy of tularemia, p. 62-79. InE. B. Balsky, I. G. Kochergin, and V. V.Porin, [ed.], Microbiology and epidemiol-ogy. English Edition, Medical Publica-tions, Ltd., London.

51. KLEPKO, G. D. 1958. Post-vaccinal reactionto anti-tularemia inoculation in patientswith influenza. Veyenno-MeditsinskiyZhurnal No. 7.

52. KOLYADITSKAYA, L. S., AND A. A. SHMURY-GINA. 1957. An improved dried live tula-remia vaccine. ZhMEI No. 10, 84-89(Pergamon, Vol. 28:1454-1459, 1957).

53. KOLYADITSKAIA, L. S., K. V. KUCHINA, ANDA. A. SHMURYGINA. 1959. Tularemia phage.ZhMEI No. 3, 13-16 (Pergamon, Vol. 30,No. 3, 14-18).

54. KOROSTOVTSEV, S. B., B. A. ONIKIYENK0,AND L. I. KHOKHLOV. 1960. The determi-nation of the maximum pulmonary ventila-tion: One of the methods of studying theside-reactions producing capacity of drylive vaccines for aerogenic immunization.Veyenno-Meditsinskiy Zhurnal No. 3.

55. KOSMACHEVSKY, V. V. 1944. Summary ofthe first clinical tests with Gaiskii's livetularemia vaccine. Klinicheskaia Medit-sina Vol. 22, No. 9, 60-68.

56. KRASOVSKIY, F. U. 1958. Interoblast scien-tific-practical conference on regional epi-demiology of natural foci diseases of theUrals, Siberia and the Far East. Zdra-vookhraneniya Rossiyskoy Federatsii No.5, 40-45.

57. KUCHEROV, P. M., L. T. BYKOV, K. S.KARPUZIDI, V. M. MERLIN, N. K. KUNITSA,M. L. KAL'IANOVA, AND M. I. PARSKIN.

1958. An attempt to prevent tularemiaduring a period of extensive epizooticamong rodents. ZhMEI No. 8, 3-7 (Perga-mon, Vol. 29:1170-1180).

58. MAISKII, I. N., AND G. K. SHIPITSINA. 1952.Cited by Verenikova et al. (100).

59. MARTINEVSKI, I. L. 1956. Characteristics ofthe NIIEG live tularemia vaccine in dryform, as related to duration of storage.ZhMEI No. 9, 20-22.

60. MATVEETS, L. S., N. G. OLSUF'EV, Y. A.IL'INSKII, AND N. M. ZHARIKOV. 1957.Immunological reactivity in persons suffer-ing from derangement of the centralnervous system to tularemia vaccination.ZhMEI No. 9, 46-51 (Pergamon, Vol.28:1263-1268, 1957).

61. MIKAT, B. AND F. KUHLMANN. 1944. UberLeingenbeteiligung bei der Tularemie.Deut. med. Wchsch. 70:302-304.

62. MIKHAILOV, I. F. 1957. Review of Practicalexperience of Soviet medicine in WorldWar II (1941 to 1945), v. 2 (Epidemiology),MEDGIZ, 1955. ZhMEI No. 8, 135-138(Pergamon, Vol. 28, No. 8, 1205-1208).

63. MUROMTSEV, S. N. 1959. Principal results ofresearches of the N. F. Gamaleia Instituteof Epidemiology and Microbiology of theAcademy of Medical Sciences of theU. S. S. R. during 1958; plan of researchfor 1959. ZhMEI No. 5, 115-119 (Perga-mon, Vol. 30, No. 5, 143-148).

64. OLSUF'EV, N. G. [ed.]. 1953. Effectivenessof vaccination against tularemia (Mono-graph). Izdatel'stvo Akademii Medit-sinskikh Nauk S. S. S. R., Moscow.

65. OLSuF'EV, N. G. 1953. Results of a study ofthe effectiveness of anti-tularemia skinvaccinations: A survey, p. 7-38. In N. G.Olsuf'ev, B. N. Pastukhov, and V. V.Kucheruck, [ed.], Problems of epidemiol-ogy and prophylaxis of tularemia.MEDGIZ, Moscow.

66. OLSUF'EV, N. G., E. M. TSVETKOVA, V. P.BORODIN, A. N. KOROLEVA, AND U. S.SIL'CHENKO. 1956. Tularin from a vaccinestrain for percutaneous use. ZhMEI No.9, 22-29.

67. OLSUF'EV, N. G., AND 0. S. EMEL'IANOVA,1957. The antigenic structure of Bacteriumtularense. Czechoslovakian Journal ofHygiene, Epidemiology, Microbiology andImmunology, Vol. 1, No. 4, 357-366.

68. OLSUF'EV, N. G. 1958. The current statusof the study of tularemia vaccine-prophy-laxis. Vestnik Akademii MeditsinskikhNauk S. S. S. R. No. 11, 63-72. (Abstracts

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of Soviet Medicine, Excerpta Medica,Part B, 1960, item 955.)

69. OLSUF'Ev, N. G., 0. S. EMEL'IANOVA, G. P.UGLOVOI, V. S. SIL'CHENKO, V. P. BORODIN,A. P. SAMSONOVA, N. S. KONKINA, G. M.SHELANOVA, Z. A. LEBACHEVA, M. I.TSAREVA, N. A. ZYKINA, AND T. F. ELBE-DEVA. 1958. Results of the mass use onhuman beings of dried tularemia vaccinefrom the Gaiskii no. 15 (restored) andEmel'ianova no. 155 strains. ZhMEI No.3, 52-57 (Pergamon, Vol. 29:386-391, 1958).

70. OLSUF'Ev, N. G., B. N. PASTUKHOV, ANDV. V. KUCHERUK [ed.]. 1958. Problems ofepidemiology and prophylaxis of tularemia(Monograph). MEDGIZ, Moscow.

71. OLSUF'EV, N. G., V. P. BORODIN, AND Y. M.TSEVTKOVA. 1958. Epicutaneous skin testwith tularin from vaccine strain to deter-mine immunity in those inoculated againsttularemia and for diagnosis of this infec-tion. In N. G. Olsuf'ev, B. N. Pastukhov,and V. V. Kucheruk, [ed.], Problems ofepidemiology and prophylaxis of tula-remia. MEDGIZ, Moscow.

72. OLSUF'Ev, N. G., 0. S. EMEL'IANOVA, ANDT. N. DUNAYEVA. 1959. Certain differ-ences in the tuleremia pathogen in the oldand new world. Vestnik Akademii Medit-sinskikh Nauk S. S. S. R. Vol. 14, No.6, 51-57. (Also in Czechoslovakian Journalof Hygiene, Epidemiology, Microbiologyand Immunology, Vol. 3:138-149 (in Eng-lish).)

73. OVASAPYAN, 0. V. 1956. Epizootics of tula-remia in the Alinsk region. IzvertiyaAkademiya Nauk Armyanskoy S. S. R.Vol. 9, No. 12, 23-31.

74. OVASAPYAN, 0. V. 1957. A tularemia epizooticin Gukasyanskiy rayon and necessaryprophylactic measures in the Leninakan-skiy Basin. Izvertiya Akademiya NaukArmyanskoy S. S. R. Vol. 10, No. 5, 77-80.

75. PANAIOTTI, A. I. 1959. The problem of activeimmuno-prophylaxis of tularemia. ZhMEINo. 5, 139 (Pergamon, Vol. 30, No. 5, 174,1959).

76. PILIPENKO, V. G., AND A. M. POLYAKOVA.1955. Problem of the simultaneous inocu-lation against tularemia and brucellosisby subcutaneous method. I. ZhMEI No.4, 52-57.

77. PILIPENKO, V. G., AND A. M. POLYAKOVA.1955. Problem of the simultaneous inocula-tion against tularemia and brucellosis bycutaneous method. II. ZhMEI No. 5,9-14.

78. PILIPENKO, V. G., A. M. POLYAKOVA, ANDT. A. SHCHEKINA. 1956. The problem ofthe possibility of simultaneous vaccina-tion against tularemia and brucellosis byintracutaneous method. III. ZhMEI Vol.27, No. 3, 79-83.

79. PIOTROVSKAIA, S. A. 1958. Certain criteriaof the condition of immunity in childrenvaccinated against tularemia (Author'ssummary). ZhMEI No. 5, 129 (Pergamon,Vol. 29:802, 1958).

80. PUCHKEVA, T. I. 1956. Investigation of theimmunogenic properties of the vaccinaltularemia strain in the process of growthand multiplication at the temperature of370. Rostov Vol. 10:197-199.

81. RODOV, M. N. 1958. The problem of tularemiain children. Pediatriia, No. 8, 39-44.

82. RUBINA, T. A. 1957. Cases of tularemiacontamination from a hare. Trudy MolotovMed. Inst. 190-191. (Abstracts of SovietMedicine, Excerpta Medica, Part B, 1958,item 3703.)

83. SHAPIRO, S. E., A. D. KALMYKOVA, 0. I.KLIMENKO, M. I. ZELENSKAIA, A. A.TIMOFEEVA, AND M. M. GARBUZOV. 1958.Cases of tularemia in the Khabarovskdistrict. ZhMEI No. 2, 21-24 (Pergamon,Vol. 29:186-189, 1958).

84. SHEVELEV, A. S. 1958. Vaccine tularemicinfection during radiation sickness ofwhite mice. Meditsinskaya Radiologiya,Vol. 3, No. 4, 50-55.

85. SHISHKIN, A. K. 1957. Rostov-on-Don Anti-Plague Research Institute of the Ministryof Public Health of the U. S. S. R. ZhMEINo. 9, 101-107 (Pergamon, Vol. 28:1316-1321, 1957).

86. SHMUTER, M. F. 1953. Relationship of theimmunological effectiveness of tularemiavaccine strains to their residual virulencefor white mice, p. 112-114. In N. G. Osuf'elv,[ed]., Effectiveness of vaccination againsttularemia (Monograph). Izdatel'stvo Aka-demii Meditsinskikh Nauk S. S. S. R.,Moscow.

87. SHMUTER, M. F. 1959. Cases of tularemia insugar factories. ZhMEI No. 3, 46-52 (Perg-amon, Vol. 30, No. 3, 57-64).

88. SHOPE, R. E. 1956. An account of the obser-vations made by the United States MedicalMission to the U. S. S. R. Mimeograph(April).

89. SHULYGINA, K. N. 1958. Experimental studyon dermal inoculation with live com-bined brucellosis and tularemia vaccine.

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99. VAKKER, V. G. 1957. Simultaneous prophy-lactic vaccination of humans by the skinmethod, against brucellosis and tularemia.Zdavookhraneniye Kazakhstana, No. 7,19-21. (Abstracts of Soviet Medicine,Excerpta Medica, Part B, 1959, item 4719.)

100. VERENIKOVA, N. K., A. A. KONTORINA, ANDE. E. BAKHRAKH. 1956. Characteristics ofthe polysaccharide fraction of Pasteurellatularensis. ZhMEI No. 2, 8-13.

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102. ZHDANOV, V. M. 1960. The role of immuniza-tion in communicable disease control.Paper A-13/Technical discussions/4, 13thWorld Health Assembly, 3 May. Mimeo-graph.

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SOVIET VIABLE PASTEURELLA TULARENSIS VACCINES · from Pasteurella tularensis strains 155 and 15 'recovered" (also translated as "renewed" or "restored"). These instructions bear an