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At least 10% of spontaneous chromosomal antibiotic resistant mutants of bacteria express a strain-dependent graded reduction of virulence; this correlates linearly with a prolonged generation time. Occasionally, these mutants are temperature sensitive or/and auxotrophe. The work described in this paper provides evidence that in such strains the resistance and the accompanying markers exist only as a functional genetic unit. In a series of transduction experiments with a pathogenic strain of Salmonella typhimurium, it was found that without exception, the resistance and the additional markers were 100% simultaneoulsy transferred. Furthermore, antibiotic-resistant Escherichia coli mutants with prolonged generation time, were isolated from faecal samples; it is thus indicated that, such innocuous mutants occur at any time in the intestine. It is concluded that concerns connecting such mutants to the possibility of resistance dissemination are unfounded; furthermore, even if transfer of resistance occurred, only attenuated strains would be disseminated.

In each of two experiments, sheep and lambs were vaccinated by a subcutaneous injection of a test vaccine (consisting of a combined serotype 1/2a and serotype 4b live Listeria monocytogenes culture) and challenged 16 days later with a mixture of the homologous wild strains. After challenge, the mortality rate of vaccinated sheep was 28.1% and that of controls 71.9%; that of lambs was 25.0%, although these had been inoculated with the LD70 dose. Furthermore, in each of two field trials in Listeria-infected flocks, primiparous pregnant ewes were vaccinated. In the first field trial, 3 or 110 lambs died of listeriosis of those born of vaccinated (n = 564) or unvaccinated (n = 3345) ewes, respectively. In the second, the perinatal mortality rate of lambs born of vaccinated or unvaccinated ewes was 7.6 or 30.3%, respectively, and the mean birth weight of lambs born of vaccinated or control ewes was 2.2 or 1.8 kg, respectively; the mean milk production of vaccinated ewes was 106 and that of controls 83 l; no Listeria was isolated from milk samples of vaccinated ewes. It is concluded that the vaccine is efficacious for the protection of sheep from listeriosis.

The vaccine candidates were constructed through stepwise incorporation of weakly attenuated purine auxotrophy with subsequent rifampicin resistance (RNA polymerase) mutation to yield optimal attenuation. These strains showed a maintained invasiveness for conjunctival epithelia. Therefore, while not causing keratoconjunctivitis, they were excreted for a short but marked period and provided partial protection in the Sereny test. Children tolerated the maximum dose of 1-3 x 10(9) colony-forming units (c.f.u.) of lyophilized vaccine without side effects. This total dose distributed among three oral inoculations, induced coproantibodies in 70% of recipients during the first three months. However, the single application of this total dose for primary immunization or booster proved to be more efficient: 90% of the subjects had markedly elevated titres for not less than six months.

A Salmonella typhimurium live vaccine with optimal level of attenuation for sheep, constructed by means of 'metabolic drift' mutations, was tested for its efficacy in preventing Salmonella abortus ovis-induced abortions. In two field trials in Kirgiziya, 78,000 to 100,000 first delivery sheep received a fully tolerated single dose of 10(9) c.f.u. live vaccine 2 months before to 4 months after insemination. Alternatively, they were immunized twice with commercial inactivated S. abortus ovis vaccine, or they served as non-immunized controls. The S. abortus ovis-induced abortion frequency in the controls was greater than or equal to 30%, in sheep immunized with inactivated vaccine greater than or equal to 11%. In flocks immunized with live vaccine, the S. abortus ovis-induced abortion frequency did not exceed 0.1%. Thus, use of 'metabolic drift' mutations for construction of stable vaccine strains optimally attenuated for the particular host species proved to be relevant to practice.

Persistence and clearance of the vaccinal strains, humoral and cell-mediated immune response and protective immunity were assessed in ICR mice, immunized intraperitoneally or intragastrically with double-marker auxotrophic mutants of Salmonella typhimurium strains 1771 and 3334 (his-pur-). Strain 3334 possesses in addition the antiepidemic Hst (high sensitivity to tensides) marker which confers decreased survival in the gut and environment. The results showed that the strains are not overattenuated. They revealed preserved multiplication capacity, established carrier state for 28-30 days and induced humoral and cell-mediated immunity as measured by passive haemolysis and microagglutination or footpad swelling tests, respectively. The immune response was determined by biological features of the strains. Strain 1771 induced synthesis of O- and H-antibodies 7-11 days earlier than strain 3334. Footpad reactions appeared 3-4 days after immunization and a week before the serum antibodies. Cell-mediated immunity showed clear correlation with the protection against challenge with a virulent strain. The present results suggest that the double-marker auxotrophic mutants of S. typhimurium might prove very useful in the development of a live vaccine in further studies on livestock.

Metabolic drift (antibiotics resistance) mutations were used to construct stable two (and three) marker vaccine candidate strains of the predominant Listeria monocytogenes serotypes 1/2a and 4b by stepwise selection. Derived from wild-type strains, spontaneous chromosomal streptomycin-resistant clones with their i.p. LD50 elevated from less than or equal to 10(5.0) c.f.u. to approximately 10(6.1) c.f.u. were used in the second step to isolate the rifampicin-resistant mutants with i.p. LD50 values ranging from 10(6.6) to 10(7.4). On i.p. immunization with fully tolerated doses (less than or equal to 1% LD50), these potential vaccine strains were found to protect not less than 95% of the mice against a lethal (approximately 100 LD50) challenge with the homologous wild-type strain. Further elevation of the i.p. LD50 to greater than 10(8.3) c.f.u. by means of a third attenuating fosfomycin-resistance marker resulted in overattenuation and reduced protective capacity.

An experimental technology for the production of live freeze-dried vaccines prepared from attenuated Shigella flexneri 2a and Shigella sonnei I strains was developed. It is based on the cultivation of bacterial strains in a fermentor under conditions which ensure high yields. The strains grow in S-form, their antigenic structure is preserved and they remain non-virulent. The attenuating markers are stable. The freeze-dried vaccines retain good immunogenicity when applied intra-intestinally to rats.

Mutants optimally attenuated for highly susceptible hosts and protecting after a single oral vaccination are often overattenuated for host species being less susceptible. Therefore, to select vaccine strains optimally attenuated for the particular host species it is essential that a range of mutants with graded levels of attenuation be provided so as to permit lesser susceptibility to be compensated for by a correspondingly lower level of attenuation. This, while guaranteeing the stability through two-marker or multi-marker attenuation, can be suitably accomplished by slightly to moderately virulence-reducing mutations. Aspartic acid auxotrophy and, in particular, 'metabolic drift' mutations, possibly by additionally incorporating antiepidemic markers, are adopted for the mouse model to demonstrate stepwise production of S. typhimurium and S. typhi vaccine candidate strains with graded attenuation or any level of attenuation desirable. It is emphasized that this basic approach is relevant to practice.

Live Shigella flexneri 2a and Shigella sonnei Phase I vaccine candidate strains with two virulence-reducing markers were constructed through stepwise incorporation of weakly attenuating purine auxotrophy with subsequent rifampicin-resistance (RNA polymerase) mutation to yield optimal attenuation. These vaccine candidate strains showed an unaltered plasmid profile; did not cause keratoconjunctivitis in the Sereny test, while being excreted for a short but still marked period and providing partial protection from wild-strain infection; exhibited for guinea-pig conjunctival epithelia, HeLa cells and rat enterocytes a maintained invasiveness with reduced intracellular multiplication with little, if any, reversible cell damage; and produced, just as their ultrasonic lysates, no exudative reaction in the rabbit gut loop test.

Shigella flexneri 2a and Shigella sonnei I vaccine candidate strains were tested for tolerance in 14 adults as well as for tolerance and immunogenicity in children aged 2-17 years. For fresh culture material, the limit for side effects fading within 36 h such as meteorism, loose stools, tenesmus and slight temperature, and tolerated by volunteers was 3 x 10(9) colony-forming units (c.f.u.). Excretion of vaccine strain germs for 2-3 days was observed only at doses of 10(10) c.f.u. and greater (2-3 x 10(10)). Children tolerated up to the maximum dose of 1-3 x 10(9) c.f.u. of the lyophilized vaccine without side effects. No vaccine strain germs were found in faeces, antigen being detected for 4-5 days. The total dose of 1-3 x 10(9) c.f.u. being distributed among three oral inoculations at intervals of 1-2 days induced coproantibodies in 70% of the vaccinees during the first 3 months, and in 30-40% up to 6 months. In contrast, the single application of this total dose for primary immunization, or booster after 10 months, proved to be more efficient. Ninety per cent of the subjects had markedly elevated titres for not less than 6 months.

Rats and rabbits were immunized intraintestinally with different doses of virulent and nonvirulent live Shigella flexneri 2a and Shigella sonnei I strains. The nonvirulent strains had one or two attenuating markers. The period of excretion with the faeces of the bacteria and of their polysaccharide antigens, the proliferation of antigen-binding and antibody-producing cells in the spleens and gut-associated lymphoid tissues and the levels of antibodies in sera and faeces were studied. Attenuated strains S. flexneri 2a 77 and S. sonnei I 3359 induced the most potent and long lasting local immune response.

The authors present the results of a comparative study of the immunogenicity of the living (Sm-d) and killed E. coli vaccine which, in determination by such methods as the Vidal test, passive hemagglutination test, bactericidal activity, establishment of passive and active defence, proved to be the same in both vaccines. However, with the aid of Rauss and Ketyi's method, modified by the authors (determination of nonsuceptibility in enteral infection of the animals with intestine sterilized with streptomycin) it was possible to reveal the capacity of the living vaccine to depress the growth and the activity (reproducible and statistically controlled) of E. coli administered into the organism of the immunized animal for the purpose of infection. They were discharged with the feces for a definite period in 90% of control animals and mice immunized with the killed vaccine; this occured in less than 10% of the animals immunized with the living vaccine.

Local and systemic immune response was studied in 3 groups of rabbits immunized and reimmunized 190 days later with S. typhimurium double-marker attenuated strain 1,771 and doses ranged from 20.10(9) to 0.2.10(9) cells. Another group of rabbits was immunized with extract (hydroxylammine) vaccine. It was found that the attenuated strain persisted for considerable time in the gut, and induced pronounced and continuous immune response as measured by the passive hemagglutination test. The serum antibody response had the character of a secondary one with switching on the synthesis of IgM to IgG already after basic immunisation. By the Coombs' technique it was shown that specific immunoglobulins demonstrated in feces were secretory antibodies of the class IgA (SIgA). The immune response developed after reimmunization was still more vigorous and prolonged pointing to an immune memory existance. It was possible to obtain well manifested immunity with the lowest dose. The extract vaccine revealed only weak and transitory serum and intestinal antibody levels without SIgA appearance. The results obtained in this setup make S. typhimurium 1771 a perspective candidate for a live vaccine.

Results of preclinical assay, preparation and conservation of a S.-dublin live vaccine based on streptomycin dependent mutants and for oral application are demonstrated and discussed in detail. The used oral vaccine is very good tolerated by calves and results in stable immunity after administration of a daily dose of 5 x 10(10) bis 1 x 10(11) living Smd.-mutants for 10 consecutive days. Complete immunity developes in calves within a period of two weeks after the last antigen administration and persists up to the age of 5 to 6 months. Conserving the vaccine at low temperatures (-15 degrees C) the number of living organism is far-reaching preserved in the first 5 months after preparation. After thawing the vaccine is to be used within a period of 3 days. With respect to the preparation of the live vaccine on semisynthetic nutritiv media some informations are given concerning the improvement of bacterial yield.

Attenuation by only one single marker with no limitation on propagation (frequency of backmutation being less than 10(-7) will not provide sufficient safety against complications along with vaccination. (This notion, derived from both theoretical considerations and practical experience, does not rule out good stability of certain one-marker mutants under practice conditions.) Two independently attenuating markers with no limitation on propagation, however, do ensure full stability on account of potentiating single frequencies of backmutation (less than 10(-14)). The second attenuating marker must be measurable, and it must not be allowed to bring about substantive reduction in the one-marker mutant's immunogenicity. The following principle was conceptualised for the purpose of resolving the problem: A pool of attenuated highly immunogenic mutants with one single marker without limitation on propagation was rendered available, such as an S-form auxotrophic set of phenotypes (attenuation by co-mutation) or R-form mutants with the potential of tissue persistence. An attenuated second marker then was rendered available which was highly immunogenic as a "one-marker mutant", for example, mutants with adenine (purine) dependence with attenuation a pleiotropic effect and limited availability of metabolities. The second marker was introduced in a one-maker mutant, with verification of additional attenuation along with remaining immunogenicity. The following results were obtained: --Experimental parameters for attenuation and immunogenicity in mice: one single intraperitoneal immunisation, using 10(5), 10(6), 10(7), and 10(8) mutant germs; challenge on 20th day from intraperitoneal immunsation with about 100 LD50; lethality of controls 95 per cent or more; --S. typhimurium his-155/ade-4 and S. dublin met-91/ade-23: LD50: 10(8) germs; Immunogenicity: 90 per cent or more survived exposure to wild strain; --S. cholerae-suis R Dessau/ade-4: LD50: about 10(9) germs; immunogenicity: about 50 per cent only within endotoxic limits, about 10(8) germs or more; --S. typhimurium his-155/marker 2 (enzyme mutation): LD50 about 10(8) germs; immunogenicity: 90 per cent or more survived exposure to wild strain.

The Salmonella example demonstrates a principle for the isolation of high-immunogenic, stable Salmonella-mutants with two independently of each other attenuating mutations as potential vaccines from bacteria capable of multiplication. The isolation of such double-marker vaccination-strains is verified by the treatment of a single-marker strain (for instance an attenuated high-immunogenic auxotrophic pheno-type) with mutagen and the following selection of clones with the marker purin-auxotrophy as a second attenuating mutation. The demonstrated double-marker strains S. typhimurium his-155 pur-4 and S. dublin met-91 pur-23 are designated by the following parameters: Stability under the conditions of the practical vaccine application; immunogenicity for mice by one immunization only, and separability from homologous wild strains with simple laboratory methods.

Mutants of Salmonella typhimurium with spontaneous chromosomal resistance against oleandomycin, streptothricin, nalidixic acid and rifampicin were investigated for their virulence behaviour with the i.p. mouse model. The strains resistant against the special antibiotic consists of a spectrum of various clones with a different behaviour of virulence: Additionally to obvious unchanged virulent strains there are such with a weak or strong attenuation. The majority of the attenuated strains protect the immunized mice against a following lethal wild strain infection. High-immunogenic attenuated double-marker mutants for application as potential vaccine strains may be isolated with the aid of a step by step introduction of a second attenuating "resistance"-marker in a one-marker strain, attenuated for another reason. These strains show the following parameters: -stability under the conditions of practical vaccine application, because a simultaneous back-mutation in both attenuating markers by reason of the unrealizable germ numbers will not occur, -immunogenicity by one immunization only, -separation from homologous wild strains of another origin with simple laboratory methods. This obvious generally acting biological principle is explained on the basis of molecular biological considerations and by referring to the literature. A test for orientation using an attenuated RNA-polymerase mutant showed, the resistance against rifampicin and attenuation are transferred together by co-transduction.

Live vaccine prepared from streptomycin-dependent mutants of E. coli was well tolerated in various doses and at various times of administration, for there were no adverse reactions. There was no evidence of reversion to a virulent form. Efficacy was closely related to frequency of application of the vaccine. Vaccination must be performed as early as possible to immunize against infection during the first few days of life. It was possible to confer protection against the diarrhoea that occurs at 3-4 weeks of age. The use of an oral vaccine under intensive husbandry systems was shown to be safe. When the piglets were kept in cages, the vaccine could be administered in the existing mixing tanks for water supply, without the need for special equipment. Oral immunization of pigs already infected or already ill did not aggravate the existing infection, whether the animals were housed individually or in pens. Because of the dilution effect of the mixing tank and water supply pipes, the dose of vaccine, given on ten successive days, should be calculated at 10(9) organisms of the E. coli mutant per animal. This should supply a number close to the desired bacterial count of 10(10) organisms per animal daily. Immunization significantly reduced the frequency of gastrointestinal diseases during the first three weeks of age in piglets.