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A total of 520 monkeys belonging to 6 species (Macaca mullata, M. fascicularis, M. nemestrina, Cercopithecus aethiops, Papio anubis, and P. hamadrias) were investigated. Total frequency of occurrence of the protozoan Cryptosporidium in the Adler apery constituted 13.8%. The majority of parasites were found in animals with intestinal disorders such as diarrhea. The lowest frequency of cryptosporidias occurrence was revealed in clinically healthy monkeys. Among sick monkeys, the invasion was most common in infants under one year of age. Cryptosporidiosis is rarely found just as it is, and, as a rule, it accompanied by other parasitogenic and bacterial infections.

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Under captive conditions, a parasite fauna connected with the changes in ecological conditions, feeding, and mode of life is usually formed in monkeys. Species composition of the intestinal parasites has been investigated in six species of the monkeys (Macaca mulatta, M. fascicularis, M. nemestrina, Ceropithecus aethiops, Papio hamadryas, and P. anubis), which were born in the Adler apery and live there for a long time. A comparison with similar investigations carried out in the Sukhumi apery, where the climatic and keeping conditions are practically identical with those in the Adler apery, was conducted. Parasite fauna of monkeys in the Adler apery was found to include three species of Nematoda (Ascaris sp., Trichocephalus sp., and Strongyloides sp.) and two species of Protozoa (Balantidium coli and Lamblia intestinalis). In our material, Trichocephalus sp. is the dominant parasite species among helminthes, and Balantidium coli is the most frequent species of Protozoa. The commonness in the transmission of these parasites and similarity in their life cycles contribute to the forming of polyinvasions in monkeys.

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The historical account and recent taxonomic consideration of the monogenean subfamily Encotyllabinae are given. Main criteria used for diagnostics of the subfamily and its genera are discussed.

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Three new species of the monogeneans of the genus Dionchus are described: Dionchus paruchini sp. n. from Rachicentron canadus and D. trachuri sp. n. from Trachurus trachurus from Indian Ocean, D. major sp. n. from R. canadus from the North Vietnamese Gulf. All species are located on gills.

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The data on an occurrence of capsalines in marine fishes are analyzed. 33 capsaline species among 55 recently known species are monoxienous, that points to the high specialization of these monogeneans. The majority of species are recorded from three subfamilies of perciform fishes: Scombridae, Istiophoridae, Xiphiidae. Possible factors causing the high specificity in monogeneans are discussed.

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A monogenean Entobdella hippoglossi (Capsalidae: Entobdellinae), a common parasite of the flat fishes (Pleuronectidae), is recorded for the first time from an ocean perch Sebastes glaucus (Scorpaenidae) in the north-east part of Pacific ocean. New data on hosts and location of Sessilorbis limopharynx (Capsalidae: Trichopodinae) are also given.

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Ribosomal fractions containing up to 72% of ribosomal material and 25% of sugars (among them, about 6% of hexose) were isolated from P.aeruginosa cells, immunotypes F-1, 2, 6 and 7, by precipitation with polyethylene glycol 6000. Lipopolysaccharide, determined in the test for ketodesoxyoctanoic acid, was not detected in these fractions, but, as determined in the passive hemagglutination test, the content of O-antigen in the preparations was 3-25%. O-antigen and ribosome present in the fractions formed a complex, disintegrating after treatment with trilon B.

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The problems of the diagnosis of deep mycoses such as candidiasis, aspergillosis and actinomycosis most frequent in this country are discussed. A classification system for deep mycoses based on the characteristics of the causative agents which is of practical value is presented. The microscopic and cultural diagnosticating of deep mycoses is described in detail. A brief information on the principles of the treatment of the mycoses is given.

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A parenteral Shigella ribosomal vaccine (SRV) was investigated in animals for safety, antibody-inducing capacity, and protective activity. Ribosomal preparations from a Shigella sonnei phase I avirulent strain were obtained and shown to possess chemical, sedimentation, and other properties typical of bacterial ribosomes. No endotoxin contamination was revealed by a ketodeoxyoctonate assay, although the presence of some kind of O antigen was evidenced by serological findings and the high activity of SRV in inducing the O-antibody response and immunological memory in animals. SRV was nontoxic in mice, guinea pigs, and monkeys and induced no local reactions when injected subcutaneously in reasonable doses. Significant protection against a local Shigella infection (Sereny test) was seen in guinea pigs injected with SRV (efficiency index, about 60%) and the specificity of the protection was evident from cross-challenge experiments. The protective efficiency of SRV was especially high in rhesus monkeys challenged orally with virulent Shigella cells (89%, as calculated from the summarized data of several experiments in 71 animals). Protection in monkeys was long lasting and could be demonstrated several months after injection of SRV. An inexpensive technique can be used for the production of SRV on a large scale. The high immunogenicity of SRV is discussed in terms of the amplifying effect of the ribosome, which serves as a delivery system for polysaccharide O antigen. Further study of SRV as a candidate vaccine for humans seems justified by the data obtained.

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The isolation and properties of endotoxin protein, or lipid A-associated protein (LAP), from Shigella sonnei were described earlier (Zh. mikrobiol. epidemiol. immunobiol., 1991, No. 4, pp. 11-17, and No. 7). In this report the data on its protective activity are presented. In experiments on mice one nanogram of LAP injected i. v. protected 50% of the animals against i. p. challenge with 40 LD50 of virulent S. sonnei. Guinea pigs injected s. c. with 10 micrograms of LAP were protected against local (keratoconjunctival) challenge with S. sonnei, the efficiency of immunization being 58%. LAP preparations containing no detectable amounts of O-antigen (less than 0.003%) were found to have a protective effect. Hyperimmune anti-LAP rabbit serum prevented local infection when incubated with S. sonnei challenge inoculum before injection into guinea pigs. Both active and passive protection induced by LAP was specific since no effect was observed in animals challenged with Shigella flexneri. In the homologous system the protective effect of anti-LAP serum was abolished by the addition of protein-free LPS. These results are compatible with the hypothesis that the protective activity of LAP depends on the presence of minute amounts of O-antigen whose immunogenic effect is greatly amplified by the protein component of the natural endotoxin complex.

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O-specific polysaccharide (L-hapten) was isolated earlier (Zh. mikrobiol. epidemiol. immunobiol., 1989, No. 11, pp. 8-11). In this paper L-hapten was shown to be unable, even at high concentrations (up to 2,000 micrograms/ml), to sensitize sheep red blood cells for passive hemagglutination by O-antibodies. At the same time classical LPS and heat-activated LPS were active at concentrations ot 32 and 8 micrograms/ml respectively. The O-antibody-neutralizing activity of L-hapten was lower than that of LPS 10(3)-10(4) times in the passive hemagglutination test and 25-50 times in competitive ELISA. The immunogenicity of isolated L-hapten was very weak: primary response in mice to the i.v. injection of 1-10 micrograms of L-hapten was similar to the effect produced by 10(-3)-10(-4) micrograms of LPS. No protective activity of L-hapten was noted in mice when the challenge dose of virulent shigellae was 16 LD50 or more, and only a weak protective effect was observed with a low challenge dose (8 LD50). The molecular basis of low serological and biological activity of L-hapten is discussed. The most probable explanation of the results obtained in this study is that L-hapten contains some nonspecific carbohydrates, inserted in or complexed with the O-side chain. Despite its low immunogenicity, L-hapten can be an important component of effective bacterial vaccines provided it is included into a suitable delivery system as is the case with Shigella ribosomal vaccine.

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Endotoxin protein or lipid A-associated protein (LAP) from Shigella sonnei was isolated and characterized earlier (Zh. mikrobiol. epidemiol. immunobiol., 1991, No. 4, pp. 47-50). In this investigation serum antibodies against LAP were studied in ELISA Anti-LAP antibodies were detected in high titers in the sera of nonimmunized mice, guinea pigs, rabbits, monkeys and healthy adults. We suppose that normal anti-LAP antibodies resulted from interaction between the immune system and environmental endotoxin. Parenteral injections of LAP to different animals induced intensive antibody response with a 100- to 1000-fold increase in the serum anti-LAP antibody level and a significant rise in the serum O-antibody level. The latter is seemingly due to the contamination of LAP with minute amounts of O-antigen (0.12% or less) and to the amplification of its immunogenicity by LAP. Both antigenic and amplifying activity of LAP was destroyed by proteinase K. The biological function of LAP and its possible use as a component of bacterial vaccines are briefly discussed.

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The scheme of the isolation of endotoxic protein from S. sonnei 9090 is presented. The isolation procedure includes the 10-minute hot (at 68 degrees C) extraction of protein from endotoxin with 45% aqueous phenol, the precipitation of protein from phenolic extract with 9.5 volumes of 95% ethanol, the purification of protein from lipid material and pigments by multiple extraction with the mixture of chloroform and ethanol in the proportion 2:1 by volume. The yield of protein obtained with the use of this isolation scheme is about 3% of the initial endotoxin preparation. Protein preparations obtained in accordance with this scheme contain 92-95% of protein (determined by Lowry's method), 2.3-3.0% of saccharides (determined by the phenol-sulfate method) and 0.02% of hexose amine, its presence indicating that the preparations contain lipid A (or its fragments) which is firmly bound with endotoxic protein and cannot be extracted with chloroform. As shown in the passive hemagglutination inhibition test, the content of endotoxin in the preparations is less than 0.003%. Out of 7-11 bands revealed by electrophoresis in 15% polyacrylamide gel in the presence of sodium dodecyl sulfate, 3 main bands have molecular weights of 43, 38 and 18 KD. Three antigens differing in their electrophoretic mobility and diffusion rate in 1% agarose gel can be detected in the preparations by the method of immunoelectrophoresis with the use of antisera to both endotoxin and endotoxic protein.

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The electrophoretic analysis of lipid A-associated protein (LAP), obtained from S. sonnei, in polyacrylamide gel in the presence of sodium dodecyl sulfate and urea has revealed the heterogeneity of the preparation; it has found to contain three main components with molecular weights of 43, 38 and 18 KD and some minor components with molecular weights of 49, 45 35, 30, 29, 27, 5, 21 and 14 KD. The electrophoretic mobility of the main protein components in the isolated preparation of LAP coincides with that of endotoxin components. The dissociation of proteins and lipopolysaccharide in the process of boiling the endotoxin in 2% sodium dodecyl sulfate is indicative of the noncovalent binding of these components. LAP contained in the endotoxin, in contrast to isolated LAP, is resistant to trypsin and proteinase K. The enzyme immunoassay (EIA) system with the use of LAP as a component of its solid phase has been developed, which makes it possible to carry out the quantitative determination of antibodies to this protein. The EIA system shows high sensitivity in the determination of anti-LAP IgG antibodies: in hyperimmune rabbit sera their titer is 1:250,000-1:800,000. As shown by the method of competitive EIA, the antigenic affinity of LAP of different origin corresponds to the degree of taxonomic propinquity of microorganisms: the maximal degree of cross reactions is observed between LAP obtained from S. sonnei, S. flexneri and Escherichia coli, while their affinity to Salmonella typhi is considerably less; remote microbial species (Bacterium bifidum and Sarcina marcescens) give practically no cross reactions.

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Ribosomal preparations from Shigella flexneri and Shigella sonnei, introduced parenterally into mice, enhance their resistance to infection with the causative agents of typhoid fever and staphylococci. This effect is considerably less pronounced than that produced by the preparation of homologous lipopolysaccharide isolated by Boivin's method. After the administration of ribosomes nonspecific resistance to bacterial infective agents lasts for a short time. Ribosomal preparations do not enhance the resistance of mice to the lethal action of endotoxin.

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Shigella ribosomal vaccine (SRV) was previously shown to be highly active in induction of mucosal and systemic O-antibody response and protection against Shigella infection in guinea pigs and monkeys. In this study, the O-specific component (OSC) was isolated from the SRV by affinity chromatography using rabbit O antibodies coupled to CNBr-Sepharose. The results of the reaction with carbocyanine dye as well as chemical data show that ribosomal OSC is devoid of lipid A and KDO, which are characteristic of classical LPS. The comparison of OSC with various LPS-related substances led to the conclusion that ribosomal OSC is similar to and probably identical with cytoplasmic O polysaccharide (L hapten), an O-side-chain polymer which accumulates in cytoplasm. It is hypothesized that the extremely high immunogenicity of SRV depends on a cooperative action of OSC, representing an epitope-specific part of the vaccine, and a ribosomal particle which serves as a vector, providing amplification of the immunogenic effect. The data obtained indicate the presence of a non-covalent link between the two components of the ribosomal vaccine.

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Along with classical lipopolysaccharide (LPS), O-specific material not precipitated by ultracentrifugation has been isolated from the water-phenol extract of S. sonnei avirulent strain 9090 possessing complete antigenic properties. The purification of O-antigen contained in the supernatant fluid has been carried out by the gel filtration of the fluid, previously treated with ribonuclease, in a column packed with Sephadex G-100. The polysaccharide nature of O-antigen thus obtained, the absence of lipid A and KDO and the low content of hexoses, or core-specific saccharides of S. sonnei LPS, in this antigen make it possible to classify this material with O-components of microbial cells, described by different authors as "native protoplasmic polysaccharide" or "L-hapten" and formed by polymers of LPS O-side chains. The content of this component in S. sonnei strains under study is, on the average, 2.5% of the weight of dry microbial substance. L-hapten preparations obtained in the course of our investigations have been found to contain two O-specific antigens detected by immunoelectrophoresis and immunodiffusion, as well as by sedimentation in saccharose gradient, where they form peaks corresponding to 4.3 S and 10.8 S. This polysaccharide O-antigen is supposed to be capable of interaction with ribosomal particles and suitable for use as a component of ribosomal dysentery vaccines.

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S. flexneri ribosomal preparations were isolated by differential centrifugation or by fractionation with polyethylene glycol-6000. Their chemical composition and spectrophotometric properties were characteristic of ribosomes, and, as shown by the results of the serological assay, the content of O-specific component was, on the average, 1.4%. The ribosomal preparations were nontoxic for mice when injected intraperitoneally and intravenously in large doses and induced systemic O-antibody response in mice and rabbits. The parenteral administration of ribosomes to guinea pigs led to the increase of resistance to Shigella keratoconjunctivitis. The results of different tests with the use of this model greatly varied. According to the summary data of several tests, the ribosomal vaccine enhanced the resistance of the eyes from 11.3% to 48.5% and the effectiveness coefficient of immunization was 42 +/- 6. Ribosomes isolated from S. flexneri avirulent strain 2a 51.6 M (Iu. A. Belaia's vaccine) showed the same activity as those isolated from virulent strains. The results obtained in this study suggest the expediency of further experimental study of ribosomal preparations obtained from S. flexneri as potential vaccine.

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