STUDIES ON THE MYXOBACTERIUM CHONDROCOCCUS COLUMNARIS' 2

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1 STUDIES ON THE MYXOBACTERIUM CHONDROCOCCUS COLUMNARIS' 2 I. SEROLOGICAL TYPING ROBERT L. ANACKER3 AND ERLING J. ORD)AL Department of Microbiology, University of Washington, Seattle, Washington Received for publication December 22, 1958 In 1922 Davis described an infectious disease which was responsible for heavy mortalities in a number of species of warm water fishes in the Mississippi Valley. Although Davis was unsuccessful in his attempts to cultivate the etiological agent, he reported that large numbers of slender, motile bacteria were characteristically present in the lesions of infected fish. He further noted that when material scraped from a lesion was placed in a wet mount, the bacteria possessed the unique property of collecting together to form columnar masses on bits of fish tissue. Because of this feature, the organism was assigned the name Bacillus columnaris, and the disease was referred to as columnaris disease. No further mention of this disease is found in the literature until the report of Nigrelli (1943) that columnaris disease had been noted in fish held at the New York Aquarium. The etiological agent of columnaris disease was isolated by Ordal and Rucker (1944) during an epizootic in young sockeye salmon (Oncorhynchus nerka) at the Leavenworth Hatchery of the U. S. Fish and Wildlife Service. Additional cultures were isolated from surface lesions and from the internal organs of adult salmon and other fishes examined at Rock Island Dam on the upper Columbia River, and from moribund adult Chinook salmon (0. tsh.awytscha) and from sockeye salmon held in large outdoor ponds at the Leavenworth Hatchery. The latter fish had been trapped at Rock Island Dam and transferred to l This investigation was supported in part by funds from the U. S. Fish and Wildlife Service and the University of Washington Fund for Biological and Medical Research. 2 Portion of a dissertation presented by the senior author as partial fulfillment of the requirements for the Ph.D. degree at the University of Washington. 3Present address: Department of Bacteriology, Montana State University, Missoula, Montana. the Leavenworth Hatchery as part of an effort to relocate runs of salmon blocked by Grand Coulee Dam. Very heavy losses occurred in these stocks of fish during the period of holding (Fish and Hanavan, 1948). Bacteriological studies were carried out on a number of isolates (Ordal and Rucker, 1944), and it was found that the organism was a myxobacterium with a complex developmental cycle. On the basis of the observed properties, the organism was renamed Chondrococcus columnaris. Experiments on transmission were carried out, and it was found that pure cultures reproduced the disease, and that the organisms could be reisolated from fish with the experimentally produced discase. The myxobacterial nature of C. columnaris was confirmed by Nigrelli and Hutner (1945) who reported on an epizootic in killifish (Fundulus heteroclitus) which was found to be due to C. columnaris. Epizooties of columnaris disease in fishes have been reported in a number of regions of the United States (Davis, 1949; Johnson and Brice, 1952). In the Pacific Northwest, C. columnaris has been found to be an important agent of disease in salmonid fishes, not only in hatcheries, but also in the natural habitat (Rucker et al., 1953). Water temperature has been found to be one of the most important factors in determining the damage to populations of fishes due to columnaris disease, and most outbreaks occur during the summer months when water temperatures reach relatively high values. It was reported by Rucker et al. (1953) that cultures of C. columnaris isolated over a period of years from fishes taken at various locations in the State of Washington exhibited striking differences in virulence for young salmon when tested at comparable water temperatures. Cultures of C. columnaris isolated from fishes in the lakes, streams, and hatcheries of Western Washington exhibited relatively low virulence. On injection 25

2 26 ANACKER AND ORDAL [VOL. 78 into experimental fish, these strains produced, at most, slowly progressing infections with death ordinarily occurring after a number of days when extensive tissue necrosis had taken place. Yet, a number of these strains had been found responsible for mass mortalities among young fishes in hatcheries and natural bodies of water in epizootics at relatively high water temperatures. Some of the strains of C. columnaris isolated from fishes from the upper Columbia River and its tributaries in Eastern Washington were classified as of low virulence, but other strains exhibited extraordinary pathogenicity. The latter strains, isolated at various times from adult or young salmonid fishes from the upper Columbia River and its tributaries were classified as high virulence strains since they killed young salmon in less than 24 hr when introduced into the water in which the fish were held. There was ordinarily little or no evidence of gross tissue damage and pure cultures of the etiological agent could be directly reisolated in almost every instance from the internal organs of the experimental fish. There are a number of possible explanations which might account for the presence of the more virulent strains of C. columnaris found in the upper reaches of the Columbia River. One possible explanation is that salmon on the spawning migration are infected by strains of C. columnaris of low virulence while in the lower part of the river, and as the fish swim upstream, mutant or recombinant strains of higher virulence arise during multiplication of the myxobacteria in the lesions of the infected fish. A second possible explanation is that there may be strains of C. columnaris which are distinct both geographically and physiologically, perhaps existing on some natural host which serves as a reservoir of infection. On the basis of qualitative observations made over a period of years, there was good reason to believe that columnaris disease represented a serious hazard to runs of salmon and steelhead trout in the Columbia River System. However, relatively little effort had been given to the study of the myxobacterium, C. columnaris, and its role as an agent of disease in fishes, and there was no quantitative data available on the impact of columnaris disease on the runs of saimonid fishes. It was suggested above that there might be strains of C. columnaris which were distinct geographically and physiologically. If this were the case, there would be good reason to believe that they might be distinct serologically. A serological study of C. columnaris was instituted not only because of this possibility, but because a method of identification of specific strains of C. columnaris was expected to be of great value in the study of this complex myxobacterium, in determining its role as an agent of disease in fishes, and in addition, in evaluating the impact of columnaris disease on the runs of salmon and steelhead trout in the Columbia River System. The present study, therefore, is concerned with the development of a tentative scheme of serological typing for the myxobacterium C. columnaris. MATERIALS AND METHODS Isolation of strains. Cultures of C. columnaris were obtained by streaking material from the surface or gill lesions of fish, or sometimes from the internal organs, on Cytophaga agar, a medium containing tryptone (Difco), 0.05 per cent; yeast extract, 0.05 per cent; sodium acetate, 0.02 per cent; beef extract, 0.02 per cent; and agar (Difco), 0.9 per cent, adjusted to ph 7.2 to 7.4. Plates made during the course of field studies were held in an ice chest and then incubated at room temperature on return to the laboratory. After incubation of the plates from 1 to 3 days, colonies of C. columnaris were transferred and checked for purity and identity. Well developed colonies of C. columnaris were ordinarily easy to recognize because of their spreading, rhizoid, greenish-yellow growth, often with a honeycomb appearance due to the peculiar swarming of the cells. Separate isolates of C. columnaris were labeled and identified by numbers or other codes and were lyophilized as soon as practical after isolation in order to preserve the natural properties of the organisms. The majority of the cultures isolated were from fishes taken in the Columbia River or its tributaries. Cultivation. Cultures of C. columnaris removed from the lyophilized state were maintained in tubes containing Cytophaga medium with 0.4 per cent agar (Difco). For preparation of antigens the organisms were grown in a liquid medium containing tryptone, 0.4 per cent; yeast infusion, 3 per cent; and Tween 80, 0.1 per cent; adjusted to a ph of 7.2 to 7.4. Preparation of antisera. Immune sera were ob-

3 1959] STUDIES ON MYXOBACTERIUM. I 27 tained from rabbits injected either intravenously with triply washed saline suspensions of cells in successive doses of 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 ml on alternate days, or subcutaneously in the axillary and inguinal regions with the antigen and killed cells of the BCG strain of Mycobacterium tuberculosis suspended in Bayol and Falba (Freund and McDermott, 1942). The cell suspensions used for the intravenous injections were prepared by resuspending 0.2 ml of packed, washed cells in 4.0 ml of saline; each rabbit injected subcutaneously with the cells in Freund's adjuvants received approximately the equivalent of 1 mg of dry cells per site or a total of 4 mg dry weight. The rabbits injected intravenously were bled 10 days after the last injection, and the rabbits injected subcutaneously were bled 3 weeks after the single injection. After the first bleeding of the rabbits injected with adjuvants, the rabbits were reinjected with the antigen preparation, and bled 10 days later. The rabbits were usually bled from the marginal ear vein, and the sera were stored in the frozen state. Agglutination and adsorption procedures. A tube agglutination procedure was used in the initial studies presented in this report. Five-tenth-ml quantities of singly washed saline suspensions of the cells, adjusted to an optical density of 0.16 in the Coleman Nepho-Colorimeter with a green filter, were added to 0.5-ml quantities of serial 2-fold saline dilutions of the sera in saline. These tubes and controls of antigen with saline and antigen with heterologous rabbit antiserum were incubated 2 hr at 52 C and approximately 18 hr at 4 C before reading. A slide agglutination procedure was used in later studies. Loopfuls of the antigen suspensions, prepared by resuspending the sedimented growth from 6 ml of a broth culture in 1 ml of saline and heating for 5 min in a 52 C water bath to eliminate nonspecific clumping, were mixed with the antisera on 1 by 3 in glass slides. The number of cells in the suspensions used for the slide agglutination tests was approximately 1.5 X 1011 cells per ml. The reactions were read after the slides had been rocked back and forth for approximately 112 min beneath a 100-watt lamp. Some of the antisera were adsorbed before they were used. Suspensions of cells used for the adsorption of sera were prepared by resuspending singly-washed cells, sedimented from 300 ml of a stationary phase broth culture, in 6 ml of saline. The concentration of cells in the suspensions was approximately 1.2 X 1012 cells per ml. One ml of each of the sera was diluted with 2 ml of saline before adsorption. These serum dilutions were then adsorbed 1 hr at 52 C with 2 ml of the appropriate antigen suspension. After at least several hr at 4 C the antigen and adsorbed antibody were removed by centrifugation. Usually 2 or 3 individual adsorptions were required to exhaust an antiserum of the antibody which reacted with the adsorbing cells. RESULTS In a preliminary experiment designed to determine whether antigenic differences in strains of C. columnaris actually existed, 4 strains from sources widely separated geographically were used to immunize rabbits by intravenous injections; and the titers of each of the antisera thus obtained against each of the 4 strains were then determined. The results, presented in table 1, indicated that there were distinct differences in the titers of the antisera and presumably qualitative antigenic differences in the strains. After the above demonstration of the existence of serological differences in strains of C. columnaris, a number of other strains were screened against the 4 sera to determine agglutination patterns and to estimate the relative frequencies of the patterns. Twenty-five strains, nearly all of them isolated during 1954 from fish taken at TABLE 1 Agglutinin titers* of 214, 128, 114, and 204 antiserat obtained from rabbits injected intravenously with saline suspensions of the antigens Cells Antiserum$ * The titer is defined as the reciprocal of the highest dilution of the antiserum which is capable of producing visible agglutination of the cells. t Each antiserum is designated by the number of the strain of Chondrococcus columnaris which was used as the antigen in the preparation of the antiserum. I The lowest serum dilution tested was 1:20.

4 28 TABLE 2 Homologous agglutinin titers of 225, 234, 235, 238, and 244 antisera obtained from rabbits injected subcutaneously with antigen and adjuvants Antiserum ANACKER AND ORDAL Titer various locations in the State of Washington, revealed 6 distinct agglutination patterns, with about one-half of the strains falling within one pattern. All of the strains reacted with one or more of the test sera. After further preliminary studies, a number of strains of C. columnaris which were considered to exhibit antigenic differences were selected for use as antigens in the preparation of new antisera. These strains had all been isolated from fishes taken in the Columbia River or its tributaries. Antisera were prepared by subcutaneous injection of the antigens with adjuvants into rabbits, rather than by intravenous injection of cells in saline. Five of these antisera were used in almost all subsequent experiments and their homologous titers are given in table 2. Each of the strains used as an antigen for immunization was now tested against each of the new antisera. It was found, in contrast to the results of the preliminary experiment described in table 1, that each of the strains was agglutinated to a greater or lesser degree by each of the antisera. Although different strains were used in this immunization schedule than in the first series, it appears from these extensive cross reactions, and from data to be presented later in this discussion, that rabbits injected subcutaneously with whole cells of C. columnaris and adjuvants produce antibody to more cellular antigens than do rabbits injected intravenously with saline suspensions of cells. This phenomenon certainly is not unique with C. columnaris. Rabbits immunized subcutaneously with streptococci produce mainly antibody against the group-specific nucleoprotein, whereas rabbits immunized intravenously produce antibody primarily against the type-specific antigens (Derick and Swift, 1929). It has also been reported that the type of antibody produced [VOL. 78 by the horse also depends upon the route of administration of certain antigens (Treffers et al., 1947). Since the new antisera reacted with all of the strains of C. colunnaris tested, simple agglutination tests with unadsorbed sera could not easily separate these strains. Therefore, in order to detect differences in the test strains, antisera 225, 234, 235, 238, and 244 were adsorbed with the heterologous strains of the series; and then each of the antigens was tested against each of the adsorbed sera by the slide agglutination test. (In this experiment cells of strain 243 were substituted for cells of strain 244 in the slide test, because the latter cells exhibited a considerable degree of nonspecific clumping. Strains 243 and 244 were considered to be antigenically identical, because in earlier tube agglutination experiments with these 2 strains, neither strain was agglutinated by its homologous antiserum after the serum had been adsorbed with the heterologous strain.) The adsorption-agglutination experiments were performed several times, and consistent results were obtained. These data are presented in table 3. From the data of table 3 it is possible to assign to the strains arbitrary symbols representing the different antigens detected by this limited screening procedure. The antigenic compositions of several strains derived from the data of table 3 are presented in table 4. Antigenic symbols 2 and higher represent the type antigens, since these antigens may be used to separate the strains. In addition to the type antigens the presence of a group antigen in the strains may be inferred from certain evidence. Before adsorption each of the test sera which were obtained from rabbits immunized with the antigen-adjuvant mixture can agglutinate all of the test strains. Adsorption of any of the test sera, serum 225 for example, with any test strain which does not possess the type-specific antigens in common with strain 225 removes the ability of the antiserum to agglutinate all other test strains which do not possess the type-specific antigens of strain 225. This evidence supports the suggestion that all strains of C. columnaris possess a common antigen which is characteristic of the species. This antigen is designated by the symbol 1. Although this hypothesis cannot be proved by agglutinin-adsorption procedures because of the ubiquity of antigen 1, indirect confirmatory evi-

5 1959] STUDIES ON MYXOBACTERIUM. I 29 dence which will be discussed later was obtained from the typing reactions. By the appropriate adsorption of the antisera, 8 different typing sera were prepared for the serological typing of the strains of C. columnaris which had been isolated during a period of several years. The method of preparation of the typing sera and the antibody content of the typing sera are indicated in table 5. These particular combinations were chosen, because it was thought that all of the known type antigens might be detected when the entire series of sera was used with the cells. In addition, 2 unadsorbed sera, 225 and 238, were used to detect the presence of the group antigen, since these sera possess only anti-1 antibody in common. TABLE 3 Slide agglutination reactions* of antisera 225, 234, 235, 238, and 244 after adsorption with heterologoius cells Serum Adsorbing Strain ii4 Antigent _ _ :1= _ I * Final serum dilution was 1:20. t = Positive reaction with large flocs; + = positive reaction with small floes; and - = negative reaction. TABLE 4 Antigenic compositions of strains 225, 234, 235, 238, and 224 determined by slide agglutinin-adsorption tests Antigen Strains A total of 325 strains of C. columnaris, most of them separate isolates from fish taken in the Columbia River or its tributaries were now subjected to serological analysis by the slide agglutination method. In view of the relatively large number of strains involved it is neither practical nor desirable to tabulate the reactions of each of the strains with the typing sera. However, the information which was obtained can be summarized. First, the strains may conveniently be separated into 4 serological groups composed of antigenically identical or antigenically related types. Two strains are placed in a fifth miscellaneous group. The antigenic types and the percentages of the strains within the groups are presented in table 6. It should be noted that reactions involving antigen 8 were often weak, and it is not certain that the presence of antigen 8 was detected in every instance. The agglutination reactions involving antigen 2, on the other hand, were easily observed; therefore, all the strains of the antigenic composition 1, 2 or 1, 2, 8 are placed together in a serological group I. The members of group III are similar in that each strain possesses antigen 6; some strains also carry antigen 7. Antigens 1, 5, and 6 were detected in 2 strains, but rather than establish a new group for just 2 strains, these strains were also classified as strains of group III. It was mentioned earlier that the reactions of the strains with the typing sera indirectly confirmed the presence of a group antigen, antigen 1. Each strain was agglutinated by both of the unadsorbed sera 225 and 238, sera which did not contain antibody against a common type antigen.

6 30 ANACKER AND ORDAL [VOL. a78 Since the remaining reactions of the antigen suspensions with the adsorbed sera demonstrated that none of the strains possessed type antigens held by both strains 225 and 238, the fact that each strain is agglutinated by both unadsorbed 225 and 238 sera substantiates the theory that all strains possess a group or species antigen. In a number of instances, questionable or weakly positive agglutination reactions occurred on the slides with adsorbed 244 serum. This pattern of agglutination response was not encountered in the preliminary studies. Perhaps significant in this matter is the fact that the final serum dilution used in typing was approximately 1:10, whereas the final serum dilution used during the preliminary investigation of antigenic composition was approximately 1:20. Since those strains which exhibited the questionable reactions otherwise fell within well defined types, the questionable reactions were ignored for purposes of grouping. Three theories may be presented to explain the reactions: (a) the antibody involved in these reactions is present in low concentration because of a weak antigenic stimulus, and the resulting weak agglutination reactions were not recognized in the preliminary studies; (b) abnormal agglutination patterns were observed because antigenic changes due to mutation and selection occurred during cultivation; or (c) the antigenic compositions of the test strains are such that it is impossible to detect all the antigens which exist in nature. Strains which differed in their antigenic composition from those of the test strains might be expected to have a modified TABLE 5 Combinations of cells and antisera used for the preparation of typing sera for analysis of strains of Chondrococcus columnaris Adsorbing Antiserum (Antibody) Remaining Antibody Cells (Antigens) 225 (1, 6, 7) 235 (1, 5, 6) (1, 3, 8) 225 (1, 6, 7) 3, (1, 3, 8) 238 (1, 2, 8) (1, 5, 6) 244 (1, 5, 9) (1, 2, 8) 225 (1, 6, 7) 2, (1, 2, 8) 234 (1, 3, 8) (1, 5, 9) 225 (1, 6, 7) 5, (1, 5, 9) 235 (1, 5, 6) 9 TABLE 6 Distributtion of str-ains of Chondrococcus columnar is within the serological groups Antigens No. of Percentage Group Antlgens Strains of Total I 1, 2 or , 2, 8 II 1, 3, III 1, 6 or , 6, 7 IV 1, Misc Total agglutination pattern. This question has not been subjected to further investigation. DISCUSSION The presence of a common antigen indicates a similarity between all of the strains of C. columnaris studied regardless of geographical source. However, the strains are not antigenically identical, and the strains were classified into 4 serological groups on the basis of agglutinin-adsorption tests. The presence of a group and type antigens among strains of C. columnaris is reminiscent of the situation which exists among the streptococci (Lancefield, 1928). However, the chemical nature of the myxobacterial antigens is still unknown, and therefore no comparison can be made of the nature of the group and type antigens of the two species. All of the strains of C. columnaris which have been examined are morphologically and antigenically similar; and, though variations in virulence do occur, there seems to be little justification for any subelassification of the strains in the collection. One significant result of the serological typing study is the fact that the 20 strains of C. columnaris which were isolated from 6 separate sources west of the Cascade Mountains, sources other than the Columbia River or its tributaries, were classified in group I. Representatives of all the serological groups, including group I, were isolated from the Columbia River and its tributaries. Though strains of C. columnaris were recovered from several fairly distinct geographical areas of the Columbia River System, no area contained a preponderance of a single antigenic type, and

7 1959] STUDIES ON MYXOBACTERIUM. I 31 representatives of each of the serological groups were found in each of the areas. This evidence indicates that specific antigenic types of C. columnaris from the Columbia River and its tributaries are not indigenous to any restricted geographical area but are distributed throughout the river system. This is a logical conclusion since salmonids might easily carry strains both up and down the river, either during their downstream migration as fingerlings or upstream migration as adults. Although evidence has been presented for the existence of 7 type antigens, it is probable that others also exist. Compared to the known antigens of the Enterobacteriaceae (Kauffman, 1954) the number of antigens of C. columnaris is indeed small. Sera have been prepared against a limited number of strains only; and, in addition, these sera have been tested and adsorbed with a restricted number of strains. Furthermore, the questionable reactions of some of the strains may indicate a wider spectrum of antigens than that which has been demonstrated. In view of these considerations it is obvious that the subject of antigens of C. columnaris deserves further study. It would be of interest to know whether strains of C. columnaris from areas outside the Pacific Northwest can be typed by the methods developed for local strains. The only available strain of C. columnaris from another area was a strain from Texas. The Texas strain is related to the local strains by virtue of the common antigen 1 and type antigen 7, but its composition differs from all the other strains tested. Therefore, until a number of strains from other sources are compared with the local strains, it is impossible to know whether the present serological typing scheme is valid only for the local strains or for the majority of strains from all sources. A number of cultures were assayed for virulence (Anacker, 1956) and strains ranging from low to moderately high degrees of virulence were found within each of the serological groups. It is apparent, therefore, that virulence cannot be associated with the presence of any of the known antigens. The only property of C. columnaris which has been correlated with virulence is the lipoidal component identified by Dyar and Ordal (1946). No direct attempt was made to detect the lipoidal component, and it is possible that this component is not associated with an antigenic determinant site on the cell surface. Some evidence has been obtained which indicates that the antisera from rabbits injected subcutaneously with antigen and adjuvants is qualitatively more complex with respect to the kinds of antibody than the antisera obtained from rabbits injected intravenously with saline suspensions of the antigen. Unadsorbed sera from rabbits injected subcutaneously agglutinate all strains, whereas sera obtained from rabbits injected intravenously agglutinate a portion of the strains only. It appears from these limited observations that rabbits injected intravenously with C. columnaris may respond only to surface type antigens before eliminating the bacteria, and that the persistence of C. columnaris in an oil emulsion enables the rabbit to react to other antigens, including the common antigen. ACKNOWLEDGMENTS The authors wish to express their sincere appreciation to Dr. R. S. Weiser for helpful discussions during the course of this work; to Messrs. R. L. Anderson, P. Clare, T. Fukuyama, and R. E. Pacha for their invaluable assistance in the isolation of the strains of Chondrococcus columnaris; and to the many individuals of the U. S. Fish and Wildlife Service, the U. S. Army Corps of Engineers, and the Oregon Fish Commission for their cooperation during the field studies. SUMMARY A serological typing procedure has been devised which separates 325 strains of Chondrococcus columnaris, isolated since 1953, from fish of the Pacific Northwest and 1 strain from Texas, into 4 serological groups and 1 miscellaneous group. All of the strains possess a common antigen which may be characteristic of the species, but the strains vary in their composition of other antigens which are called the type antigens. Each of the serological groups is composed of strains containing the identical type antigens or of strains which possess at least one type antigen in common. Because of the serological relationship which exists among the strains and the morphological similarities of all the strains, there is no justification at present for a taxonomic division of these strains. It is not yet possible to draw firm conclusions

8 32 ANACKER AND) ORDAL [VOL. 78 concerning the geographic distribution of serological types of C. columnaris. All of the strains of C. columnaris isolated from lakes and hatcheries of western WAashington and the Rogue River of western Oregon were placed in serological group I. However, representatives of all 4 serological groups were found in the Columbia River and its tributaries, most of which are located in eastern WVashington. 'lthe Texas strain, the only strain from outside the Pacific Northwest, is related to the other strains but nevertheless differs from the others in antigenic composition. It must be concluded that it is difficult to logically explain the occurrence of serological types from the evidence which has been accumulated thus far. REFERENCES ANACKER, R. L Studies on the identification, virulence, and distribution of strains of (hondrococcus columnnaris isolated from fish in the Columbia River System. Ph.D). Thesis, University of Washirngton. D)AVIS, H. S A new bacterial disease of fresh-water fishes. U. S. Butr. Fisheries Bull., 38, DAVIs, H. S (Cytophaga columnaris as a cauise of fish epidemics. Trans. Am. Fisheries Soc., 77, DERICK, C. L. AND SUIFT, H. F Iteactions of rabbits to nonhemolytic streptococei. I. General tubercullin-like hypersensitiveness allergy, or hyperergy following the secondary reactioni. J. Exptl. Med., 49, DxYAR, MI. T. AND ()RDAL, E. J Electrokinietic studies on bacterial surfaces. I. The effects of surface-active agents on the electrophoretic mobilities of bacteria. J. Bacteriol., 51, FISH, F. F. AND HANAVAN, M. G A report upon the Grand Coulee fish maintenance project of U. S. Fish and Wildlife Serv. Spec. Sci. Rept. No. 55. FREULND, J. AND MCI)ERMOTT, K Sensitization to horse serum by means of adjuvants. Proc. Soc. Exptl. Biol. MIed., 49, JOHNSON, H. E. AND BRICE, R. F Observations on columnaris in salmon and trout. Progressive Fish Culturist, 14, KAU.FFMANN, F Enterobacteriaceae. Collected studies Salmlonella, Arizona, Escherichia, Klebsiella, Cloaca, Hafnia, Shigella, Proteus, and Providencia. Ejnar Munksgaard, Copenhagen. LANCEFIELD, R. C The antigenic complex of Streptococc us haemitolyticus. I. Demonstration of a type-specific stubstance in extracts of Streptococcus haeuiolyticits. J. Exptl. Med., 47, NIGRELLI, R. F Causes of disease and death of fishes in captivity. Zoologica, 28, NIGRELLI, R. F. AND HUTNER, S. H The presence of a myxobacterium, Chondrococculs columnaris (Davis) Ordal and Rucker (1944) on Futnduluts heteroclitius (Linn.). Zoologica, 30, ORDAL, E. J. AND RU CKER, R. R Pathogenic myxobacteria. Proc. Soc. Exptl. Biol. Med., 56, RUCKER, R. R., EARP, B. J., AND ORDAL, E. J Infectious diseases of Pacific salmon. Trans. Am. Fisheries Soc., 83, TREFFERS, H. P., HEIDELBERGER, M., AND FREUND, J Anti-proteins in horse sera. III. Antibodies to rabbit serum-n and their reaction with antigen. J. Exptl. MIed., 86,