ALBERT

Notes: Six integral membrane proteins of bacterial, animal, and plant origin, which are believed to function in solute transport, share sequence identity and are grouped together as members of the MIP family. These include the Escherichia coli glycerol facilitator, the major intrinsic protein from bovine lens fibre junction membranes, a plant tonoplast membrane protein, a soybean protein from the peribacteroid membrane, and a Drosophila neurogenic protein. These proteins, each of which appears to consist of six transmembrane helical segments per subunit, apparently arose by internal duplication of a three-transmembrane segment. Phylogenetic‘trees’interrelating these proteins and segments are presented.

Notes: We recently described the use of selective transposon mutagenesis to generate a series of a virulent mutants of a pathogenic strain of Salmonella typhimurium. Cloning and sequencing of the insertion sites from two of these mutants reveals that both have identical locations within an open reading frame that is highly homologous to a gene, htrA, encoding a heat-shock protein in Escherichia coli. DNA sequence analysis of S. typhimurium htrA reveals the presence of a gene capable of encoding a protein with a calculated Mr of 49316 that has 88.7% protein:protein homology with its E. coli counterpart. In E. coli, lesions in this gene, also known as degP, reduce proteolytic degradation of aberrant periplasmic proteins. Characteristics of the S. typhimurium htrA mutants, 046 and 014, in vivo and in vitro suggested that they are avirulent because of impaired ability to survive and/or replicate in host tissues. In vitro, the S. typhimurium htrA mutants 046 and 014 are not temperature-sensitive but were found to be more susceptible to oxidative stress than the parent, suggesting that they may be less able to withstand oxidative killing within macrophages.

Notes: Germination and growth of Bacillus cereus spores from emetic and diarrheal strains were measured in Trypticase soy broth (TSB) and in autoclaved rice/beef extract from 5°– 55°C. Growth for some strains occurred from 15°– 50°C, and little difference was noted between responses of diarrheal and emetic types or between media, except a higher maximum population was achieved in rice. Germination was more extensive in rice than in TSB at 〈15°C and was generally more extensive for diarrheal strains in either medium.

Notes: In a field population of the protogynous red grouper Epinephelus morio in the eastern Gulf of Mexico, females with oocytes at all stages of development were collected during the spawning season suggesting that several batches of oocytes may be released over the spawning period. Plasma oestradiol (E2) levels were highest in ripe females whose gonads contained both cortical alveoli and vitellogenic oocytes during the breeding season. Males were still spermiating as late as August, although levels of androgens 11-ketotestosterone (11-KT) and testosterone (T) had declined from their peaks in March. A few red grouper with either perinucleolar or cortical alveoli stage oocytes were undergoing sex change both during and after the spawning period. Low levels of E2, T and 11-KT were detected in transitionals. Proliferation of male tissue was not restricted to any specific area of the gonad but occurred in pockets within the ovarian lumen. The sequence of an increase in gonial cells along the periphery of the lamellae, increase in interstitial tissue, degradation of female elements, and formation of a sperm duct seemed to be concurrent with spermatocyte proliferation and the process of preparing the gonad to function as a testis.

Notes: Soymilk was produced by (A) heating of intact soybeans after soaking but before disruption, (B) heating of soaked soybeans to 80°C during disruption, and (C) disruption of soaked soybeans with no prior heating. The results show that process A has low yields of solids and low recoveries of protein in comparison with processes B and C. Heating of soybeans preceding disruption seems to keep protein bodies intact when soybean cells are disrupted. Homogenization will redisperse protein bodies that have been fixed by heat, and homogenization at high pressure (8,000 psi) and high temperature (75°C) is more effective than lower pressures and temperatures. Data are presented on rates of water or buffer uptake by steeping soybeans, on times and temperatures needed to eliminate the green-beany flavor and on the solids analysis by spectrophotometry.

Notes: Abstract. Protection against Aeromonas salmonicida was determined by passive immunization and with various bacterin preparations. Rabbit antiserum was prepared against a rough, virulent strain of A. salmonicida (AS-1R), the same strain boiled (AS-1R, boiled), and an avirulent, smooth strain of this same isolate (AS-1S). Cross-absorption, cross-passive protection and analysis by counter immunoelectrophoresis of various extraction methods were studied. It was shown that AS-1R cells contained an additional antigen not present in AS-1R (boiled) and AS-1S cells. Antiserum to the AS-1R antigen passively protected sockeye salmon, Oncorhynchus nerka (Walbaum), against a virulent challenge, and antisera to AS-1R (boiled) and AS-1S were not protective. The antigen was not destroyed by formalin or heat at 5°C for 60 min, but it appeared to be partially inactivated with proteolytic enzymes. The antigen was produced in casein yeast beef (CYB) broth up to 32 h but not thereafter, and low yields were obtained in tryptic soy or brain heart infusion (BHI) broth. It was extracted from cells with ethylenediamine tetraacetic acid (EDTA) and especially alkaline hydrolysis, but not with proteolytic enzymes or detergents. The detergents appeared to destroy the antigen. We concluded that the antigen was protein and is most likely the external A-protein (AP) reported for rough, virulent strains of A, salmonicida. Various methods of preparing A. salmonicida bacterins were evaluated by determining the level of protective immunity induced in intraperitoneally (i.p.) vaccinated fish. Growth of cells in CYB or BHI broth resulted in production of only rough (autoagglutinated in saline) variants of A. salmonicida. Although only rough variants were associated with protective immunity, one strain was not protective, it was avirulent by bath challenge. Bacterins prepared in CYB were more efficacious than those grown in BHI, but inactivation with formalin, iodine, or glutaraldehyde worked equally well. However, boiling the bacterin or filtering the cells from the bacterin removed its efficacy. Methods of releasing the AP were evaluated by sonification, pH-lysis, disaggregation and treatment with EDTA, and all treatments worked equally well. Also, precipitation on to aluminium or use of Freund's complete adjuvant did not significantly improve the protection. In parenterally vaccinated fish, protection was demonstrated by challenging the fish at various levels by bath, injection or cohabitation with infected fish. The best protection was demonstrated using the cohabitation challenge method. The potency and field efficacy of an A. salmonicida bacterin prepared in CYB broth and extracted with 5 mM EDTA was evaluated. Fish were vaccinated by i.p, injection and potency was determined in the laboratory by experimental challenge and in the field by natural challenge. Chinook salmon, O.ishawytschu (Walbaum), developed immunity within seven days at 10°C. The bacterin could be diluted up to 1:2000 without loss of potency. The field tests results were equivocal; however, (he prevalence of infection was lower in vaccinated fish.

Notes: Abstract. The comparative efficacy of several methods of vaccinating rainbow trout, Salmo gairdneri Richardson, with Yersinia ruckeri bacterins was evaluated. In general, the protective immunity was best by intraperitoneal injection followed by direct immersion, shower and spray, respectively. The bacterin was effective by all methods when diluted 1:20 or less, but there was a trend towards less efficacy with higher dilutions and shorter exposure time.

Notes: Abstract. The level of protective immunity was determined for several salmonid species following vaccination by the direct immersion method with commercial Vibrio anguillarum (two serotypes) and Yersinia ruckeri (Hagerman strain) bacterins. The duration of protective immunity varied with the bacterin concentration, size and species of fish, but the duration between the two bacterins was comparable. In fish under 1 g duration of protective immunity was longest when the most concentrated bacterin was used. Generally, immunity lasted in 1-g fish for about 120 days, in 2-g fish for about 180 days, but in 4-g fish and larger immunity lasted for a year or longer. Coho salmon (Oncorhynchus kisutch) and sockeye salmon (O. nerka) retained immunity for a longer time and pink salmon (O. gorbuscha) the shortest time. Chinook salmon (O. tshawytscha) and rainbow trout (Salmo gairdneri) were intermediate. Field data generally followed the laboratory tests, but the duration appeared somewhat shortened. In one test 20-g rainbow trout were vaccinated by the shower method and no loss of immunity occurred after 311 days.