Immunodiffusion Analysis Research Articles

The effect of maternal supply of rumen-protected protein to Holstein Friesian cows during the dry period on the transfer of passive immunity and colostral microbial composition

The objective of this study was to analyze if maternal supply of rumen-protected protein during the dry period can affect the IgG concentration and microbial composition of colostrum and the IgG absorption and fecal microbial composition in the calf. Seventy-four multiparous Holstein Friesian (HF) dairy cows were stratified per parity and randomly assigned to one of 2 different dry period diets, a diet with a low crude protein (CP) level (LP) and a diet with a high CP level (HP) by addition of rumen-undegraded protein (RUP; formaldehyde-treated soybean meal, Mervobest, Nuscience, Drongen, Belgium). Colostrum was collected within 1 h after calving and IgG concentration was quantified by radial immunodiffusion analysis. Forty-nine calves (23 female and 26 male) were enrolled in the trial with a 2 × 2 factorial design, with prenatal and postnatal treatment as the 2 independent variables. This led to 4 experimental groups: LPLP, LPHP, HPLP, and HPHP, in which the first 2 letters refer to the prenatal treatment (diet of the dam) and the last 2 refer to the postnatal treatment (diet of the colostrum-producing cow). Calves received 3× 2 L of colostrum within 2, 6, and 24 h after birth. Meconium and feces were collected solely from female calves (n = 18) by digital palpation of the rectum, immediately after birth and before colostrum administration and at d 3 of age. Microbial DNA was extracted from meconium (n = 9), feces (n = 15), and colostrum (n = 49). Amplicon sequencing of the bacterial V3-V4 region of the 16S rRNA gene was performed for characterization of the bacterial communities. Colostrum IgG concentration was higher in cows that were supplemented with RUP, especially in cows entering their second lactation (LSM ± SEM 61.3 ± 2.3 vs. 55.2 ± 2.8 g of IgG/L). Calves born out of LP cows that received colostrum from HP cows (LPHP) had a lower serum IgG level compared with HPHP and LPLP calves (LSM ± SEM 14.2 ± 1.3 vs. 18.8 ± 1.2 and 20.9 ± 1.3 g of IgG/L in HPHP and LPLP, respectively). The most abundant phyla in colostrum were Proteobacteria (48.2%), Firmicutes (24.8%), Bacteroidetes (9.5%), and Actinobacteria (5.0%). The most abundant phyla in calf meconium and feces were Firmicutes (42.5 and 47.5%), Proteobacteria (21.7% and 33.7%), Bacteroidetes (16.8% and 15.7%), and Actinobacteria (2.9% and 3.1%). There was no difference in the overall microbial communities between colostrum from HP and LP cows. However, 2 genera (both members of the family Lachnospiraceae) were more abundant in colostrum from HP cows compared with LP cows. The microbial composition of meconium, feces and colostrum differed from each other. Fecal samples were more similar to each other and are characterized by a lower intersample diversity compared with colostrum and meconium samples. To conclude, increasing the CP level by addition of RUP in the dry period diet affected the colostrum IgG concentration and the transfer of passive immunity, but did not change the overall microbial composition of colostrum nor of meconium and feces in the calf.

Supplementing fresh bovine colostrum with gut-active carbohydrates reduces passive transfer of immunoglobulin G in Holstein dairy calves

High concentrations of coliform bacteria in maternal colostrum (MC) have been associated with reduced IgG absorption in calves. Mannan-oligosaccharide, a gut-active carbohydrate (GAC) derived from yeast cell wall, has been shown to adsorb pathogens expressing type-1-fimbriae, reducing their ability to colonize the gastrointestinal tract. The objective of this study was to investigate if addition of a GAC to colostrum would result in increased IgG absorption in newborn calves. Newborn Holstein heifer and bull calves were enrolled in summer 2012 at a commercial transition cow facility in western Wisconsin. Each day, 7.6-L pools of fresh, first milking MC were created, split into 3.8-L aliquots, and refrigerated until feeding. Eligible newborn calves were removed from the dam 30 to 60min after birth, weighed, and randomly assigned to be fed either 3.8 L of the MC pool (control) or 3.8 L of the MC pool with 30 g of GAC mixed in immediately before feeding. Duplicate 10-mL samples of colostrum were collected and frozen at −20°C before feeding (and before addition of GAC) for bacterial culture and IgG determination. A 10-mL venous blood sample was collected from calves before feeding colostrum and 24 h after colostrum feeding, for laboratory determination of serum IgG using radial immunodiffusion analysis. Colostrum and calf characteristics, including colostrum IgG concentration (g/L), colostrum bacteria counts (log10, cfu/mL), calf dystocia scores (1 to 4), birth weights (kg), and age at first feeding (min) were not different between the group fed GAC (n=47) and the control group (n=48). Mixed linear regression analysis showed that calves fed colostrum supplemented with 30 g of GAC had lower mean (standard error) apparent efficiency of absorption of IgG and lower serum IgG concentrations at 24 h [23.9% (1.0); IgG=24.0 (1.1) g/L] than did control calves [30.4% (1.0); IgG=30.8 (1.0) g/L]. Given the negative effect observed in this study, it is not recommended that fresh colostrum be supplemented with 30 g of GAC.

Immunochemical detection of Azospirilla in soil with genus-specific antibodies

Immunoelectrophoresis and immunodiffusion analysis with antibodies to whole intact cells of the type strain of nitrogen-fixing soil bacteria Azospirillum brasilense Sp7 revealed at least three conservative surface immunogenic proteins of azospirilla. Cross-reactions with these proteins made it possible to use the above antibodies for detection of azospirilla as a genus-specific probe conjugated with horseradish peroxidase as an enzymatic label. Direct immune-enzyme analysis of soil suspensions (typical chernozem, Saratov oblast) confirmed applicability of the conjugates based on genus-specific antibodies to the surface proteins of azospirilla for direct detection of this bacterial genus in environmental samples. These results provide a basis for broad application of this method for analysis of Azospirillum occurrence in soil.

Heat-treated (in single aliquot or batch) colostrum outperforms non-heat-treated colostrum in terms of quality and transfer of immunoglobulin G in neonatal Jersey calves

The objective of this randomized clinical trial was to describe the effect on colostrum characteristics and passive transfer of IgG in neonatal calves when using the Perfect Udder colostrum management system (single-aliquot treatment; Dairy Tech Inc., Greeley, CO) compared with a negative control (fresh refrigerated or fresh frozen colostrum) and a positive control (batch heat-treated colostrum). First-milking Jersey colostrum was pooled to achieve 31 unique batches with a minimum of 22.8L per batch. The batch was then divided into 4 with 3.8L allocated to each treatment group: (1) heat-treated in Perfect Udder bag at 60°C for 60min and then stored at −20°C (PU); (2) heat-treated in a batch pasteurizer (Dairy Tech Inc.) at 60°C for 60min and then stored at −20°C in Perfect Udder bag (DTB; positive control); (3) fresh frozen colostrum stored at −20°C in Perfect Udder bag (FF; negative control); and (4) fresh refrigerated colostrum stored at 4°C in Perfect Udder bag (FR; negative control). Colostrum from all treatments was sampled for analysis of IgG concentration and bacterial culture immediately after batch assembly, after processing, and before feeding. Newborn Jersey calves were randomly assigned to be fed 3.8L of colostrum from 1 of the 4 treatment groups. A prefeeding, 0-h blood sample was collected, calves were fed by esophageal tube within 2h of birth, and then a 24-h postfeeding blood sample was collected. Paired serum samples from 0- and 24-h blood samples were analyzed for IgG concentration (mg/mL) using radial immunodiffusion analysis. The overall mean IgG concentration in colostrum was 77.9g/L and was not affected by treatment. Prefeeding total plate counts (log10 cfu/mL) were significantly different for all 4 treatments and were lower for heat-treated colostrum (PU=4.23, DTB=3.63) compared with fresh colostrum (FF=5.68, FR=6.53). Total coliform counts (log10 cfu/mL) were also significantly different for all 4 treatments and were lower for heat-treated colostrum (PU=0.45, DTB=1.08) compared with fresh colostrum (FF=3.82, FR=4.80). Mean 24-h serum IgG concentrations were significantly higher for calves in the PU (41.0mg/mL) and DTB (40.6mg/mL) groups compared with FF (35.1mg/mL) and FR (35.5mg/mL) groups. Mean apparent efficiency of absorption of IgG was significantly higher for the PU (37%) and DTB (37%) groups compared with the FF (32%) and FR (32%) groups. Calves fed heat-treated colostrum (PU or DTB) experienced significantly improved AEA and serum IgG concentrations.

Serological relationships of azospirilla revealed by their motility patterns in the presence of antibodies to lipopolysaccharides

Motility of the serologically different Azospirillum brasilense strains Sp245 (serogroup I) and Sp7 (serogroup II) was studied in the presence of antibodies to their lipopolysaccharides (LPS). A procedure was proposed in order to determine the motility patterns indicating the specificity of the interaction between the anti-LPS antibodies and bacteria. Analysis of the effect of such antibodies on motility of 25 strains (A. brasilense, A. lipoferum, A. irakense, and Azospirillum sp.) revealed bacteria exhibiting antigenic cross reactions with A. brasilense Sp7 or Sp245. The effect of anti-LPS antibodies on motility of azospirilla was in agreement with the results of immune agglutination analysis of bacterial cells and of immunodiffusion analysis of the LPS preparations. According to our results, strains Azospirillum sp. SR81 and A. brasilense SR14 should be included into serogroups I and II, respectively.

The antigenic character of zinc-binding proteins and their localization in boar reproductive tract--a preliminary study.

In this study immunoelectrophoretic and double immunodiffusion analyses were used to investigate the antigenic character of zinc-binding proteins (ZnBPs), whereas the indirect immunofluorescence technique was used to identify their origin in boar reproductive tract. The mmunoelectrophoretic analysis of ZnBPs of the seminal plasma resulted in the appearance of three antigenic protein complexes, while specific immunoreactivity patterns of the anti-ZnBP serum were detected by double immunodiffusion analysis. Indirect immunofluorescence technique confirmed that ZnBPs were secreted by different reproductive tract tissues, suggesting their contributions to the seminal plasma.

Immunological detection of keratan sulfate in meat products with and without mechanically separated chicken meat

Keratan sulfate is a glycosaminoglycan found in the structure of cartilage proteoglycans, aggrecan and fibromodulin. This study was undertaken to detect this glycosaminoglycan in meat products containing mechanically separated chicken meat (MSCM) having cartilage particles. Dry-defatted samples of MSCM and meat products with or without MSCM were digested with papain, and a non-dialyzable fraction from each papain digest was examined by immunodiffusion analysis using anti-keratan sulfate monoclonal antibody (IgM). No precipitine line was formed with the antibody for all samples of meat products without MSCM, while a sample of MSCM and all samples of meat products with MSCM gave clear precipitine lines with the antibody. The immunodiffusion test described here appears to be a simple sensitive specific method for qualitative analysis of keratan sulfate, which in combination with other methods may be useful for detection of MSCM in meat products.

Dissolvable Microneedle Patches for the Delivery of Cell-Culture-Derived Influenza Vaccine Antigens

Microneedle patches are gaining increasing attention as an alternative approach for the delivery of vaccines. In this study, a licensed seasonal influenza vaccine from 2007 to 2008 was fabricated into dissolvable microneedles using TheraJect's microneedle technology (VaxMat). The tips of the microneedles were made of antigens mixed with trehalose and sodium carboxymethyl cellulose. The patches containing 15 μg per strain of the influenza antigen were characterized extensively to confirm the stability of the antigen following fabrication into microneedles. The presence of excipients and very low concentrations of the vaccine on the microneedle patches made it challenging to characterize using the conventional single radial immunodiffusion analysis. Novel techniques such as capture enzyme-linked immunosorbent assay and enzyme digestion followed by mass spectroscopy were used to characterize the antigens on the microneedle patches. The in vivo studies in mice upon microneedle administration show immunogenicity against monovalent H1N1 at doses 0.1 and 1 μg and trivalent vaccine at a dose of 1 μg. The initial data from the mouse studies is promising and indicates the potential use of microneedle technology for the delivery of influenza vaccine.

Composition and immunochemical characteristics of exopolysaccharides from the rhizobacterium Paenibacillus polymyxa 1465

Exopolysaccharides (EPS) synthesized by Paenibacillus polymyxa 1465 in the course of batch cultivation were proven to contain neutral and acidic fractions. EPS are heterogeneous polysaccharides, represented by a complex of macromolecules with molecular mass of 7 x 10(4) to 2 x 10(6) Da. The acidic component was shown to be predominant in EPS preparations isolated from bacteria cultivated on glucose, which corresponds to a higher viscosity of EPS water solutions. The exoglycans were shown to contain glucose, mannose, galactose, and uronic acids. Polyclonal rabbit antibodies against the isolated P. polymyxa 1465 EPS preparations were used in a comparative immunodiffusion analysis of a number of P. polymyxa strains.

Detection of a sheath on Azospirillum brasilense polar flagellum

The presence of a polysaccharide sheath on the surface of the polar flagellum of Azospirillum brasilense was revelted by immunoelectron microscopy and immunodiffusion analysis with strain-specific antibodies to lipopolysaccharides (LPS). The antigenic identity of A. brasilense Sp245 sheath material and one of the two O-specific polysaccharides of its somatic LPS was demonstrated. The screening effect of the sheath in respect to flagellin was determined by agglutination tests and by the inhibition of azospirilla motility in liquid and semisolid agarized media caused by strain-specific antibodies to LPS; no pronounced effect of genus-specific antibodies to flagellin was observed.

Evaluation of electroelution and immunodiffusion as methods for purification and identification of the allergenic proteins of Ailanthus excelsa Roxb. pollen

The total soluble proteins from the pollen of Ailanthus excelsa Roxb., a potentially allergenic plant, was extracted and separated on a 12% SDS‐Polyacrylamide gel with an aim to identify the allergenic determinants responsible for causing respiratory disorders. The protein profile revealed nine prominent bands, which were designated as AE1 to AE9 lying within a short molecular weight range of 42.7 kDa to 97.4 kDa. Each protein fraction was recovered from the gel by electroelution and sent for skin tests to identify the allergenic fractions. All the protein fractions except AE3 (85.3 kDa), were found to be capable of inducing reactivity in sensitive patients. However, AE6 (59.7 kDa) and AE8 (45.0 kDa) proved to be the major allergenic determinants, while AE1 (97.4 kDa), AE2 (92.6 kDa), AE4 (73.3 kDa), AE5 (64.8 kDa), AE7 (52.4 kDa) and AE9 (42.7 kDa) are the minor contributors of allergenicity. Immunodiffusion analysis with pooled sera of patients sensitive to the total extract also revealed similar results.

Isolated central nervous system histoplasmosis in immunocompetent hosts: A series of 11 cases

Histoplasmosis of the central nervous system occurs in a significant percentage of patients with Histoplasma capsulatum infection, but has usually been described in association with immunosuppression and/or disseminated histoplasmosis. We aim to review the clinical and laboratory features of isolated histoplasmosis of the central nervous system in the immunocompetent host by presenting a series of 11 cases with this condition. Most of these patients presented with headache, meningeal irritation signs and mental status changes, comprising a somewhat different picture from that described in immunosuppressed patients. Moreover, almost all patients had signs of ventricular dilatation in neuroimaging studies, and 8 of the 11 patients had a ventriculoperitoneal shunt at the time of diagnosis, suggesting hydrocephalus to be an important feature of this condition and/or the possibility of shunt infection by the fungus. Immunodiffusion analysis of the cerebrospinal fluid appeared to be the most efficient way to reach the diagnosis and should be considered in immunocompetent patients with chronic lymphocytic meningitis, especially in those who have ventricular shunt or live in endemic areas of Histoplasma capsulatum.

Demonstration of Streptococcus mutans with a cell wall polysaccharide specific to a new serotype, k, in the human oral cavity.

Streptococcus mutans organisms are occasionally isolated from the blood of patients with infective endocarditis, though the mechanisms of invasion and survival remain to be elucidated. Two of four blood isolates from patients with bacteremia or infective endocarditis (strains TW295 and TW871) were serologically untypeable by immunodiffusion testing, which was due to a lack of the glucose side chain of the serotype-specific polysaccharide antigen of S. mutans. Immunodiffusion analyses using antisera against these strains demonstrated that 2 of 100 isolates from 100 subjects showed a positive reaction, while further analysis of 2500 isolates from 50 subjects revealed that all 50 isolates from a single subject were not reactive with anti-c, -e, and -f antisera, though they were reactive with anti-TW295 and -TW871 antisera. The oral isolates showed biological properties similar to those of the reference S. mutans strain MT8148, including high levels of sucrose-dependent adhesion and cellular hydrophobicity, along with expression of glucosyltransferases and a protein antigen, PA. We designated these organisms serotype k. A glucose side chain-defective mutant strain was then constructed by insertional inactivation of the gluA gene of strain MT8148, which showed biological properties similar to those of serotype k of S. mutans. Serotype k oral isolates were less susceptible to phagocytosis, as were the gluA-inactivated mutant of strain MT8148 and blood isolates. These results indicate that S. mutans serotype k strains are present in the oral cavity in humans and may be able to survive longer in blood owing to their low susceptibility to phagocytosis.

PROPERTIES OF A TOMBUSVIRUS THAT INFECTS COCONA (SOLANUM SESSILIFLORUM) IN THE PERUVIAN JUNGLE

SUMMARY Cocona (Solanum sessiliflorum) is a native plant of the Amazon basin that is cultivated in Ecuador, Venezuela, Brazil and Peru. From cocona plants in the Peruvian highland jungle showing stunting, leaf deformation and necrotic spots a virus was recovered after mechanical inoculation exhibiting isometric particles approximately 30 nm in diameter. This virus was biologically, serologically and partly molecularly characterized. It was transmitted by mechanical inoculation to 44 of 49 species in six different botanical families. Symptoms were primarily localized infections that resembled those elicited by tombusviruses. The virus was found by immunodiffusion analysis to be serologically related to several definitive tombusvirus species. A reaction of identity was found with Pear latent virus (PeLV), a recently described tombusvirus from Italy. The coat protein genes of the cocona virus isolate and PeLV showed a high degree of identity, i.e. 94% at the nucleotide level and 96% at the amino acid level.

Identification of the allergenic proteins of Cassia siamea pollen

Total soluble proteins from the pollen of Cassia siamea Lamk. were extracted and subjected to 80% ammonium sulphate precipitation. The total protein was then separated on a 12% SDS-Polyacrylamide gel which revealed 11 bands between molecular weight range of 45 kDa and 181kDa with another band having molecular weight less than 29kDa. Each band was recovered from the gel by electroelution and sent for skin tests. CS4 (97kDa). CS7 (63kDa), CS9 (53 kDa) and CS10 (45kDa) were the major allergenic components while CS2 (148kDa), CS3 (116kDa), CS6 (75kDa) and CS8 (60kDa) also induced reactivity in some patients. Immunodiffusion analysis with pooled sera of patients sensitive to the total extract confirmed the allergenicity of the bands.

Purification and characterization of isoforms of β-galactosidases in mung bean seedlings

Five isoforms of β-galactosidase (EC 3.2.1.23), designated as β-galactosidases I–V, were isolated from five-day-old mung bean ( Vigna radiata) seedlings. β-Galactosidases II and III were purified to electrophoretic homogeneity by a procedure involving acid precipitation, ammonium sulfate fractionation, chromatography on diethylaminoethyl-cellulose (DEAE-Cellulose) and con A-Sepharose, and chromatofocusing. β-Galactosidases I, II and III have the same molecular mass of 87 kDa, comprising two non-identical subunits with molecular masses of 38 and 48 kDa, while β-galactosidases IV and V have molecular masses of 45 and 73 kDa, respectively. All the enzymes were active against p-nitrophenyl-β- d-galactoside, and to a lesser extent, p-nitrophenyl-α- l-arabinoside and p-nitrophenyl-β- d-fucoside. The enzymes were inhibited by d-galactono-1,4-lactone, d-galactose, Hg 2+, Ag + and sodium dodecyl sulfate (SDS). β-Galactosidases I, II and III were shown to be competitively inhibited by either d-galactono-1, 4-lactone or d-galactose. Isoforms I, II and III have a common optimal pH of 3.6, while isoforms IV and V have pH optima at 3.8 and 4.0, respectively. Isoelectric points of isoforms I, II and III were 7.7, 7.5 and 7.3, respectively. Double immunodiffusion analysis indicated that β-galactosidases I, II, III and V are immunologically similar to each other, while β-galactosidase IV shares partially identical antigenic determinants with the other four isoforms. The purified β-galactosidases II and III were capable of releasing d-galactose residue from the hemicellulose fraction isolated from mung bean seeds.

Absence or reduction of carbonic anhydrase II in the red cells of the beluga whale and llama: implications for adaptation to hypoxia.

Carbonic anhydrase (CA) expression was examined in the red cells of two mammals that have adapted to low oxygen stress: the llama, which has adapted to high altitudes, and the beluga (or white) whale, which routinely dives for extended periods. Immunodiffusion analyses of their Hb-free hemolysates and partial amino acid sequencing of their HPLC-separated nonheme proteins indicate that the low-activity CA I isozyme is the major nonheme protein in erythrocytes of both the beluga whale and the llama. The high-activity CA II isozyme was not detected in the whale red cells but was present at low levels in erythrocytes of the llama. These results suggest that the absence or decrease in the expression of the high-activity CA II isozyme may be advantageous under hypoxic conditions.

Identification and partial characterization of the allergenic proteins of Ricinus communis L. pollen - a new approach

The pollen of Ricinus communis L., a potentially allergenic plant, was extracted to identify the allergenic determinants responsible for causing respiratory disorders. The soluble proteins were extracted and subjected to ammonium sulphate precipitation at 80% saturation and the total protein separated on 12% SDS-Polyacrylamide gel. In order to avoid the time consuming and expensive biochemical methods of column chromatography, each band was directly recovered from the gel by electroelution and the allergenic proteins identified directly by skin tests, without the necessity of Phadezym RAST or ELISA inhibition by reaction with serum IgE, the general procedure to identify the allergens. The fourth and the fifth band in the protein profile of R. communis pollen, RC4 (77 kD) and RC5 (66 kD) were the two major allergenic components. RC3 (91 kD) also induced a considerable amount of reactivity in sensitive patients. Contrary to the earlier reports of protein bands of R. communis ranging from 14 kD to 70 kD, 4 bands above 70 kD i.e. RC1 (123 kD), RC2 (97 kD), RC3 (91 kD) and RC4 (77 kD) are reported here for the first time. Immunodiffusion analysis with pooled sera of patients sensitive to the total extract also revealed similar results.

Identification of glutathione S-transferase p-1 as the class pi form dominantly expressed in mouse hepatic adenomas.

To clarify which of the two genes for pi class glutathione S‐transferases (GSTs) (p‐1 and p‐2) is dominantly expressed in mouse hepatic adenomas, the relative mRNA levels were examined by means of the reverse transcription‐polymerase chain reaction (RT‐PCR). Hepatic adenomas were induced in male and female B6C3F1 mice by diethylnitrosamine treatment. Northern blot analysis revealed that pi class mRNA levels were decreased in adenomas of male mice, but increased in those of females, with reference to the respective surrounding non‐adenoma tissues. In contrast to the marked sex difference in surrounding tissues, pi class GST mRNA levels in adenomas were almost the same in both males and females. To evaluate p‐1 and p‐2 mRNA levels separately, the products of RT‐PCR employing primers common for both cDNAs were digested with the endonuclease BanI (specific for p‐2) and then resolved by electrophoresis. The p‐1 mRNA was thus found to be dominant in adenomas of both female and male mice. The p‐2 mRNA levels were increased in the lesions as compared with those in the surrounding non‐adenoma tissues. Recombinant p‐1 and p‐2 proteins were expressed in Escherichia coli. Unlike p‐1, the p‐2 protein did not show any significant activity towards 1‐chloro‐2,4‐dinitrobenzene and did not bind to S‐hexylglutathione‐Sepharose despite immunological cross‐reactivity. The dominant pi class form in adenomas could also be identified as p‐1 by its binding to S‐hexylglutathione‐Sepharose. Single radial immunodiffusion analyses confirmed that the p‐1 protein levels were in line with the mRNA findings, i.e., 1.9±0.3 mg/g adenoma as compared to 6.5±1.2 mg/g non‐adenoma tissue for males and 2.2±0.6 mg/g as compared to 0.7±0.2 mg/g for females. The results thus indicated that the change of pi class forms in adenomas is caused mainly by alteration in the p‐1 level and the contribution of p‐2 is minimal.

Serological Differences among Photobacterium damsela subsp. piscicida Isolates.

Serological differences ofPhotobacterium damsela subsp. piscicida (=Pasteurella piscicida) isolates were determined using yellowtail (Seriola quinqueradiata) and rabbit antisera. In Oita, Japan, from June to October, 1995, Ph. damsela piscicidawas isolated from net-pen cultured diseased yellowtail and sera were collected from fish cultured in the same net-pen. Antisera against formalin-killed cells of three representative isolates were raised in yellowtail and rabbits. The yellowtail antisera were absorbed with homologous or heterologous isolates and used for the agglutinating antibody absorption test. Crude lipopolysaccharides were extracted from the isolates and analyzed by immunodiffusion with rabbit antisera. As a result, three types of antigenic differences were revealed among the isolates by the agglutinating tests with the sera of naturally infected fish and by the absorption tests using yellowtail antisera. Some common and distinctive antigens of the isolates were found by immunodiffusion analysis with rabbit antisera.