Formalin-killed Cells Research Articles

Immunogenicity of Pressure Inactivated Edwardsiella tarda Bacterin to Anguilla japonica (Japanese Eel)

Japanese eel Anguilla japonica were immunized with inactivated Edwardsiella tarda bacterin preparations (formalin-killed cells, FKC (0.4%), formalin with heat-killed cells, FHKC (0.1% and 70 degreesC for 10 min), heat-killed cells, HKC (70 degrees C for 15 min), potassium chloride-killed cells, KKC (0.6%), tannic acid-killed cells, TKC (0.9%), citric acid-killed cells, CAKC (0.9%), pressure-killed cells, PKC (600 psi for 5 min) and electric current-killed cells, ECKC (100 mA at 12 v DC for 5 sec) via intraperitoneal injection in order to develop adequate inactivating method. Immune parameters in the immunized eel were measured to compare responses to different bacterins. Generally, eel rose agglutinating antibody titer in the serum within 2 week and the maximum titer occurred at 6 weeks post immunization. Elevated and significantly higher titer was produced with the PKC of E. tarda than other bacterin preparations. An Enzyme Linked Immunosorbent Assay (ELISA), to determine specific anti-E. tarda antibody in the serum, also showed significantly higher antibody titer with PKC than the other antigen preparations. Bacteriostatic assay with serum and live E. tarda indicated significantly higher activity in the PKC-immunized fish. Immunization with PKC also showed the increased level ofphagocytosis. PKC-inactivated vaccine at an immunization dose of 10(6) cells/fish induced high protection against experimental infection. Coincident with higher immune parameters and protection in the fish immunized with the PKC bacterin strongly suggested that pressure-killing is an effective inactivating method to develop an effective vaccine against edwardsiellosis.

Effect of Lipopolysaccharide (LPS) and Outer Membrane Protein (OMP) Vaccines on Protection of Grass Carp (Ctenopharyngodon idella) against Aeromonas hydrophila

The gram-negative bacterium, Aeromonas hydrophila, causes high mortality and economic losses to the aquaculture industry. We investigated whether lipopolysaccharide (LPS) or outer membrane proteins (OMP) from A. hydrophila can enhance specific and/or non-specific immunity in grass carp (Ctenopharyngodon idella). Fish were injected intraperitoneally with LPS, OMP, or formalin-killed cells (FKC) from A. hydrophila. The control group was injected with phosphate buffered saline (PBS). All three antigens elicited strong immune responses. Respiratory burst and phagocytic activities in head kidney leukocytes and serum lysozyme activity peaked on day 21 after vaccination. Heavy chain gene transcription of immunoglobulin M and Z in the head kidney in vaccinated fish peaked on day 28. Relative percent survival was 83.3%, 72.2%, and 55.6% in the LPS, OMP, and FKC groups, respectively, but only 10% in control fish. Results suggest that LPS and OMP isolated from A. hydrophila can enhance specific immunity, non-specific immunity, and protection against A. hydrophila in fish. Thus, LPS and OMP could be important antigens for development of vaccines to control diseases caused by A. hydrophila in grass carp and other aquatic animals.

THE EFFECT OF VITAMIN C AND AEROMONAS VACCINE ON THE IMMUNE RESPONSE AND DISEASE RESISTANCE OF GROUPER (Epinephelus fuscoguttatus)

We evaluated the effectiveness of vitamin C and Aeromonas salmonicida vaccine in grouper (Epinephelus fuscoguttatus) for increasing immune responses and protection against A .salmonicida. The vitamin C used was polyethoxylated ascorbic and tocopherol. The vaccine was prepared from formalin-killed cells and concentrated extracellular products of a single isolate A. salmonicida. Bath immersion vitamin C and vaccine trials were conducted for 60 min. Fish used had a mean weight 25 g. Control groupers were injected with tryptic soy broth. The results showed that vitamin C enhanced phagocytic activity in head kidney leucocytes of grouper 7, 14, 28 and 36 days after treatments. A significant different of the antibody titre was found between control fish and the treated fish at 42 days after treatments. In addition, at day 42, Relative Percent Survival (RPS) for control group was 53.3 %, vitamin C-treated group was 80.0 % and vaccinated group was 90.0 %. The results of this study suggest that bath immersion of vitamin C provided an increasing of phagocytic activity (non-specific immune responses), titre antibody (specific immune responses) and protection against A. salmonicida infection in grouper. A. salmonicida vaccine also en-hanced titre antibody and protection against A. salmonicida infection in grouper.

Production of recombinant ghost bacterial vaccine against streptococcal disease of olive flounder

Abstract The simple fed-batch fermentation was carried out to produce E. coli XL1-Blue/pHCE-InaN-GAPDH Ghost 37 SDM as a ghost bacterial vaccine (GBV). The fermentation was carried out in four phases of batch fermentation (phase 1), fed-batch fermentation with intermittent feeding strategy (phase 2), thermal induction by temperature increase to 42 °C for the expression of lysis E gene (GBV formation, phase 3) and high temperature holding phase to increase the efficiency of GBV formation (phase 4). After the high temperature holding phase at 47 °C, efficiency of the GBV formation reached 99.7% with the culture OD600 of 57.9. The maximum GBV of 22 g dcw/l was obtained. The protective efficacy of GBV was determined by a challenge test to immunized olive flounder using live Streptococcus iniae. In 14 days of challenge test, the positive and E. coli strain control groups showed 100% cumulative mortalities. Test groups immunized by formalin killed cell (FKC) vaccine, GBV with 42 °C and 47 °C heat shock showed 66%, 54% and 54% of cumulative mortalities, respectively. These results suggest that GBV showed the effectiveness for the protection from the streptococcal infection and had higher potential to induce protective antibodies than FKC vaccine.

Evaluation of Optimal Culture Conditions for Recombinant Ghost Bacteria Vaccine Production with the Antigen of Streptococcus iniae GAPDH

For the production of ghost bacteria vaccine to prevent the streptococcal disease in aquaculture fish species, a double cassettes vector was constructed and cloned in Escherichia coli DH5alpha. Ghost bacteria vaccine production from Escherichia coli DH5alpha/pHCE-InaN-GAPDH-Ghost 37 SDM (SIG) was maximized at a glucose concentration of 1 g/l, agitation of 300 rpm, and aeration of 1 vvm. The maximal efficiency of ghost bacteria formation was obtained at the mid-exponential phase (OD600=2.0) with the concentration of 0.77 g/l for SIG. The molecular mass of GAPDH was detected at 67 kDa with the insoluble fraction, by SDS-PAGE and Western blot. The protective efficacy of ghost bacteria vaccine was evaluated by challenge test using olive flounder. The cumulative mortalities of the positive control, formalin-killed cell (FKC) vaccine, and SIG vaccine immunized groups were 91% , 74% , and 57% , respectively. These results suggest that SIG vaccine showed efficacy as a vaccine and had a higher potential to induce protective antibodies than did FKC vaccine.

BCG vaccine confers adaptive immunity against Mycobacterium sp. infection in fish

Mycobacteriosis, caused by Mycobacterium sp., results in severe loss of fish production in Japan's aquaculture industry. In this study, the effects of two vaccine candidates, Bacillus Calmette and Guèrin (BCG, an attenuated strain of Mycobacterium bovis) and formalin-killed cells of Mycobacterium sp. were evaluated in Japanese flounder, Paralichthys olivaceus. In the immediate response and tuberculin response, BCG-vaccinated fish showed higher gene expression levels of inflammatory cytokines such as IL-1β, IL-6, IFN-γ and TNFα. Furthermore, BCG vaccination conferred protective efficacy against Mycobacterium sp. infection in Japanese flounder. Transcriptome analysis using a Japanese flounder cDNA microarray revealed that BCG vaccination induced not only adaptive immunity against Mycobacterium sp. antigen but also the expression of non-specific bactericidal proteins such as lysozyme. These data suggest that BCG confers immunity to Mycobacterium sp. infection and is a potent vaccine candidate for fish mycobacteriosis.

Induction of Myocarditis Lesions in Lewis Rats by Formalin-Killed Cells of Group a Streptococcus

The feasibility of inducing rheumatic myocarditis lesions in Lewis rats by immunization with formalin-killed group A Streptococcus was evaluated. The rats were divided into three groups. Group A was immunized by injecting formalin-killed group A Streptococcus suspended in complete Freund's adjuvant (CFA) into the hind-foot pads and repeat immunization was given 1 week later by subcutaneous injection into the belly. The rats were then sacrificed 3 weeks after the initial immunization. In group B, the rats received the same initial immunization as group A, but the repeat immunization was carried out at 1, 2 and 3 weeks after the initial immunization and the rats were sacrificed 6 weeks after the initial immunization. Group C was a control group with the rats injected with saline/CFA and sacrificed on the same schedule as group A. Heart pathology sections showed that myocarditis lesions, focal inflammatory cell infiltration in interstitial near small vessels and valvulitis were induced in Lewis rats following immunization with formalin-killed group A Streptococcus.

Identification of novel genes in Japanese flounder ( Paralichthys olivaceus) head kidney up-regulated after vaccination with Streptococcus iniae formalin-killed cells

Streptococcosis caused by Streptococcus iniae, a Gram-positive bacterium, is one of the diseases often found in the culture of Japanese flounder ( Paralichthys olivaceus). Inactivated bacterial-whole cells of S. iniae have been used as a prophylactic treatment to prevent the disease. To understand the molecular mechanisms involved in activation of the fish immune system upon formalin-killed cell (FKC) treatment, we have investigated the gene transcription profile of Japanese flounder head kidney cells after intraperitoneal injection of S. iniae FKC. Three known genes (C3 complement, calmodulin, microtubule aggregate protein) and 6 unknown genes were specifically up-regulated more than 10-fold by S. iniae FKC. RT-PCR analysis of differential expression of the 3 unknowns and interleukin-1β confirmed the result of the microarray analysis. From this study, we conclude that 6 up-regulated unknown clones represent novel genes that are involved against Gram-positive FKC immune response.

Vaccination of rainbow trout against Streptococcus iniae infection: comparison of different routes of administration and different vaccines

Antibody production and clinical efficacy (relative percent survival RPS) were measured in 40±5g rainbow trout after immunization with two types of Streptococcus iniae vaccines consisting of formalin killed cells (FKC) and FKC enriched with the bacterial extracellular products (ECP) administered by intraperitoneal (i.p), immersion and oral routes at 16±1°C for 18 weeks. No significant difference was found in antibody levels among the fish i.p immunized with FKC enriched with ECP plus Freunds' adjuvant (FA), FKC plus FA and FKC vaccines (P>0.05), whilst the antibody production was significantly higher in these three groups than fish immunized by immersion and oral routes of FKC and FKC enriched ECP (P 0.05). The RPSs ranging 82.6-100, 73.9-95 and 73.9-91.7% were obtained in the fish intraperitoneally immunized with FKC enriched ECP plus FA, FKC plus FA and FKC vaccines, respectively, compared to 0% survival for the control fish. Also, RPS in fish vaccinated by the immersion route was in the range 45.8-30.4% after 18 weeks post-vaccination. Efficacy of oral vaccination of fish with FKC plus ECP was in range of 8.7-29% and that of fish orally vaccinated with FKC resulted in 8.7-20.8% protection.

Stationary phase culture supernatant containing membrane vesicles induced immunity to rainbow trout Oncorhynchus mykiss fry syndrome

Flavobacterium psychrophilum is the causative agent of bacterial cold water disease (BCWD) and rainbow trout ( Oncorhynchus mykiss) fry syndrome (RTFS). Logarithmic phase formalin-killed cells (FKC) of F. psychrophilum induced immunity to BCWD in ayu ( Plecoglossus altivelis) by using an oral administration. In this study, we investigated the effective antigens of logarithmic phase cells in rainbow trout. Rainbow trout fry immunized with logarithmic phase FKC resulted in near complete protection, but the vaccine effect was low in fry immunized with stationary phase FKC. Scanning electron microscopy showed that logarithmic phase cells had many membrane vesicles (MVs) on the surface of F. psychrophilum cells. The MVs were released into medium at the stationary phase. MVs rich supernatant was collected from the stationary phase culture supernatant by using an ammonium precipitation method. Immunization with MVs rich supernatant combined with stationary phase FKC resulted in a relative percentage survival (RPS) of 94–100%, but immunization with MVs rich supernatant only resulted in no protection against F. psychrophilum infection. These data show that MVs have an adjuvant efficacy and suggest that combination of MVs and cells is necessary to obtain efficient protection.

Application of attenuated Lactococcus garvieae strain lacking a virulence-associated capsule on its cell surface as a live vaccine in yellowtail Seriola quinqueradiata Temminck and Schlegel

Summary An attenuated Lactococcus garvieae strain lacking a virulence-associated capsule on its cell surface was evaluated for application as a live vaccine. The attenuated strain (MS93003A) was obtained from the parent strain (MS93003V), which produced a well-developed capsule, by culturing on an agar medium supplemented with 2,3,5-triphenyltetrazolium chloride. When live cells of L. garvieae (MS93003A) or formalin-killed cells (MS93003A) were used as an injectable vaccine, protection against virulent L. garvieae (MS93003V) was conferred on Seriola quinqueradiata. The MS93003A cells did not recover their virulence even after in vivo passages in fish. MS93003A live cells also conferred long-lasting protective immunity to S. quinqueradiata against virulent L. garvieae infection.

Serum antibody response induced in mice after oral administration of three different antigens of enterotoxigenic Escherichia coli in enteric coated microparticles.

Gastric digestion of these antigens plays an important role, decreasing the ability to deliver antigens to the gut-associated lymphoid tissue. To overcome this obstacle, microencapsulated antigens from enterotoxigenic Escherichia coli (ETEC) were evaluated for oral immunization of mice. Four groups of 10 each received 3 series of 3 doses each of (1) B subunit of cholera toxin (CTB), similar to heat-labile toxin of ETEC, (2) formalin-killed whole cell ETEC H10407 (FK-ETEC), (3) crude preparation of colonization factor antigen I (CFA/I), or (4) placebo. Serum antibody was measured on day 0 and 60 by ELISA. In group 1 a CTB antibody response was induced in all mice, 3 with 1:105 titer and 7 with 1:106. These antibodies neutralized cholera toxin-induced steriodogenesis of Y-1 adrenal cells. In group 2, 8 mice developed a whole H10407 bacteria antibody titer of 1:100, one 1:200 and one showed no immune response. In the same group, an anti-CFA/I response was observed in 6 mice and anti-LPS in 4 mice as determined by Western blot. All mice in group 3 showed > 1:104 anti-CFA/I antibody titer. Group 4 mice did not develop an immune response to any ETEC antigens. Microencapsulation appears to be a suitable approach for oral vaccination against ETEC and Vibrio cholerae.

Immune response of sea bass Dicentrarchus labrax to Tenacibaculum maritimum antigens

Seawater fishes are affected by a pathology commonly called ‘myxobacteriosis’, caused by Tenacibaculum maritimum (formerly Flexibacter maritimus). The disease is characterized by fin erosion and necrotic ulcers of skin and muscle, and by low but constant mortality in cultured marine fish; in Italy is one of the most important and widely spread diseases affecting sea bass Dicentrarchus labrax, gilthead seabream Sparus aurata, sharp-snouted bream Diplodus puntazzo, white bream Diplodus sargus, and six-tooted bream Dentex dentex. In order to obtain an effective vaccine against the disease, formalin killed cells (FKC), extracellular products (ECPs) and crude lipopolysaccharide (LPS) preparations were obtained from the T. maritimum strain SPVId and injected intraperi-toneally twice into the sea bass. The fish immune response to the preparations was studied: agglutinating antibody titer and in vitro phagocytosis were determined after the first and second injection in order to evaluate whether the preparations are immunogenic or not and if the booster effect took place. The results show that FKC and LPS preparations increased the antibody titer after the first injection when compared to the control sea bass. Moreover, all the preparations stimulated a secondary (booster) response. In vitro phagocytosis of the total blood was significantly higher for all the preparations when compared to the controls, but the crude LPS immunized sea bass showed the highest activity.

Antigenicity of Streptococcus agalactiae extracellular products and vaccine efficacy

Streptococcus agalactiae is a major bacterial pathogen that is the cause of serious economic losses in many species of freshwater, marine and estuarine fish worldwide. A highly efficacious S. agalactiae vaccine was developed using extracellular products (ECP) and formalin-killed whole cells of S. agalactiae. The vaccine efficacy following storage of S. agalactiae ECP and formalin-killed S. agalactiae cells at 4 degrees C for 1 year was determined. The stored ECP containing S. agalactiae formalin-killed cells failed to prevent morbidity and mortality among the vaccinated fish, and the relative percentage survival was 29. Serum antibody responses of the stored ECP and freshly prepared ECP against soluble whole cell extract of S. agalactiae indicated that significantly less antibody was produced in fish immunized with stored ECP and S. agalactiae cells than in those fish immunized with freshly prepared ECP and S. agalactiae cells at day 31 post-vaccination. Silver staining of sodium dodecyl sulphate-polyacrylamide gels and immunostaining of Western blots with tilapia antiserum to S. agalactiae revealed that predominant 54 and 55 kDa bands were present in the freshly prepared ECP fraction. The 55 kDa band was absent from the stored ECP and new bands below 54 kDa appeared on the Western blot. The results of this study on S. agalactiae ECP provide evidence for a correlation between protection and antibody production to ECP and for the importance of the 55 kDa ECP antigen for vaccine efficacy.

Development of an ELISA to measure the humoral immune response of hybrid striped bass Morone chrysopsxM. saxatilis to Streptococcus iniae

A previously developed monoclonal antibody (mAb) specific for the heavy chain of hybrid striped bass (HSB) Morone chrysops×M. saxatilis immunoglobulin was used in an assay to detect the humoral response to antigens of Streptococcus iniae. In order to validate this assay, an anti-S. iniae antibody was produced in HSB by immunization with formalin-killed cells of S. iniae mixed with Freund's complete adjuvant (FCA). After boosting with cells mixed with Freund's incomplete adjuvant (FIA), fish were challenged with live S. iniae by i.p. injection. This resulted in mortalities of 100%, 13% and 7% for non-immunized, immunized and non-challenged fish respectively. Live S. iniae were recovered only from moribund non-immunized challenged fish, indicating that the immunization conferred protection against S. iniae. Sera were taken at 28 and 42 days post challenge and anti-S. iniae antibody was measured by both agglutination and indirect ELISA. Titres of anti-S. iniae antibody increased only in the immunized group as measured by agglutination and ELISA. Serum complement measured in the final bleeding was significantly higher in the immunized group. Western blotting using immune HSB serum indicated that the predominant antigen in this case was the high molecular weight polysaccharide of S. iniae.

Efficacy of Streptococcus agalactiae (group B) vaccine in tilapia ( Oreochromis niloticus) by intraperitoneal and bath immersion administration

We evaluated the effectiveness of a Streptococcus agalactiae vaccine in tilapia ( Oreochromis niloticus) for prevention of streptococcal disease. The vaccine was prepared from formalin-killed cells and concentrated extracellular products (greater than 3 kDa) of a single isolate of S. agalactiae (ARS-KU-MU-11B). Intraperitoneal (IP) and bath immersion (BI) vaccine trials were conducted at two temperatures, 32 and 26 °C, and mean fish weights, 5 and 30 g. Control tilapia were injected with tryptic soy broth. Thirty gram tilapia vaccinated and challenged by IP injection with 1.5×10 4 colony-forming units (CFU)/fish of Streptococcus agalactiae at 30 days post-immunization had a relative percent survival (RPS) of 80. Smaller tilapia vaccinated and challenged under similar conditions had an RPS of 25. An RPS of zero was noted in 30 g fish IP vaccinated with Streptococcus iniae and IP challenged with S. agalactiae. The 5 and 30 g tilapia bath immunized with S. agalactiae and IP challenged with 3.6×10 5 and 1.7×10 6 CFU/fish of S. agalactiae had RPS values of 34. Intraperitoneal administration of the vaccine provided efficacious protection only in the 30 g tilapia regardless of whether the fish were immunized and challenged at 26 or 32 °C. Bath immunization of both 5 and 30 g tilapia resulted in RPS values that were two times lower than those achieved with IP vaccination. The results of this study suggest that there is a lack of cross-protection of S. iniae bacterins against S. agalactiae challenge. Protection against S. agalactiae infection is, however, provided through vaccination with a S. agalactiae modified bacterin vaccine.

The rise and fall of Salmonella Enteritidis in the UK.

After rising in the early 1980s, the number of recorded human cases of Salmonella enterica subsp. enterica in the UK has fallen in the last 5 years, with a particular decline in cases of infection with serovar Enteritidis. This decline has been concomitant with the introduction of vaccination of egg-laying hens against serovar Enteritidis. It is likely that other factors such as improved biosecurity in egg-laying flocks, a build-up of immunity in other animals and the rise in the number of livestock infections with host-adapted serovars of Salmonella have also played a part in this decline. Although human Salmonella cases are currently at their lowest level since 1987, it is important to remember that the reasons for the dominance of Enteritidis in human infection are poorly understood and it is possible that other serovars could share similar properties and the eradication of Enteritidis may leave a niche for them to fill.

Efficacy of oral vaccine against bacterial coldwater disease in ayu Plecoglossus altivelis.

The development of a practical vaccination method against bacterial coldwater disease (BCWD) in ayu Plecoglossus altivelis and the efficacy of oral administration of formalin-killed cells (FKCs) of Flavobacterium psychrophilum was investigated. The FKC was administrated at a dose of 0.1-0.2 g kg(-1) body weight to juvenile ayu (0.5 g body weight) every day for 2 wk or on 5 days over 2 wk. Experimental immersion challenge at 3 and 7 wk after vaccination showed significantly higher survival rates than the controls. The results show the effectiveness of oral vaccination against BCWD in ayu.

Simple and rapid detection of serum antibody to periodontopathic bacteria by dot blotting

The aim of this study was to detect the specific immunoglobulin G antibodies against periodontopathic bacteria by dot blotting. In the procedure used, bacterial preparations were blotted on a nitrocellulose membrane. After blocking the nonspecific binding sites, the diluted serum was blotted onto the preparations. The membrane was immersed in secondary antibodies and then in substrate buffer. The colored blots were then evaluated. To test the reliability of this procedure, 20 serum samples were examined for antibody: ten for anti-Porphyromonas gingivalis antibody, and the other ten for anti-Actinobacillus actinomycetemcomitans antibody. Five samples out of each set of ten had previously been confirmed as having high enzyme-linked immunosorbant assay (ELISA) titers to the antigen, while the other five had been confirmed as having average titer levels. Both whole-cell sonic extracts and fimbriae of P. gingivalis were used as antigens in the dot blotting, in order to compare their use as antigens in assays of the patients' sera. ELISA was also used to measure anti-P. gingivalis antibody titers. For the measurement of IgG antibodies against A. actinomycetemcomitans, formalin-killed whole cells were used. Fifty serum samples were examined for IgG antibodies against A. actinomycetemcomitans by dot blotting and ELISA. With both antigens, after 4 h, coloration of blots was more clearly visible for the high-titer sera than for the average-titer sera. The intensity of coloration of the blots for P. gingivalis and A. actinomycetemcomitans showed correlation with the ELISA titers. A particularly significant correlation was shown when P. gingivalis fimbriae were used as antigen. These results suggest that this dot blot method is a simple and rapid means of detection of serum antibodies, and that it shows promise as a chair-side assay method.

A Vaccination Trial Using Live Cells of Edwardsiella tarda in Tilapia.

In this study, we conducted a vaccination trial using live attenuated cells of Edwardsiella tarda in tilapia Oreochromis niloticus. A mutant strain (SPM31) with lowered siderophore production was constructed from a wild-type E. tarda FPC498 using transposon Tn5. FPC498 had a calculated LD50 of 1.8 ×107CFU/100 g body weight, while SPM31 had a 2.9×108CFU/100 g body weight. Formalin-killed cells or live bacteria of SPM31 were intraperitoneally injected to tilapia at a dose of 0.1 mg/100 g (about one third of the LD50 value) for vaccination. Both the formalin-killed and the live cells activated the antibody production. The vaccinated fish were challenged with the parent strain FPC498 at the 2nd, 3rd and 4th weeks of postvaccination. There were no deaths due to the challenge with the bacterium in the fish immunized with the live cells. On the contrary, the vaccination with formalin-killed cells resulted in 80100% mortality. These results indicate that the live cells with lowered siderophore-production are capable of conferring protective immunity against edwardsiellosis.