Serum Levels Of Specific Antibodies Research Articles

Insight into the allergenicity of digestion-resistant epitopes in Antarctic krill (Euphausia superba) tropomyosin

The allergenic potential of four digestion-resistant linear epitopes (EADRKYDE, ERAEERAEAG, RSVQKLQKEVDR, and VNEKEKYKGI) in Antarctic krill tropomyosin was investigated through in vivo and in vitro experiments. Mice induced by the four epitopes exhibited obvious clinical allergic symptoms, with significant increases in serum specific antibody levels and blood lymphocytes, neutrophils, and eosinophils (p<0.05). Additionally, mast cells exhibited pronounced degranulation, as well as increased release of histamine and mMCP-1, and enhanced vascular permeability. Mice challenged with the four epitopes further displayed severe pathological damage. In contrast to the antagonistic effect of interleukin-4 and interferon-γ in the plasma of mice in previous studies, stimulation of the epitopes resulted in immune system dysregulation in mice, with significant increases in the levels of interleukin-4 and interferon-γ to 110.03 pg/mL and 103.59 pg/mL, respectively. The enhanced binding ability of the four epitopes to IgE was also indicated in vitro. The four digestion-resistant epitopes are the major factors to the allergenicity of tropomyosin and have strong sensitizing abilities. This study provides a theoretical basis for using site directed mutagenesis to reduce the allergenicity of tropomyosin in Antarctic krill.

Comparison of the efficacy of Aeromonas veronii ΔhisJ vaccine in Carassius auratus via different immunization routes.

Aeromonas veronii is a significant pathogen to various aquatic life. Infections in fish can lead to high mortality rates, causing substantial economic losses in aquaculture. Vaccination is proposed as a substitute for antibiotics in aquaculture to decrease disease-related mortality and morbidity. Our study previously constructed a hisJ-deleted strain of A. veronii, which provided protective effect to Loach. To further assess the vaccine's applicability, this study evaluated its genetic stability and safety, and the immune protective effects in Carassius auratus through four distinct administration routes: intraperitoneal injection, intramuscular injection, oral administration, and immersion, to determine the efficacy of these administration routes. The results showed that the vaccine remained genetically stable after 45 generations. Immunization via these administration routes was safe for Carassius auratus, with intraperitoneal and intramuscular injections causing stronger adverse reactions. Immersion immunization resulted in mild adverse reactions, and no significant adverse reactions were observed following oral immunization. Immunizing Carassius auratus at safe concentrations via these routes enhanced the phagocytic activity in serum, increased the levels of non-specific immune-related enzymes (ACP, AKP, C3, C4, LZM, SOD, and IgM), and improved specific serum antibody levels. It also elevated levels of cytokines related to inflammatory responses (IL-1β, IL-10, TNF-α, TGF-β) in organ tissues (liver, spleen, kidney, mid-post intestine, and gills). The survival rates of Carassius auratus were measured after challenging with the virulent strain A. veronii TH0426, resulting in the relative survival rates of 64% for Intraperitoneal vaccine group, 56% for Intramuscular vaccine group, 52% for oral vaccine group, and 48% for immersion vaccine group. Analysis of bacterial load in the liver, spleen, and kidney post-challenge showed a decreasing trend in the control group, indicating that the vaccine strain ΔhisJ could gradually restrict the rapid proliferation of bacteria in these tissues, thereby providing a certain level of immune protection against A. veronii. In brief, the vaccine strain ΔhisJ can serve as a safe live attenuated vaccine for Carassius auratus, and this study lays the foundation for the development of live attenuated vaccines against Aeromonas veronii.

Evaluation of immunogenicity and protective efficacy of the outer membrane porin F (OprF) against Pseudomonas plecoglossicida in large yellow croaker (Larimichthys crocea)

Large yellow croaker (Larimichthys crocea) farming dominates the marine aquaculture industry in China. However, the epidemic outbreaks of visceral white nodules disease (VWND), caused by bacterial pathogen Pseudomonas plecoglossicida, have emerged as a significant concern within the large yellow croaker industry. Although vaccination is considered to be an effective method for preventing and controlling P. plecoglossicida infection, there is currently no commercially available vaccine targeting this bacterium. In the present study, the outer membrane porin F (OprF) of P. plecoglossicida was characterized and revealed a high sequence similarity with that of other Pseudomonas species. The recombinant OprF protein (rOprF) produced in Escherichia coli was then evaluated for its immunogenicity and protective role against P. plecoglossicida in large yellow croaker. The rOprF was identified to have immunogenicity by Western blot using large yellow croaker anti-P. plecoglossicida sera. Additionally, the indirect immunofluorescence assay (IIFA) provided evidence indicating the surface exposure of OprF in P. plecoglossicida. Fish vaccinated twice via intraperitoneal (IP) injection with the purified rOprF combined with commercial adjuvant ISA 763A VG exhibited a relative percent survival (RPS) of 70.60% after challenge with virulent P. plecoglossicida strain through immersion. The administration of rOprF resulted in a notable increase in specific serum antibody levels and serum lysozyme activity compared to the control groups. The immune-related genes in the spleen and head kidney of rOprF-vaccinated fish were remarkably upregulated compared with the PBS-vaccinated sham group after the P. plecoglossicida challenge. In summary, the findings of this study suggest that rOprF exhibits considerable potential in inducing a robust immune response, making it a viable candidate for vaccination against P. plecoglossicida infection in large yellow croaker.

Protective efficacy of recombinant proteins AMA1 and IMP1 in rabbits infected with Eimeria intestinalis

Eimeria intestinalis is one of the most pathogenic rabbit coccidia species causing severe intestinal damage and increased risk of secondary infection from opportunistic pathogens, which results in huge economic losses to the rabbit industry. Anticoccidial drugs are currently the main method to control coccidiosis; however, increasing resistance and drug residues have fueled research on anticoccidial vaccines. Apical membrane antigen 1 (AMA1) and immune mapped protein 1 (IMP1), as surface proteins, are associated with host invasion and might have the potential as candidate vaccine antigens. In the present study, recombinant IMP1 (rEiIMP1) and AMA1 (rEiAMA1) from E. intestinalis were expressed using Escherichia coli BL21. The immunoreactivity and immunoprotective effects of rEiIMP1 and rEiAMA1 were then analyzed. Fifty rabbits were grouped randomly (n = 10 per group): The unimmunized-unchallenged control group (sterilized phosphate-buffered saline (PBS)), the unimmunized-challenged control group (sterilized PBS), the vector protein-challenged control group (100 μg of pET-32a vector protein per rabbit), the rEiIMP1 immunized group (100 μg of rEiIMP1 per rabbit), and the rEiAMA1 immunized group (100 μg of rEiAMA1 per rabbit). After two immunizations, the rabbits were challenged with homologous oocysts (except for the unimmunized-unchallenged group). Serum specific antibody levels were assessed weekly throughout the experimental period; and the levels of different cytokines in the serum before the challenge were detected. The clinical symptoms, oocysts output, weight gain, feed conversion ratio (FCR), and lesion scores were recorded after experimental infection, and the anticoccidial indexes (ACIs) were calculated. The results showed that both rEiIMP1 and rEiAMA1 had good immunoreactivity. Rabbits immunized with rEiIMP1 and rEiAMA1 displayed 66.74 % and 63.14 % oocyst reduction, respective land 81.79 % and 78.87 % body weight gain, respectively. The rEiIMP1 and rEiAMA1 groups had lower FCRs (3.77:1 and 4.06:1, respectively) and lesion scores (P = 0.00). The rEiIMP1 and rEiAMA1 showed moderate effects, with an ACI of 152.09 and 147.17, respectively. Immunization induced high levels of anti-rEiIMP1 and -rEiAMA1 antibodies. Rabbits immunized with rEiIMP1 and rEiAMA1 displayed significantly increased interleukin (IL)− 2 (P = 0.00), interferon gamma (IFN)− γ (P = 0.00), and IL− 4 (P = 0.00) levels. Therefore, this study provided potential candidate vaccine antigens for E. intestinalis.

Type I interferon induced by polyinosinic-polycytidylic acid does not contribute to the efficacy of a formalin-killed cell vaccine against Edwardsiella piscicida in the Japanese flounder (Paralichthys olivaceus)

Polyinosinic-polycytidylic acid (poly I:C) is a type of pathogen-associated molecular pattern that can strongly induce the expression of type I interferon (I-IFN). Our previous study has demonstrated that the combination of poly I:C with a recombinant protein antigen not only stimulated the expression of I-IFN but also conferred protection against Edwardsiella piscicida in the Japanese flounder (Paralichthys olivaceus). In this study, our aim was to develop a better immunogenic and protective fish vaccine, for which we intraperitoneally coinjected P. olivaceus with poly I:C and formalin-killed cells (FKCs) of E. piscicida and compared the efficiency of protection against E. piscicida infection with that of FKC vaccine alone. Results showed that the expression levels of I-IFN, IFN-γ, interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and the interferon-stimulated genes (ISGs) ISG15 and Mx were significantly increased in the spleen of fish inoculated with poly I:C + FKC. The results of ELISA showed that the levels of specific serum antibodies in the FKC and FKC + poly I:C groups were gradually increased until 28 days postvaccination and were significantly higher than those in the PBS and poly I:C groups. At 3 weeks after vaccination in the challenge test, the respective cumulative mortality rates of fish in the PBS, FKC, poly I:C, and poly I:C + FKC groups were 46.7%, 20.0%, 33.3%, and 13.3% under low-concentration challenge and 93.3%, 46.7%, 78.6%, and 53.3% under high-concentration challenge. This study showed that poly I:C may not provide an effective adjuvant effect with FKC vaccine for intracellular bacterial infections.

Field Efficacy of a Feed-Based Inactivated Vaccine against Vibriosis in Cage-Cultured Asian Seabass, Lates calcarifer, in Malaysia.

Vibrio spp. are important aquaculture pathogens that cause vibriosis, affecting large numbers of marine fish species. This study determines the field efficacy of a feed-based inactivated vaccine against vibriosis in cage-cultured Asian seabass. A total of 4800 Asian seabass, kept in a field environment, were separated equally into two groups (vaccinated and non-vaccinated) in duplicate. Fish of Group 1 were orally administered the feed-based vaccine on weeks 0 (prime vaccination), 2 (booster), and 6 (second booster) at 4% body weight, while the non-vaccinated fish of Group 2 were fed with a commercial formulated pellet without the vaccine. Fish gut, mucus, and serum were collected, the length and weight of the fish were noted, while the mortality was recorded at 2-week intervals for a period of 16 weeks. The non-specific lysozyme activities were significantly (p &lt; 0.05) higher in the fish of Group 1 than the non-vaccinated fish of Group 2. Similarly, the specific IgM antibody levels in serum and mucus were significantly (p &lt; 0.05) higher in Group 1 than in Group 2, as seen in the second week, with the highest level 8 weeks after primary immunization. At week 16, the growth performance was significantly (p &lt; 0.05) better in Group 1 and showed lower bacterial isolation in the gut than Group 2. Despite the statistical insignificance (p &gt; 0.05), the survival rate was slightly higher in Group 1 (71.3%) than Group 2 (67.7%). This study revealed that feed-based vaccination improves growth performance, stimulates innate and adaptive immune responses, and increases protection of cultured Asian seabass, L. calcarifer, against vibriosis.

Immune responses and protective immunity in Pangasius pangasius (Hamilton, 1822) as induced by outer membrane proteins of Edwardsiellatarda and aluminium hydroxide adjuvant complex.

Edwardsiella tarda is considered one of the important bacterial fish pathogens. The outer membrane proteins (OMPs) of E. tarda are structurally and functionally conserved, and immunogenic. This study assessed the effects of the OMPs of E. tarda CGH9 as a vaccine without aluminium hydroxide [AH] (T1) and with AH adjuvant (T2) on the respiratory burst (ROB) activity, lymphocyte proliferation of head kidney (HK) leukocytes, and serum antibody production in pangas catfish Pangasius pangasius. The ROB activity and lymphocyte proliferation of HK leukocytes increased in both vaccinated groups compared to the control. Nonetheless, the T2 group showed a gradual increase in ROB activity and lymphocyte proliferation of HK leukocytes up to 3-weeks post-vaccination (wpv). The serum antibody production in the T1 group decreased initially for up to 2-wpv and increased from 3-wpv; whereas, in the T2 group, the serum-specific antibody levels were significantly high from 1-wpv compared to control. Simultaneously, the protective efficacy in terms of relative percentage survival in the T2 group after injecting with a lethal dose of E. tarda CGH9 was high (89.00±15.56) compared to the T1 group (78.00±0.00). Furthermore, the catfish administered with a booster dose of E. tarda OMPs with or without AH adjuvant showed no additional increase in immune response or protective immunity. These results suggested that E. tarda OMPs and AH adjuvant complex has a higher potential to induce protective immunity, which may be a good choice as a vaccine to combat E. tarda infection in catfish.

Immune Evaluation of Recombinant Lactobacillus plantarum With Surface Display of HA1-DCpep in Mice.

Avian influenza viruses can be efficiently transmitted through mucous membranes, and conventional vaccines are not effective in protecting against mucosal infection by influenza viruses. To induce multiple immune responses in an organism, we constructed a recombinant Lactobacillus plantarum expressing the influenza virus antigen HA1 with the adjuvant dendritic cell-targeting peptide (DCpep). The recombinant L. plantarum strains NC8Δ-pWCF-HA1 and NC8Δ-pWCF-HA1-DCpep were used to immunize mice via oral administration, and the humoral, cellular and mucosal immune responses were evaluated. In addition, the serum levels of specific antibodies and hemagglutination inhibition (HI) levels were also measured. Our results showed that recombinant L. plantarum activated dendritic cells in Peyer’s patches (PPs), increased the numbers of CD4+IFN-γ+ and CD8+IFN-γ+ cells in the spleen and mesenteric lymph nodes (MLNs), and affected the ability of CD4+ and CD8+ cells to proliferate in the spleen and MLNs. Additionally, recombinant L. plantarum increased the number of B220+IgA+ cells in PPs and the level of IgA in the lungs and different intestinal segments. In addition, specific IgG, IgG1 and IgG2a antibodies were induced at high levels in the mice serum, specific IgA antibodies were induced at high levels in the mice feces, and HI potency was significantly increased. Thus, the recombinant L. plantarum strains NC8Δ-pWCF-HA1 and NC8Δ-pWCF-HA1-DCpep have potential as vaccine candidates for avian influenza virus.

Immunoadjuvant activity of marine bacteria exopolysaccharides in normal and immunosuppressive conditions

Aim. To assess the mechanisms of immunoadjuvant activity of marine bacteria exopolysaccharides (PS) in normal and immunosuppressive conditions in vivo. Material and methods. Experimental studies were carried out on male ВALB/c mice, immunized with compositions of PS or aluminum hydroxide and ovalbumin (OVA). Immunosuppression was induced by triple intraperitoneal administration of dexamethasone (2 mg/kg). Serum levels of specific antibodies (IgG, IgG1, and IgG2a) and cytokines (IFN-γ, IL-2, IL-10) were determined. Results. The inclusion of PS to the immune compositions led to the formation of an enhanced specific IgG, IgG1, and IgG2a to OVA, comparable to the effect of aluminum hydroxide. The studied PS also contributed to an increase in the level of both pro- and anti-inflammatory cytokines. Conclusion. PS from marine bacteria act as adjuvants in normal and immunosuppressive conditions, stimulating a mixed Th1 (IgG2a, INF-γ) and Th2 (IgG1, IL-10) immune response to OVA.

Immune adjuvant effects of interferon-gamma (IFN-γ) of flounder (Paralichthys olivaceus) against Edwardsiella tarda

IFN-γ plays a key role in T-cell activation and the establishment of the adaptive immune response, which has a potential as a cytokine adjuvant in the context of vaccination. In this study, we evaluated the immune adjuvant effects of two forms of flounder (Paralichthys olivaceus) IFN-γ, including pcDNA3.1-IFN-γ (pcIFN-γ) and recombinant IFN-γ (rIFN-γ), and comparatively analyzed the immune responses of flounder to E. tarda subunit vaccine rOmpV. The results showed that vaccination with rOmpV plus pcIFN-γ or rIFN-γ produced a relative percent survival of 57% and 71%, respectively, which were significantly higher than that of the control groups, rOmpV plus pcN3 (36%) or rHis (40%). Compared with the two control groups, vaccination with rOmpV plus pcIFN-γ or rIFN-γ could induce significantly higher levels of specific serum antibodies and sIg + lymphocytes in peripheral blood, spleen and head kidney, and significantly higher upregulated expressions of CD4-1, CD8α, IgM, MHC Ⅰα, MHC Ⅱα, IL-1β and TNF-α were also detected in rOmpV plus pcIFN-γ or rIFN-γ vaccinated fish. In addition, compared with pcIFN-γ, rOmpV co-vaccination with rIFN-γ elicited higher levels of sIg + lymphocytes, specific serum antibodies and several immune-related genes expressions in vaccinated flounder. These results demonstrated that rOmpV co-vaccination with rIFN-γ or pcIFN-γ could both boost the immune responses and evoke highly protective effects against E. tarda, indicating that flounder IFN-γ is a promising adjuvant candidate for fish vaccination via an injection administering route.

Comparison of the roles of IgM in systemic and mucosal immunity via tissue distribution analysis in largemouth bass (Micropterus salmoides)

Immunoglobulin M (IgM), an important immune effector, plays a crucial role in protecting fish against various pathogens. Largemouth bass (Micropterus salmoides) is an economically important farmed fish, but the understanding of IgM characteristics and function in largemouth bass is still lacking. In the present study, the largemouth bass IgM heavy chain gene was first cloned. Sequence analysis showed that cDNA of the IgM heavy chain contains an open reading frame (ORF) of 1740 bp. The ORF of the largemouth bass IgM heavy chain encodes a polypeptide of 579 amino acids that consists of a signal peptide, a variable region (VH) and four constant regions (CH1, CH2, CH3 and CH4). Next, largemouth bass IgM was purified from serum, and polyclonal antibodies against IgM were developed using purified IgM as an antigen. Western blotting analysis showed that the polyclonal antibodies reacted with the heavy and light chains of largemouth bass IgM. Indirect immunofluorescence assay (IFA) analysis suggested that the polyclonal antibodies could also react with membrane-bound IgM (mIgM) on the surface of B cells. Flow cytometric analysis revealed that the percentages of mIgM+ B lymphocytes in the peripheral blood, spleen and head kidney were higher than those in the gill in largemouth bass. qRT-PCR analysis showed that the gene expression of the IgM heavy chain in the head kidney and spleen was significantly higher than that in the gill, intestine and skin. Moreover, indirect ELISA showed that the IgM contents in serum were higher than those in mucus and that specific serum antibody levels were significantly increased after Aeromonas hydrophila immunization. These results demonstrated that the largemouth bass IgM gene was first cloned and characterized in this study and that IgM may play a more important role in systemic immunity than in mucosal immunity.

Immune responses induced by inactivated vaccine against Aeromonas hydrophila in pacu, Piaractus mesopotamicus

Aeromonas hydrophila is responsible for outbreaks of a severe infectious disease in fish farms around the world and is one of the major causes of economic losses to the neotropical fish farmers. This study assessed the induction of immune responses and protection against A. hydrophila in pacu, Piaractus mesopotamicus, vaccinated through intraperitoneal and immersion route with inactivated virulent strain. Fish were randomly distributed in three vaccinated groups: intraperitoneal (i.p.) route; immersion; and immersion + booster; and control group (unvaccinated). All vaccination protocols used the concentration of 1.7 × 108 CFU mL−1 of inactivated A. hydrophila., and an oil adjuvant was used for vaccine prepararion for i.p. route vaccination. Blood and skin mucus from 9 fishes per treatment were collected at 14, 28, 42 and 84 days post-vaccination (DPV) for determination of lysozyme concentration in skin mucus, as well as antibodies anti-A. hydrophila in blood serum and skin mucus. Fish were challenged at 84 DPV with homologous and virulent strain of A. hydrophila for evaluation of resistance against bacterial infection. The results demonstrated that vaccination with inactivated A. hydrophila suspension by i.p. or immersion resulted in significant increase of skin mucus lysozyme and specific antibody levels in serum and skin mucus, at 28 and 42 DPV, and this increase in innate and adaptive immunity remained significant in pacu vaccinated through i.p. route up to 84 DPV. Although no significant differences were observed in the survival study, pacu vaccinated through i.p. route presented 31,33% of relative percentage survival (RPS) in LD50–96h when compared unvaccinated fish challenged at 84 DPV. The results observed in this study indicate that vaccination programs with inactivated A. hydrophila, including booster doses by i.p. or immersion routes, could result in more effective protection in pacu against this bacteriosis, by increasing innate and adaptive mucosal and systemic immune responses.

Interleukin-2 (IL-2) of flounder (Paralichthys olivaceus) as immune adjuvant enhance the immune effects of E. tarda subunit vaccine OmpV against Edwardsiellosis

In our previous study, we cloned and explored the biological functions of flounder (Paralichthys olivaceus) interleukin-2 (poIL-2), and showed that poIL-2 might have adjuvant potential for fish vaccines. In this study, the adjuvant effects of recombinant and molecular forms of poIL-2 (rIL-2 and pcIL-2) were comparatively analyzed and evaluated in flounder from several aspects by co-vaccination with the recombinant E. tarda OmpV (rOmpV). The results showed that co-vaccination with rOmpV plus rIL-2 or pcIL-2 resulted in a relative percent survival of 71% and 57% respectively, which was significantly higher than the control groups, rOmpV plus rHis (40%) or pcN3 (36%). Immunological analysis showed that: (1) the levels of specific serum antibodies and sIg + lymphocytes in head kidney, spleen and peripheral blood induced by rOmpV plus rIL-2 or pcIL-2 were significantly higher than that in the two control groups; (2) Compared to the two control groups, CD4-1, CD4-2, CD8α, CD8β, MHCIα, MHCIIα, IgM and IFN-γ mRNA levels were also significantly induced by rOmpV plus rIL-2 or pcIL-2; (3) the rOmpV plus rIL-2 could induce higher levels of sIg + lymphocytes, specific serum antibodies and the expressions of all investigated genes than rOmpV plus pcIL-2. These results demonstrated that co-vaccination with rOmpV with rIL-2 or pcIL-2 could induce stronger humoral and cellular immune responses, and evoked higher immune protective efficacy against E. tarda infection, suggesting that poIL-2 could be served as a promising candidate adjuvant and have a potential application in the control of flounder diseases.

Vaccination at different anatomic sites induces different levels of the immune responses

This study was to evaluate the effects of anatomical sites for vaccination on the immune responses. In experiment A, rats were subcutaneously (s.c.) immunized with a quintuplet vaccine twice at houhai acupoint, underjaw, popliteal fossa or back with a two weeks interval. The serum specific antibody levels were determined 2, 4 and 6 weeks after second immunization. Splenocytes were separated for detection of lymphocyte proliferation and cytokine mRNA expression. In experiment B, 10 female Rottweiler puppies at their age of 34 ± 2 days were subcutaneously injected with a bivalent vaccine Nobivac® Puppy DP containing live attenuated canine distemper virus (CDV) and parvovirus (CPV) for primary vaccination, and a quadrivalent vaccine Nobivac® DHPPI containing live attenuated canine distemper virus (CDV), adenovirus type 2 (CAV-2), parvovirus (CPV) and parainfluenza virus (CPIV) for subsequent vaccination at houhai acupoint (4 dogs), the shoulder (3 dogs) or the nape (3 dogs) region. Blood samples were collected at 0, 2, 4 and 6 weeks after vaccination for determination of serum specific antibody responses by ELISA. The results showed that injection of a vaccine in houhai acupoint induced the highest antibody responses in both rats and dogs. When a vaccine was injected in houhai acupoint, significantly increased proliferative responses to Con A and LPS as well as mRNA expression of IL-4, IL-10, IL-12 and IFN-γ of splenocytes were detected in rats. Therefore, houhai acupoint is recommended for injection of a vaccine to improve the immune response in dogs.

Establishment of an animal model of Sparganum mansoni infection and study on therapeutic methods Ⅰ Establishment of an animal model of Sparganum mansoni infection in mice and changes of serum specific antibody levels post-infection

To establish an animal model of Sparganum mansoni (plerocercoid larva of S. mansoni) infection in mice and observe the changes of blood routine examinations and serum anti-sparganum antibody levels after the infection. The spargana tapeworms were collected from frogs, and 25 Kunming mice were orally infected with the Sparganum tapeworms (3 tapeworms/mouse). Two days before the infection and 2, 7, 14, 21, 28, 35, 42 days and 49 days after the infection, the peripheral blood samples of mice were collected for the blood routine examinations and the detections of anti-S. mansoni IgG antibody with ELISA. Forty-nine days after the infection, all the mice were sacrificed to find out the Sparganum tapeworms in the bodies of mice. The count of the total white blood cells was significantly elevated on the second day of the mice infected with Sparganum. The serum anti-Sparganum antibody was detected on the 14th day of the infection in some mice, and on the 21st day of the infection, the serum anti-Sparganum antibody was detected in all the mice. After the mice were sacrificed, the Sparganum tapeworms were found out in many tissues and organs, and especially in the subcutaneous tissues and muscle. The establishment of animal model of Sparganum infection is successful in mice with the oral method, and white blood cells and serum specific IgG antibody detection can be used as auxiliary diagnosis methods of S. mansoni infection.

Comparative study of the adjuvant potential of four Th0 cytokines of flounder (Paralichthys olivaceus) on an E. tarda subunit vaccine

Cytokines have the potential as adjuvants for the application of vaccines in mammals. However, the adjuvant potential of teleost cytokines was limited. In the present work, the adjuvant effects of four recombinant cytokines including IL-1β, IL-8, TNF-α and G-CSF on E. tarda subunit vaccine rOmpV were comparatively investigated in flounder (Paralichthys olivaceus). Compared with control, the levels of specific serum antibodies and IgM + B lymphocytes were significantly enhanced by rIL-1β, rIL-8 and rG-CSF, whereas rIL-1β and rIL-8 induced significantly higher levels than rG-CSF. All four cytokines enhanced the expression of genes involved in humoral and/or cellular immunities, whereas rIL-1β and rIL-8 induced highest levels of genes involved in humoral immunities and cellular immunities, respectively. Compared to the relative percent survivals (RPS) of control group (40%) and rOmpV plus rG-CSF group (54%), rOmpV plus rIL-1β or rIL-8 produced higher RPS of 75% and 68%, respectively. Our results indicated that rIL-1β and rIL-8 are promising adjuvants for subunit vaccines against E. tarda.

Outer membrane protein A: An immunogenic protein induces highly protective efficacy against Vibrio ichthyoenteri

Vibrio ichthyoenteri was an important causative agent of bacterial enteritis in flounder (Paralichthys olivaceus). Outer membrane protein A (OmpA) of Gram-negative pathogen was a major cell surface antigen. In the present study, OmpA of V. ichthyoenteri was recombinantly expressed in Escherichia coli, and the immunogenicity of OmpA was identified by western blotting using flounder anti-rOmpA and anti-V. ichthyoenteri antibodies. The vaccine potential of rOmpA was tested in a flounder model, and a high relative percentage of survival rate was obtained with 73.1% after challenge with V. ichthyoenteri. Meanwhile, the immune response of flounder induced by rOmpA was also investigated, and the results showed that the sIg + lymphocytes in blood, spleen, and pronephros significantly proliferated, and the peak levels occurred at week 4 after immunization. Moreover, rOmpA could induce higher levels of specific serum antibodies than the control group after immunization, and the peak level occurred at week 5 after immunization. Meanwhile, qRT-PCR analysis showed that the expressions of CD4-1, CD8α, IL-1β, IFN-γ, MHCIα and MHCIIα genes were significantly up-regulated after immunization with rOmpA. Taking together, these results demonstrated that rOmpA could evoke highly protective effects against V. ichthyoenteri challenge and induce strong immune response of flounder, which indicated that OmpA was a promising vaccine candidate.

The Immune Adjuvant Effects of Flounder (Paralichthys olivaceus) Interleukin-6 on E. tarda Subunit Vaccine OmpV.

Interleukin-6 (IL-6) as a pleiotropic cytokine was widely used as an effective adjuvant for vaccines in mammals. In this study, the immune adjuvant effects of two forms of flounder (Paralichthys olivaceus) IL-6, including recombinant IL-6 (rIL-6) and pcDNA3.1-IL-6 (pcIL-6), were evaluated and comparatively analyzed on E. tarda subunit vaccine recombinant outer membrane protein V (rOmpV). The results showed that the relative percent survivals of flounder vaccinated with rOmpV plus rIL-6 or pcIL-6 were significantly higher than that in the two control groups, rOmpV plus recombinant 6× histidine-tag (rHis) or empty expression vector pcDNA3.1 (pcN3). The levels of specific serum antibodies and surface membrane immunoglobulin-positive (sIg+) lymphocytes in peripheral blood, spleen, and head kidney in the two adjuvant groups were also much higher than that in the two control groups. Compared with the two control groups, higher upregulated expressions of major histocompatibility complex class Iα (MHCIα), cluster of differentiation 8α (CD8α), MHCIIα, CD4-1, interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) were detected in flounder vaccinated with rOmpV plus rIL-6 or pcIL-6 after challenge. In addition, the rOmpV plus rIL-6 could induce significant higher levels of specific serum antibodies, sIg+ lymphocytes and four genes expressions than rOmpV plus pcIL-6. These results demonstrated that both rIL-6 and pcIL-6 used as adjuvants could enhance the immune response and evoke immune protections against E. tarda infection, which has a significant value in controlling diseases using vaccines in flounder.

Adaptive immune response to whole cell pertussis vaccine reflects vaccine quality: A possible complementation to the Pertussis Serological Potency test

Whole cell Bordetella pertussis (wP) vaccines are still used in many countries to protect against the respiratory disease pertussis. The potency of whole-cell pertussis vaccine lots is determined by an intracerebral challenge test (the Kendrick test). This test is criticized due to lack of immunological relevance of the read-out after an intracerebral challenge with B. pertussis. The alternative in vivo test, which assesses specific antibody levels in serum after wP vaccination, is the Pertussis Serological Potency test (PSPT). Although the PSPT focuses on a parameter that contributes to protection, the protective immune mechanisms after wP vaccination includes more elements than specific antibody responses only. In this study, additional parameters were investigated, i.e. circulating pro-inflammatory cytokines, antibody specificity and T helper cell responses and it was evaluated whether they can be used as complementary readout parameters in the PSPT to assess wP lot quality. By deliberate manipulation of the vaccine preparation procedure, a panel of high, intermediate and low quality wP vaccines were made. The results revealed that these vaccines induced similar IL-6 and IP10 levels in serum 4h after vaccination (innate responses) and similar antibody levels directed against the entire bacterium. In contrast, the induced antibody specificity to distinct wP antigens differed after vaccination with high, intermediate and low quality wP vaccines. In addition, the magnitude of wP-induced Th cell responses (Th17, Th1 and Th2) was reduced after vaccination with a wP vaccine of low quality. T cell responses and antibody specificity are therefore correlates of qualitative differences in the investigated vaccines, while the current parameter of the PSPT alone was not sensitive enough to distinguish between vaccines of different qualities. This study demonstrates that assessment of the magnitude of Th cell responses and the antigen specificity of antibodies induced by wP vaccination could form valuable complementary parameters to the PSPT.

Edwardsiella tarda Outer Membrane Protein C: An Immunogenic Protein Induces Highly Protective Effects in Flounder (Paralichthys olivaceus) against Edwardsiellosis.

Outer membrane protein C of Edwardsiella tarda is a major cell surface antigen and it was identified to be an immunogenic protein by Western blot using flounder (Paralichthys olivaceus) anti-recombinant OmpC (rOmpC), and anti-E. tarda antibodies. rOmpC tested the immune protective effect against E. tarda challenge in a flounder model and produced a relative percentage of survival rate of 85%. The immune response of flounder induced by rOmpC was investigated, and the results showed that: (1) the levels of specific serum antibodies induced by rOmpC were significantly higher than the control group after the second week after immunization, and the peak level occurred at week five after immunization; (2) rOmpC could induce the proliferation of sIg+ lymphocytes, and the peak levels of sIg+ lymphocytes in blood, spleen, and pronephros occurred at 4–5 weeks after immunization; and (3) the MHCIIα, CD4-1, IL-1β, IL-6 and TNF-α genes were significantly induced after being injected with rOmpC. Taken together, these results demonstrated that rOmpC could evoke highly protective effects against E. tarda challenge and induce strong innate immune response and humoral immune response of flounder, which indicated that OmpC was a promising vaccine candidate against E. tarda infection.