Non-immunized Group Research Articles

Post-immunization immunohistochemical expression of Caspase 3 and p53 apoptotic markers in experimental hydatidosis.

The aim of this study was to investigate post-immunization apoptotic changes in experimental hydatidosis, using Caspase 3 and p53 immunohistochemical markers. Two groups of rabbits were immunized with a crude antigen (group 1) or a partially purified antigen (group 2) and were compared to an infected non-immunized control group. More effective immune responses were obtained in group 2 than group 1, signified by fewer and smaller cystic lesions and more severe destructive changes. Normal growth of cysts was attained in the control group, with no expression of apoptotic markers. Significantly higher expression of Caspase 3 and p53 were observed in group 1 compared to group 2, as indicated by OD and area percentage, respectively (Group 1 Caspase 3: 0.89±0.21, 93.5%±6.2; Group 1 p53: 0.46±0.18, 53.26%±11.6; Group 2 Caspase 3: 0.52±0.15, 49.23%±11.7; Group 2 p53: 0.19±0.4, 18.17%±7.3). Vaccine-induced immune responses and cellular damage may underlie the expression of apoptotic markers that appeared to result in a degenerative and atrophic course of action upon immunization. The results of the current study emphasize the importance of immunization for the stimulation of protective immune responses and in preventing mechanisms of evasion to ensure normal cell growth. A cost/benefit control program that implements proper vaccine preparations should be further assessed for complete elimination of severe infections in endemic areas.

Capsular polysaccharide from Mycoplasma mycoides subsp. mycoides shows potential for protection against contagious bovine pleuropneumonia

Contagious Bovine Pleuropneumonia (CBPP) is a severe respiratory disease caused by Mycoplasma mycoides subsp. mycoides (Mmm) which is widespread in Africa. The capsule polysaccharide (CPS) of Mmm is one of the few identified virulence determinants. In a previous study, immunization of mice against CPS generated antibodies, but they were not able to prevent multiplication of Mmm in this model animal. However, mice cannot be considered as a suitable animal model, as Mmm does not induce pathology in this species. Our aim was to induce antibody responses to CPS in cattle, and challenge them when they had specific CPS antibody titres similar or higher than those from cattle vaccinated with the live vaccine. The CPS was linked to the carrier protein ovalbumin via a carbodiimide-mediated condensation with 1-ethyl-3(3-imethylaminopropyl) carbodiimide (EDC). Ten animals were immunized twice and challenged three weeks after the booster inoculation, and compared to a group of challenged non-immunized cattle. When administered subcutaneously to adult cattle, the vaccine elicited CPS-specific antibody responses with the same or a higher titre than animals vaccinated with the live vaccine. Pathology in the group of immunized animals was significantly reduced (57%) after challenge with Mmm strain Afadé compared to the non-immunized group, a figure in the range of the protection provided by the live vaccine.

Efficacy of a potential DNA vaccine encoding Cryptosporidium baileyi rhomboid protein against homologous challenge in chickens

The parasite Cryptosporidium baileyi can infect the larynx, trachea, bursa and cloaca of poultry, causing high mortality during severe infection and leading to substantial economic losses of the poultry industry. The rhomboid protein is very important in Cryptosporidium infection. In this study, a nucleic acid based vaccine candidate pEGFP-CbROM was constructed. After orally challenging with C. baileyi oocysts, the corresponding immune responses induced were analyzed and the immunoprotective effect evaluated in chickens. Obtained results revealed that this nucleic acid based vaccine could induce antibody responses and peripheral blood T lymphocytes proliferation significantly (P<0.05), while the peripheral blood B lymphocyte proliferation increased significantly (P<0.05) only at a high dose of 100μg of pEGFP-CbROM, compared with the PBS control group. After C. baileyi infection, the duration of oocysts shedding was shortened by 2days in the 100μg pEGFP-CbROM group, and the rate of reduction could reach to around 71.3%. While no significant difference in body weight gain was observed among the immunized groups (P>0.05), the differences between the immunized and the non-immunized groups were found to be significant (P<0.05). Our data provides a useful basis for further work in cryptosporidiosis prevention and treatment.

Induction of humoral immune response against Pseudomonas aeruginosa flagellin(1-161) using gold nanoparticles as an adjuvant

Flagellin of Pseudomonas aeruginosa is an important vaccine candidate. N-terminal domains are highly conserved in both type a and type b flagellins. The efficacy of gold nanoparticles (AuNPs) conjugated to N-terminal domains of P. aeruginosa flagellin (flagellin(1-161)), as an immunogen in mice, has been assessed. The nanoparticles were conjugated to the recombinant protein through direct interaction of thiol molecules of the cysteines with AuNPs and formation of AuS bond. Flagellin(1-161), AuNP-flagellin(1-161), and flagellin(1-161) emulsified in Freund's adjuvant (FA: complete/incomplete Freund's adjuvant formulation) were administered subcutaneously to BALB/c mice. Mice given AuNP-flagellin(1-161) elicited high titers of anti-flagellin(1-161) antibodies compared with non-immune group and/or mice which received flagellin(1-161) without adjuvant. In whole cell ELISA, these antibodies effectively recognized the native flagellin on the bacteria. Opsonophagocytosis assay demonstrated the functional activity and specificity of anti-flagellin(1-161) antibodies raised by AuNP-flagellin(1-161) against homologous strain. All of the results were comparable with those obtained by use of FA. Taken together, this is the first report of conjugation of AuNPs to flagellin and evaluating its immune response against P. aeruginosa.

Comparison of the Effect of Two Purification Methods on the Immunogenicity of Recombinant Outer Membrane Protein H of Pasteurella multocida Serovar A:1.

Recombinant outer membrane protein H (rOmpH) of Pasteurella multocida strain X-73 can be purified using affinity chromatography but this adversely affects its immunogenicity. The current study presents the results from an intervention study comparing the immunogenicity of rOmpH purified using electroelution with rOmpH purified using affinity chromatography and native OmpH purified using electroelution and a nonimmunized control group. Chickens immunized with rOmpH purified using electroelution produced the highest ELISA antibody levels against P. multocida strains. Chickens in each of the 5 treatment groups were split into two subgroups for challenge with two different P. multocida strains. The average number of adhesions to CEF cells was statistically significantly lower in sera from chickens immunized with rOmpH or native OmpH purified using electroelution than in those of the three other treatment groups. The survival amongst chickens immunized with rOmpH or native OmpH purified using electroelution indicated high levels of protection. In contrast, survival probability was zero or low in the groups immunized with rOmpH purified using affinity chromatography and in the nonimmunized group. These findings show that the rOmpH purified using electroelution retains its immunogenicity and stimulates high levels of protection in chickens against P. multocida infection.

Potency of Attenuated Eimeria tenella in Protective Immunity Induction on Homologous and Heterologous Challenges

The aim of this study was to know the protective immunity of broilers immunized with attenuated Eimeria tenella (E. tenella) on homologous and heterologous challenges. The protective immunity was represented by oocyst production and histopathological changes of cecum. The previous experiment already produced attenuated E. tenella through serial passages of precocious lines in naive chicken. Four groups of chickens were separately divided into two non-immunized control groups and two immunized with attenuated E. tenella groups, and each group was challenged with homologous and heterologous strains. The results showed significant difference in oocysts production (p<0.01) and histopathological changes between control and immunized groups.

A Live Salmonella Gallinarum Vaccine Candidate Secreting an Adjuvant Protein Confers Enhanced Safety and Protection Against Fowl Typhoid.

Live attenuated vaccines are used for effective protection against fowl typhoid (FT) in domestic poultry. In this study, a lon/cpxR/asd deletion mutant of Salmonella Gallinarum expressing the B subunit of a heat labile toxin (LTB) from Escherichia coli, a known adjuvant, was cloned in a recombinant p15A ori plasmid, JOL1355, and evaluated as a vaccine candidate in chickens. The plasmid was shown to be stable inside the attenuated Salmonella Gallinarum cell after three successive generations. Moreover, from an environmental safety point of view, apart from day 1 the JOL1355 strain was not detected in feces through day 21 postinoculation. For the efficacy of JOL1355, a total of 100 chickens were equally divided into two groups. Group A (control) chickens were intramuscularly inoculated with phosphate-buffered saline at 4 and 8 wk of age. Group B chickens were primed and boosted via the intramuscular route with 200 μL of a bacterial suspension of JOL1355 containing 1 × 10(8) colony forming units. All the chickens in Group A and B were challenged at 3 wk postbooster by oral inoculation with a wild-type Salmonella Gallinarum strain, JOL420. The JOL1355-immunized group showed significant protection and survival against the virulent challenge compared to the nonimmunized group. In addition, Group B exhibited a significantly higher humoral immune response, and the chickens remained healthy without any symptoms of anorexia, diarrhea, or depression. Group B also exhibited a significantly lower mortality rate of 4% compared to the 46% of the control group, which can be attributed to higher immunogenicity and better protection. The Group B chickens had significantly lower lesion scores for affected organs, such as the liver and spleen, compared to those of the control chickens (P < 0.01). These findings suggest that JOL1355 is a promising candidate for a safe and highly immunogenic vaccine against FT.

Resistance to cecal coccidiosis following sonicated oocysts immunization of Eimeri tenella in broilers

Seventy five broiler chicks were used to determine the immunization of in ovo inoculation or intramusclular (I/M) injection of sonicatedoocyts (SO) antigen against E. tenella manifestation. The chicks were divided in to 3 equal groups compromising of 25 chicks each. The 1st group inoculated in ovowithSO of E. tenella and repeated by I/M injection at 21 days old. The 2nd group injected I/M withSO at day old and repeated at 21 days old, The birds of the 3rd group remains as control. All birds were challenge by 50,000 sporulatedoocysts of E. tenellaat day 28. No significant (P &gt; 0.05) group difference was detected between the immunized and non-immunized groups for mean, WBC count, H/L ratio, lesion score, mortality and oocysts shedding until E. tenella challenge at day 28. After challenge test the in ovo and I/M immunized groups showed a great protective immunity against E. tenellainfection documented by significantly (P &lt; 0.05) reduced mortality, lesion score and decreased fecal oocyst shedding, compared with non-immunized group. It is concluded that in ovo and I/M immuinzation of sonicatedoocyst stimulates a remarkable protection in broilers following E. tenellainfectioncomparing with non-immunized groups. There was no significance difference between the two procedures of immunization.

Generation of a Novel Staphylococcus aureus Ghost Vaccine and Examination of Its Immunogenicity against Virulent Challenge in Rats.

Staphylococcus aureus is a Gram-positive pathogen that causes a wide range of infections in humans and animals. Bacterial ghosts are nonliving, empty cell envelopes and are well represented as novel vaccine candidates. In this study, we examined the immunogenicity and protective efficacy of S. aureus ghosts (SAGs) against a virulent challenge in rats. Nonliving SAGs were generated by using the MIC of sodium hydroxide. The formation of a transmembrane lysis tunnel structure in SAGs was visualized by scanning electron microscopy. To investigate these SAGs as a vaccine candidate, rats were divided into four groups, A (nonimmunized control), B (orally immunized), C (subcutaneously immunized), and D (intravenously immunized). The IgG antibody responses were significantly stronger in the SAG-immunized groups than in the nonimmunized control group (P < 0.05). Moreover, a significant increase in the populations of CD4(+) and CD8(+) T cells was observed in all three immunized groups (P < 0.05). We also found that serum bactericidal antibodies were significantly elicited in the SAG-immunized groups (P < 0.05). Most importantly, the bacterial loads in the immunized groups were significantly lower than those in the nonimmunized control group (P < 0.01). These results suggest that immunization with SAGs induces immune responses and provides protection against a virulent S. aureus challenge.

Recombinant influenza A virus hemagglutinin HA2 subunit protects mice against influenza A(H7N9) virus infection.

A novel avian influenza A(H7N9) virus has emerged to infect humans in eastern China since 2013. An effective vaccine is needed because of the high mortality despite antiviral treatment and intensive care. We sought to develop an effective vaccine for A(H7N9) virus. The HA2 subunit was chosen as the vaccine antigen because it is highly conserved among the human A(H7N9) virus strains. Moreover, in silico analysis predicted two immunogenic regions within the HA2 subunit that may contain potential human B-cell epitopes. The HA2 fragment was readily expressed in Escherichia coli. In BALB/c mice, intraperitoneal immunization with two doses of HA2 with imiquimod (2-dose-imiquimod) elicited the highest geometric mean titer (GMT) of anti-HA2 IgG (12699), which was greater than that of two doses of HA2 without imiquimod (2-dose-no-adjuvant) (6350), one dose of HA2 with imiquimod (1-dose-imiquimod) (2000) and one dose of HA2 without imiquimod (1-dose-no-adjuvant) (794). The titer of anti-HA2 IgG was significantly higher in the 1-dose-imiquimod group than the 1-dose-no-adjuvant group. Although both hemagglutination inhibition titers and microneutralization titers were below 10, serum from immunized mice showed neutralizing activity in a fluorescent focus microneutralization assay. In a viral challenge experiment, the 2-dose-imiquimod group had the best survival rate (100 %), followed by the 2-dose-no-adjuvant group (90 %), the 1-dose-imiquimod group (70 %) and the 1-dose-no-adjuvant group (40 %). The 2-dose-imiquimod group also had significantly lower mean pulmonary viral loads than the 1-dose-imiquimod, 1-dose-no-adjuvant and non-immunized groups. This recombinant A(H7N9)-HA2 vaccine should be investigated as a complement to egg- or cell-based live attenuated or subunit influenza vaccines.

Comparative effect of Freund's adjuvant andAloe veraL. gel on the expression of TNF-α and IL-1β in vaccinatedCyprinus carpioL. withAeromonas hydrophilaC. bacterin

The comparative effects of Freund's and Aloe vera gel as adjuvants on the expression of IL-1β and TNF-α genes were studied in vaccinated common carp (Cyprinus carpio) with Aeromonas hydrophila bacterin. Fishes were intraperitoneally immunized with A. hydrophila bacterin in combination with Aloe vera gel or Freund's and also without any adjuvant. At day 28 after immunization, all groups were challenged by lethal dose of A. hydrophila (107 cells/fish). Changes in the expression of IL-1β and TNF-α genes were evaluated in anterior kidney before challenge and 12, 24, 72 and 7 days postchallenge using quantitative real-time PCR. Higher expression levels of both genes were observed in all vaccinated groups compared with non-immunized group. Fishes which received Aloe vera gel showed higher expression of IL-1β and TNF-α in relation to animals which vaccinated with or without Freund's adjuvant. We concluded that Aloe vera gel in compared with Freund's adjuvant had a more stimulatory effect on the expression of immune-related genes in vaccinated common carp and it can use as a novel adjuvant in aquaculture.

Phlebotomus papatasi exposure cross-protects mice against Leishmania major co-inoculated with Phlebotomus duboscqi salivary gland homogenate

Leishmania parasites are inoculated into host skin together with sand fly saliva and multiple exposures to uninfected sand fly bites protect mice against Leishmania infection. However, sand fly vectors differ in composition of the saliva and therefore the protection elicited by their salivary proteins was shown to be species-specific. On the other hand, the optimal vaccine based on sand fly salivary proteins should be based on conserved salivary proteins conferring cross-reactivity. In the present study we therefore focused on cross-protective properties of saliva from Phlebotomus papatasi and Phlebotomus duboscqi, the two natural vectors of Leishmania major. Two groups of mice exposed to bites of P. papatasi and two control, non-immunized groups were infected with L. major promastigotes along with either P. papatasi or P. duboscqi salivary gland homogenate. All mice were followed for the development of Leishmania lesions, parasite burdens, specific antibodies, and for production of NO, urea, or cytokines by peritoneal macrophages. Protection against Leishmania infection was observed not only in exposed mice challenged with homologous saliva but also in the group challenged with P. duboscqi saliva. Comparing both exposed groups, no significant differences were observed in parasite load, macrophage activity, or in the levels of anti-L. major and anti-P. papatasi/P. duboscqi antibodies. This is the first study showing cross-protection caused by salivary antigens of two Phlebotomus species. The cross-protective effect suggests that the anti-Leishmania vaccine based on P. papatasi salivary proteins might be applicable also in areas where L. major is transmitted by P. duboscqi.

Protection against salmonella typhimurium, salmonella gallinarum, and salmonella enteritidis infection in layer chickens conferred by a live attenuated salmonella typhimurium strain.

In the present study, we investigated the protection conferred by a live attenuated Salmonella enterica serovar Typhimurium (ST) strain against Salmonella Typhimurium, Salmonella Gallinarum (SG), and Salmonella Enteritidis (SE) infection in layer chickens. Birds were orally primed with the attenuated ST strain at 7 days of age and then boosted at 4 weeks post prime immunization (PPI). Sequential monitoring of plasma IgG and mucosal secretory IgA (sIgA) levels revealed that inoculation with ST induced a significant antibody response to antigens against ST, SE, and SG. Moreover, significant lymphoproliferative responses to the 3 Salmonella serovars were observed in the immunized group. We also investigated protection against virulent ST, SE, and SG strain challenge. Upon virulent SG challenge, the immunized group showed significantly reduced mortality compared to the non-immunized group. The reduced persistence of the virulent ST and SE challenge strains in the liver, spleen, and cecal tissues of the immunized group suggests that immunization with the attenuated ST strain may not only protect against ST infection but can also confer cross protection against SE and SG infection.

Development of Rapid Diagnostic Kit for Identification of Hanwoo (Korean Native Cattle) Brand Meat by Detecting BIO-TAG.

This study was performed to develop a rapid immuno-assay kit, by using a specific antigen to detect Hanwoo brand meat. We selected a synthetic antigen specific to our target antibody, named BIO-TAG (Tyr-D-Ala-Phe), by utilizing a computer-based analysis and literature review. BIO-TAG tagged with adjuvant was subcutaneously injected in sheep and Hanwoo. The serum and meat juice of the immunized or non-immunized animal were then analyzed, to measure the titer of antibody by ELISA and Western blot. The amount of antibodies against the BIO-TAG increased (p<0.05) in serum by vaccination. Furthermore, meat juice from the immunized Hanwoo showed greater (p<0.05) antibody titer, compared with those from non-immunized groups. To optimze the dilution factor, we performed dot-ELISA, with various combination levels of BIO-TAG. Results from dot-ELISA showed that 2 mg/mL BIO-TAG was sufficient to distinguish the immunized meat from non-immunized groups. These results support our hypothesis that simple immunization of Hanwoo generates a sufficient amount of antibodies to be detectable in the meat juice by means of the immune-assay. Therefore, specific Hanwoo brand meat can be more precisely identified by our rapid diagnostic kit. This technology can deter possible fraud of counterfeit meat brands in the Korean domestic market with ease and rapidity; and offers a new tool that guarantees consumers high quality Hanwoo brand beef.

Characterization of adaptive immune responses induced by a new genetically inactivated Salmonella Enteritidis vaccine.

The superior conservation of antigenic determinants on the surface of genetically inactivated bacterial ghosts makes them attractive immunogenic inactivated vaccine candidates. The efficacy of Salmonella Enteritidis (SE) ghost vaccination was evaluated in chickens by characterizing the nature of the adaptive immune response. Chickens from the immunized group demonstrated significant increases in SE-specific plasma IgG, intestinal secretory IgA, and lymphocyte proliferative response. The populations of CD4, CD8, and TCR γδ T-cells in immunized chickens were significantly greater than in the controls. Increased levels of IFN-γ, IL-2, IL-6 and IL-10 were observed in peripheral blood mononuclear cells stimulated with SE specific antigen. After virulent SE challenge, the immune system of immunized chickens was rapidly stimulated, as indicated by significantly increased population of CD4 and CD8 T-cells. Furthermore, the immunized group exhibited decreased challenge strain recovery of the internal organs compared to the non-immunized group. Together, these data indicate that the immunization induced humoral and cell-mediated immunity might be responsible for significant reduction of the virulent challenge strain load in the internal organs of immunized chickens.

Tightly regulated bacteriolysis for production of empty Salmonella Enteritidis envelope

To avoid leaky expression of the bacterial host-toxic PhiX174 lysis gene E from the λpR promoter, a convergent promoter construct was made in which gene E was placed between a sense λpR promoter and an anti-sense ParaBAD promoter. In the presence of l-arabinose, leaky transcription of lysis gene E at 28°C from the sense λpR promoter was repressed by an anti-sense RNA simultaneously expressed from the ParaBAD promoter. The stringent repression of lysis gene E in the absence of induction temperature resulted into higher concentration of bacteria in culture suspension, and consequently higher and stable production of a Salmonella Enteritidis (S. Enteritidis) ghost. The immunogenicity of the S. Enteritidis ghost was evaluated by immunizing chickens. Chickens from the immunized group demonstrated a significant increase in the levels of S. Enteritidis-specific plasma IgG, intestinal sIgA, and lymphocyte proliferative response. After virulent S. Enteritidis challenge, the immunized group exhibited decreased bacterial recovery from organs compared with the non-immunized group. Together, these results demonstrate that the stringent molecular control over leaky transcription of lysis gene E enabled the stable production of S. Enteritidis ghost, and immunization with the S. Enteritidis ghost can protect chickens by inducing robust humoral and cellular immune responses.

Rubella Immunity among Pregnant Women in Jeddah, Western Region of Saudi Arabia.

To determine the presence of rubella immunity among pregnant women attending their first prenatal visit in Jeddah, Saudi Arabia, a retrospective, descriptive, cross-sectional, hospital-based study (prevalence study) was undertaken. A total of 10276 women attending prenatal clinics between January 1, 2008, and December 31, 2011 were included. Rubella screening tests (immunoglobulins: IgG and IgM), rubella antibody titer levels, patient age, gravidity, parity, and the number of previous abortions were analyzed. No patients tested IgM positive, and 9410 (91.6%) were immune (IgG positive); the remaining 866 (8.4%) were susceptible. There were no significant differences in gravidity, parity, or the number of previous abortions between immune and nonimmune groups. In contrast, the immunity rate decreased with increasing age, with a significant difference between the youngest age group (15–19 years) and the oldest age group (40–49 years) (P = 0.0005; odds ratio, 2.86; 95% confidence interval, 1.7–4.7). Rubella immunity among pregnant women was high (91.6%) but decreased significantly with increasing age. A possible explanation for this is the change in the rubella vaccination policy in Saudi Arabia in 2002, from 1 dose to 2 doses. In addition, antibody levels begin to decline after vaccination and natural infection.

Immunotherapy Targeting Tau and Amyloid Aβ Pathology in AD Animal Models

Immunotherapy Targeting Tau and Amyloid Aβ Pathology in AD Animal Models

Development of a Biosafety Enhanced and Immunogenic Salmonella Enteritidis Ghost Using an Antibiotic Resistance Gene Free Plasmid Carrying a Bacteriophage Lysis System

In the development of genetically inactivated bacterial vaccines, plasmid retention often requires the antibiotic resistance gene markers, the presence of which can cause the potential biosafety hazards such as the horizontal spread of resistance genes. The new lysis plasmid was constructed by utilizing the approach of balanced-lethal systems based on auxotrophic gene Aspartate semialdehyde dehydrogenase (asd). The PhiX174 lysis gene E and λPR37-cI857 temperature-sensitive regulatory system was cloned in the asd gene positive plasmid and this novel approach allowed the production of antibiotic resistance marker free Salmonella Enteritidis (S. Enteritidis) ghost. The immunogenic potential of the biosafety enhanced antibiotic resistance gene free S. Enteritidis ghost was evaluated in chickens by employing the prime-boost vaccination strategy using a combination of oral and intramuscular routes. A total of 75 two-week-old chickens were equally divided into five groups: group A (non-immunized control), group B (intramuscularly primed and boosted), group C (primed intramuscularly and boosted orally), group D (primed and boosted orally), and group E (primed orally and boosted intramuscularly). Chickens from all immunized groups demonstrated significant increases in plasma IgG, intestinal secretory IgA levels, and antigen-specific lymphocyte proliferative response. After a virulent S. Enteritidis challenge, all immunized groups showed fewer gross lesions and decreased bacterial recovery from organs in comparison with the non-immunized control group. Among the immunized chickens, groups B and D chickens showed optimized protection, indicating that the prime-booster immunization with the ghost via intramuscular or oral route is efficient. Taken together, our results demonstrate that an antibiotic resistance gene free lysis plasmid was successfully constructed and utilized for production of safety enhanced S. Enteritidis ghost, which can be used as a safe and effective vaccine against virulent S. Enteritidis infections.

Vaccine potential of recombinant saposin-like protein 2 against Fasciolosis gigantica in mice

Saposin-like protein 2 (SAP-2) is a protein that adult of Fasciola spp. use to lyse plasma membrane of red blood cells, so that their contents can be digested by proteases for the parasites’ nutrients. Thus SAP-2 is a plausible target for vaccination against these parasites. Recombinant Fasciola gigantica saposin-like protein 2 (rFgSAP-2) was expressed in Escherichia coli BL21 (DE3). A vaccination was performed in ICR mice (n=10) by subcutaneous injection with 50μg of rFgSAP-2 combined with Freund's adjuvant. At 2 weeks after the second boost, mice were infected with 30 F. gigantica metacercariae by oral route. The percentages of protection of rFgSAP-2 vaccine against F. gigantica were estimated to be 76.4–78.5% when compared with non vaccinated-infected and adjuvant-infected controls, respectively. The antibodies in immune sera of vaccinated mice were shown by immuno-blotting to react with native FgSAP-2 in the extract of 2- and 4-week-old juvenile parasites. By determining the levels of IgG1 and IgG2a in the immune sera, which are indicative of Th2 and Th1 immune responses, it was found that both Th1 and Th2 humoral immune response were significantly increased in rFgSAP-2 immunized group compared with the control groups, with higher levels of Th2 (IgG1) than Th1 (IgG2a). The levels of serum aspartate aminotransferase (AST) and alanine transaminase (ALT) in rFgSAP-2-immunized group showed no significant difference from those of the non-immunized and infected group, indicating that early juvenile parasites induced liver parenchyma damage, even though the numbers of worm recoveries were significantly different. This study indicates that rFgSAP-2 has a high potential as a vaccine candidate against F. gigantica in mice, and this potential will be tested in larger economic animals.