Hematological parameters, antioxidant status, and gene expression of γ-INF and IL-1ß in vaccinated lambs fed different type of lipids.

This study was aimed to evaluate the effects of vegetable oils as calcium salt on immune responses and the expression of immune-related genes in vaccinated lambs. Twenty-four lambs (35 kg body weight, 6 months old) were assigned to four treatments with six replicates in a completely randomized design for 40 days. Four concentrates were formulated in which the calcium salts of palm oil, canola oil, corn oil, and flaxseed oil were used. On day 30 of the experiment, lambs were vaccinated by a dose of foot-and-mouth disease virus. The blood samples were collected from jugular vein 10 days after vaccination. The level of malondialdehyde and the activity of liver enzymes were the highest in lambs receiving corn oil and the lowest in lambs receiving flaxseed oil. The highest lymphocytes and the lowest neutrophil percentages were observed in lambs receiving flaxseed oil. There was a significant difference among treatments for the relative genes expression. Flaxseed oil significantly upregulated interferon-γ and corn oil upregulated interleukin-1ß. The highest titer against foot-and-mouth disease virus was related to lambs receiving flaxseed oil, and the lowest titer was related to lambs that received corn oil. Flaxseed oil had more beneficial effects on immune response than other oils.

Effect of gamma-irradiated honey bee venom on gene expression of inflammatory and anti-inflammatory cytokines in mice.

In this study, the effect of gamma-irradiated honey bee venom (doses of 0, 2, 4, 6, and 8 kGy, volume of 0.1 ml and concentration of 0.2 mg/ml) was evaluated on the reduction of allergen compounds and the gene expression of inflammatory and anti-inflammatory cytokines in mice. Hence, edema activity induced by the bee venom irradiated at 4, 6, and 8 kGy was reduced, compared with the control group and that irradiated at 2 kGy. In contrast, the paw edema induced by the bee venom irradiated at 8 kGy increased, compared with 4 and 6 kGy. At all the time periods, there was a significant decrease in the gene expression of interferon gamma (IFN-γ), interleukin 6 (IL-6), and interleukin 10 (IL-10) in the bee venoms irradiated at 4, 6, and 8 kGy, compared with the control group and that irradiated at 2 kGy. In contrast, there was an increase in the gene expression of IFN-γ and IL-6 in the bee venom irradiated at 8 kGy, compared with those irradiated at 4 and 6 kGy. Therefore, gamma irradiation at 4 and 6 kGy reduced the gene expression of cytokines at each time period by decreasing the allergen compounds of honey bee venom.

The effects of thymol, oxalic acid (Api-Bioxal) and hops extract (Nose-Go) on viability, the Nosema sp. spore load and the expression of vg and sod-1 genes in infected honey bees.

This study was done to investigate the effects of thymol, fumagillin, oxalic acid (Api-Bioxal) and hops extract (Nose-Go) on Nosema sp. spore load, the expression of vitellogenin (vg) and superoxide-dismutase-1 (sod-1) genes and mortality of bees infected with N. ceranae. Five healthy colonies were assigned as the negative control, and 25 Nosema sp. infected colonies were assigned to five treatment groups including: the positive control: no additive to sirup; fumagillin 26.4 mg/L, thymol 0.1 g/L, Api-Bioxal 0.64 g/L and Nose-Go 5.0 g/L sirup. The reduction in the number of Nosema sp. spores in fumagillin, thymol, Api-Bioxal and Nose-Go compared to the positive control was 54, 25, 30 and 58%, respectively. Nosema sp. infection in all infected groups increased (p < .05) Escherichia coli population compared to the negative control. Nose-Go had a negative effect on lactobacillus population compared to other substances. Nosema sp. infection decreased vg and sod-1 genes expression in all infected groups compared to the negative control. Fumagillin and Nose-Go increased the expression of vg gene, and Nose-Go and thymol increased the expression of sod-1 gene than the positive control. Nose-Go has the potential to treat nosemosis if the necessary lactobacillus population is provided in the gut.

Improved Whole Gamma Irradiated Avian Influenza Subtype H9N2 Virus Vaccine Using Trehalose and Optimization of Vaccination Regime on Broiler Chicken.

Gamma (γ)-radiation can target viral genome replication and preserve viral structural proteins compared to formalin inactivation. Thus, a stronger immunity could be induced after the inoculation of the irradiated virus. In this study, γ-irradiated low-pathogenic avian influenza virus-H9N2 (LPAIV-H9N2) was used to immunize the broiler chicken in two formulations, including γ-irradiated LPAIV-H9N2 with 20% Trehalose intranasally (IVT.IN) or γ-irradiated LPAIV-H9N2 plus Montanide oil adjuvant ISA70 subcutaneously (IV+ISA.SC) in comparison with formalin-inactivated LPAIV-H9N2 vaccine intranasally (FV.IN) or formalin-inactivated LPAIV-H9N2 plus ISA70 subcutaneously (FV+ISA.SC). Two vaccination regimes were employed; the first one was primed on day 1 and boosted on day 15 (early regime), and the second one was primed on day 11 and boosted on day 25 (late regime). A challenge test was performed with a live homologous subtype virus. Virus shedding was monitored by quantifying the viral load via RT-qPCR on tracheal and cloacal swabs. Hemagglutination inhibition (HI) antibody titration and stimulation index (SI) of the splenic lymphocyte proliferation were measured, respectively, by HI test and Cell Proliferation assay. Cytokine assay was conducted by the RT-qPCR on antigen-stimulated spleen cells. The results of the HI test showed significant increases in antibody titer in all vaccinated groups, but it was more evident in the IVT late vaccination regime, reaching 5.33 log2. The proliferation of stimulated spleen lymphocytes was upregulated more in the IVT.IN vaccine compared to other vaccines. The mRNA transcription levels of T-helper type 1 cytokines such as interferon-gamma (IFN-γ) and interleukin 2 (IL-2) were upregulated in all vaccinated groups at the late regime. Moreover, IL-6, a pro-inflammatory cytokine was upregulated as well. However, upregulation was more noticeable in the early vaccination than the late vaccination (p< 0.05). After the challenge, the monitoring of virus shedding for the H9 gene represented an extremely low viral load. The body weight loss was not significant (p > 0.05) among the vaccinated groups. In addition, the viral load of <100.5 TCID50/ml in the vaccinated chicken indicated the protective response for all the vaccines. Accordingly, the IVT vaccine is a good candidate for the immunization of broiler chicken via the intranasal route at late regime.

Carboxymethyl chitosan bounded iron oxide nanoparticles and gamma-irradiated avian influenza subtype H9N2 vaccine to development of immunity on mouse and chicken.

BACKGROUND: Avian influenza virus (AIV) subtype H9N2 is a low pathogenic avian influenza virus (LPAIV). OBJECTIVE: This study aims to evaluate the humoral and cellular immunity in vaccinated mice and broiler chicken by irradiated AIV antigen plus carboxymethyl chitosan bounded iron oxide nanoparticles (CMC-IO NPs) as an adjuvant. METHODS: AIV subtype H9N2 with 108.5 EID50 /ml and haemagglutinin antigen assay about 10 log2 was irradiated by 30 kGy gamma radiation dose. Then, the gamma-irradiated AIV was used as an inactivated vaccine and conjugated with CMC-IO NPs to improve immune responses on mice. IO NPs must be applied in all activated tests using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysulfosuccinimide sodium salt (sulfo-NHS), and then functionalized by CMC as IO-CMC. Fourier transform infrared (FTIR) spectra on functionalized IO-CMC showed a peak of 638 cm-1 which is a band between metal and O (Fe-O). RESULTS: Based on the comparison between the two X-ray diffraction (XRD) patterns on Fe2 O3 -NPs and IO-CMC, the characteristics of IO-NPs did not change after carboxymethylation. A CHN Analyzer was applied to measure the molecular weight of IO-CMC that was calculated as 1045 g. IO-CMC, irradiated AIV-IO-CMC and formalin AIV-IO-CMC were injected into 42 BALB/c mice in six groups. The fourth group was the negative control, and the fifth and sixth groups were inoculated by irradiated AIV-ISA70 and formalin AIV-ISA70 vaccines. An increase in haemagglutination inhibition (HI) antibody titration was observed in the irradiated AIV-IO-CMC and formalin AIV-IO-CMC groups (p < 0.05). In addition, increases in the lymphoproliferative activity of re-stimulated splenic lymphocytes, interfron-γ (IFN-γ) and interleukin-2 (IL-2) concentration in the irradiated AIV-IO-CMC group demonstrated the activation of Type 1 helper cells. The concentration of IL-4 was without any significant increases in non-group. CONCLUSIONS: Accordingly, Th2 activation represented no increase. Finally, the finding showed that AIV-IO-CMC was effective on enhancing immunogenicity as irradiated AIV antigen administered with a clinically acceptable adjuvant (i.e. IO-CMC).

Immune Response of Gamma-Irradiated Inactivated Bivalent Polio Vaccine Prepared plus Trehalose as a Protein Stabilizer in a Mouse Model.

INTRODUCTION: Poliovirus causes paralysis by infecting the nervous system. Currently, 2 types of polio vaccine are given in many countries in polio eradication program including inactivated polio vaccine (IPV) and oral polio vaccine (OPV). Because of OPV-related paralysis, OPV should be replaced by IPV. METHODS: The aim of this study was to prepare the gamma-irradiated IPV and determine its effectiveness compared with the commercial vaccine (OPV) in the mouse model. The virus titration of OPV was determined and then inactivated by the appropriate dose of gamma radiation into an irradiated vaccine formula. The vaccine was inoculated in BALB/c mice in 2 different formulations of intramuscular injection with 2-week intervals. The level of anti-polio-neutralizing antibody and polio-specific splenocyte proliferation assay were evaluated by collecting the blood samples and spleens of the vaccinated groups with conventional vaccine and irradiated vaccine. RESULTS: There was a significant increase in the neutralizing antibody titration between all of the vaccinated groups and negative control group (A) (p < 0.05). And it shows that the IPV by gamma irradiation has the highest antibody titration. Also, the increasing of stimulation index value in the B* group, F group, and G group was the most against other groups. Furthermore, the neutralizing anti-serum titer and splenic lymphocyte proliferation assay show humoral and cellular immunity were significantly increased in the irradiated vaccine group as compared with conventional group. CONCLUSION: According to the results, gamma-irradiated IPV could induce humoral and cellular immunity in vaccinated mouse groups, so the irradiated poliovirus could be recommended as a good candidate vaccine to prevent the transport of poliovirus to the central nervous system and thus protect against paralysis.

Protective cellular and mucosal immune responses following nasal administration of a whole gamma-irradiated influenza A (subtype H1N1) vaccine adjuvanted with interleukin-28B in a mouse model.

The use of gamma-irradiated influenza A virus (γ-Flu), retains most of the viral structural antigens, represent a promising option for vaccine development. However, despite the high effectiveness of γ-Flu vaccines, the need to incorporate an adjuvant to improve vaccine-mediated protection seems inevitable. Here, we examined the protective efficacy of an intranasal gamma-irradiated HIN1 vaccine co-administered with a plasmid encoding mouse interleukin-28B (mIL-28B) as a novel adjuvant in BALB/c mice. Animals were immunized intranasally three times at one-week intervals with γ-Flu, alone or in combination with the mIL-28B adjuvant, followed by viral challenge with a high lethal dose (10 LD50) of A/PR/8/34 (H1N1) influenza virus. Virus-specific antibody, cellular and mucosal responses, and the balance of cytokines in the spleen IFN-γ, IL-12, and IL-4) and in lung homogenates (IL-6 and IL-10) were measured by ELISA. The lymphoproliferative activity of restimulated spleen cells was also determined by MTT assay. Furthermore, virus production in the lungs of infected mice was estimated using the Madin-Darby canine kidney (MDCK)/hemagglutination assay (HA). Our data showed that intranasal immunization with adjuvanted γ-Flu vaccine efficiently promoted humoral, cellular, and mucosal immune responses and efficiently decreased lung virus titers, all of which are associated with protection against challenge. This combination also reduced IL-6 and IL-10 levels in lung homogenates. The results suggest that IL-28B can enhance the ability of the vaccine to elicit virus-specific immune responses and could potentially be used as an effective adjuvant.

Evaluation of Immune Responses and Histopathological Effects against Gamma Irradiated Avian Influenza (Sub type H9N2) Vaccine on Broiler Chicken

Abstract Vaccination is a good strategy for the prevention of avian influenza virus. In this research Gamma Irradiated Avian Influenza (Sub type H9N2) Vaccine (GAIV) was prepared by 30 kGy irradiation and used for vaccination of broiler chickens. The purpose was a comparison of immune responses in the two routes of administration for the GAIV vaccine; intranasal and subcutaneously, use of Montanide ISA70 and Trehalose accompanied with irradiated vaccine and compare with formalin vaccine. The Influenza Virus A/Chicken/IRN/Ghazvin/2001/H9N2 was irradiated and used for vaccine formulation, and formalin inactivated AIV was used as conventional vaccine. Chickens were vaccinated by GAIV with and without Trehalose, GAIV and formalin vaccines with ISA70, two routes of administration were intranasal and subcutaneously. All the vaccinated chickens showed a significant increase in antibody titration. The most significant increase of antibody titration was in irradiated vaccine plus Trehalose groups intranasal and subcutaneously. After the first and second intranasal vaccination, the amount of IFN-gamma increased in the irradiated vaccine plus Trehalose group compared to other groups. However, most of the vaccinated groups did not show any significant increase of IFN-α concentration. Histopathological examination revealed lymphocyte infiltration (++), foci dispersed of hemorrhage and edema in intranasal vaccination groups and in addition to these, thickening of alveolar septa was observed in the injection groups. GAIV vaccine can be a good candidate for vaccine preparation, and Trehalose as a stabilizer protects viral antigenic proteins, also makes more absorbance of antigen by the inhalation route. In vaccinated chickens the ulcers in injected vaccines were lower than intranasal vaccines.

Analysis of antigen conservation and inactivation of gamma-irradiated avian influenza virus subtype H9N2.

Avian influenza (AI) A subtype H9N2 virus belongs to Orthomyxoviridae family and causes low-pathogenic disease AI. The use of gamma-irradiated viral antigens has been developed in the production of effective vaccines. In this research, LPAIV H9N2 strain, A/Chicken/IRN/Ghazvin/2001, was multiplied on SPF eggs and irradiated by a Nordian gamma cell instrument. Irradiated and non-irradiated AI virus (AIV) samples were titrated by EID50 method and hemagglutinin (HA) antigen was analyzed by HA test as the WHO pattern method. Infectivity of irradiated virus was determined by egg inoculation method during four blind cultures. The results showed that after increasing the dose of gamma radiation, virus titer gradually decreased. D10 value and optimum dose for complete virus inactivation were calculated by dose/response curve, 3.36 and 29.52 kGy, respectively. In addition, HA antigenicity of gamma-irradiated virus samples from 0 to 30 kGy was not changed. The results of safety test for gamma-irradiated AIV samples showed complete inactivation with gamma ray doses 30 and 35 kGy, without any multiplication on eggs after four blind cultures. According to the results of HA antigen assay and safety test, the gamma-irradiated and complete inactivated AIV subtype H9N2 is a good candidate as an inactivated immunogenic agent for poultry vaccination.

Development of Protective Immunity against Inactivated Iranian Isolate of Foot-and-Mouth Disease Virus Type O/IRN/2007 Using Gamma Ray-Irradiated Vaccine on BALB/c Mice and Guinea Pigs.

OBJECTIVES: Foot-and-mouth disease virus (FMDV) causes a highly contagious disease in cloven-hoofed animals and is the most damaging disease of livestock worldwide, leading to great economic losses. The aim of this research was the inactivation of FMDV type O/IRN/1/2007 to produce a gamma ray-irradiated (GRI) vaccine in order to immunize mice and guinea pigs. METHODS: In this research, the Iranian isolated FMDV type O/IRN/1/2007 was irradiated by gamma ray to prepare an inactivated whole virus antigen and formulated as a GRI vaccine with unaltered antigenic characteristics. Immune responses against this vaccine were evaluated on mice and guinea pigs. RESULTS: The comparison of the immune responses between the GRI vaccine and conventional vaccine did not show any significant difference in neutralizing antibody titer, memory spleen T lymphocytes or IFN-γ, IL-4, IL-2 and IL-10 concentrations (p > 0.05). In contrast, there were significant differences in all of the evaluated immune factors between the two vaccinated groups of mice and negative control mice (p < 0.05). The protective dose 50 for the conventional and GRI vaccines obtained were 6.28 and 7.07, respectively, which indicated the high potency of both vaccines. CONCLUSION: GRI vaccine is suitable for both routine vaccination and control of FMDV in emergency outbreaks.