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The grass carp reovirus (GCRV) is the most major pathogen that has threatened the grass carp (Ctenopharyngodon idella) industry of China for years. Though the oral vaccine has many advantages, the current vaccines still do not provide complete protection. Therefor the exploration of new preventive strategies is urgently needed. In this study, heat-labile enterotoxin B subunit of Escherichia coli (LTB) was combined with VP6 from GCRV type II (GCRV-II) via Lactococcus lactis expression system to form a potent oral vaccine and determines if fusion of LTB to the protective vaccine antigen can enhance protection in the fish. The expression of recombinant protein was confirmed by Western-blotting and enzyme-linked immunosorbent assay. The rare minnow was set as the model for the evaluation of the experiment administrated orally. The immune response including the antibody titer and the immune-related gene expression, and the protective efficacy which included the virus loaded and the relative protection, were thoroughly investigated after the trial. The results indicated that LTB can significantly elicit a higher neutralizing antibody responses and enhanced T-cell priming, activities and proliferation in mononuclear cells from intestine, spleen and kidney tissues when compared to the VP6 vaccine alone. Moreover, the combined adjuvant can significantly up-regulate type I interferon signaling in different immune organs, especially the mucosa associated lymphoidtissue which could not be induced by VP6 along, result in the contribution of the improvement in adaptive immune responses of the fish. In addition, challenge study showed that LTB combined VP6 could greatly improve the relative percent survival of the fish during the virus infection. These results highlight that LTB has the potential value to be a mucosal adjuvant of the fish, approaching for improving the efficacy of vaccination against GCRV-II, which does elicit both non-specific and specific immune responses., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Grass carp reovirus (GCRV) is a severe virus that causes great losses to grass carp culture every year, and GCRV-II is the current popular and fatal strain. VP56, fibrin on the outer surface of GCRV-II, mediates cell attachment. In this study, we firstly divided the VP56 gene into four fragments to screen the optimal antigen by enzyme-linked immunosorbent assay and neutralizing antibody methods. The second fragment VP56-2 demonstrates the optimal efficiency and was employed as an antigen in the following experiments. Bacillus subtilis were used as a carrier, and VP56-2 was expressed on the surface of the spores. Then, we performed the oral immunization for grass carp and the challenge with GCRV-II. The survival rate was remarkably raised, and mRNA expressions of IgM were significantly up-regulated in spleen and head kidney tissues in the B. s -CotC-VP56-2 group. Three crucial immune indexes (complement C3, lysozyme and total superoxide dismutase) in the sera were also significantly enhanced. mRNA expressions of four important genes ( TNF-α , IL-1β , IFN1 and MHC-II ) were significantly strengthened. Tissue lesions were obviously attenuated by histopathological slide examination in trunk kidney and spleen tissues. Tissue viral burdens were significantly reduced post-viral challenge. These results indicated that the oral recombinant B. subtilis VP56-2 subunit vaccine is effective for controlling GCRV infection and provides a feasible strategy for the control of fish virus diseases.

Vaccination is the most effective way to control the grass carp haemorrhagic disease (GCHD) with the primary pathogen grass carp reovirus genotype II (GCRV-II). However, due to the large difference in breeding conditions and unclear genetic background of grass carp, the results of the experiment were not reliable, which further hinders the effective prevention and control of GCHD. The rare minnow (Gobiocypris rarus) is highly sensitive to GCRV. Its small size, easy feeding, transparent egg membrane, and annual spawning are in line with the necessary conditions for an experimental aquatic animals culture object. In this study, immunogenicity and protective effects of attenuated and inactivated viruses for grass carp and rare minnow were evaluated in parallel. The expression of immune-related genes increased statistically significant after immunization. With the rise of specific serum antibody titers, the results of rare minnow and grass carp were consistent. In addition, there was no significant residue of adjuvant observed in both fish species injected with an adjuvanted and inactivated virus. Challenge of immunized grass carp and rare minnow with the isolate HuNan1307 resulted in protection rates of 95.8% and 92.6% for attenuated virus, 81.4% and 77.7% for inactivated virus, respectively, as well as the viral load changed consistently. The results indicated that rare minnow can be used as a model for evaluation of experimental vaccines against GCHD., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Grass carp reovirus (GCRV) is the main viral pathogen that endangers grass carp seriously. Application of vaccine has been considered to be the most effective way to prevent virus infection. VP56 is a protein encoded by gene segment 7 of grass carp reovirus, and is predicted to share homology with fiber protein of mammalian reovirus (MRV). In our study, the immunogenicity of VP56 was evaluated by neutralization test. GCRV was incubated with mouse anti-VP56 antibody, and then was injected into grass carp. Results showed that disease progress and death occurrence was hindered in the experimental group compared with the control group. For further study, the recombinant VP56 protein (rVP56) expressed by pET-32a (+) vector was purified, and was used as subunit vaccine to immunize grass carp. After each fish (15 ± 1.5 g) was injected with 30 μg purified rVP56 intraperitoneally, the immune protective efficacy of recombinant VP56 protein was assessed by a series of immune parameters. The population of red blood cells in immunized fish increased significantly after 5 d post injection (dpi), and reached a peak with (2.98 ± 0.17) × 10 9 /ml at 7 dpi (p < 0.05). The numbers of white blood cells peaked with (8.42 ± 1.01) × 10 7 /ml at 7 dpi (p < 0.05). Additionally, the percentage of monocytes and neutrophils rose to a peak with (9.05 ± 0.92)% and (25.93 ± 2.60)% respectively at 5 dpi (p < 0.05 or p < 0.01), whereas lymphocytes reached the highest value of (85.81 ± 2.73) % at 14 dpi (p < 0.01). Serum antibody titer in the vaccinated fish increased significantly and reached a peak at 21 dpi (p < 0.01). The mRNA expression levels of type I interferon (IFN1), major histocompatibility complex class I (MHC I), Toll-like receptor 22 (TLR22), and immunoglobulin M (IgM) were significantly up-regulated in head kidney and spleen (p < 0.05 or p < 0.01). The GCRV challenge test showed that the relative survival rate in immunized group was 71%-75%. Collectively, the results indicated that rVP56 protein can induce immune protection in grass carp, and can be consider as a candidate vaccine against GCRV infection., (Copyright © 2019. Published by Elsevier Ltd.)

Grass carp reovirus (GCRV) is one of the most serious pathogens threatening grass carp (Ctenopharyngodon idellus) production and results in high mortality in China. VP7 from GCRV is involved in viral infection and could be suitable for developing vaccines for the control of GCRV infection. To obtain a genetically engineered vaccine and a plant-based oral vaccine and to evaluate their immune efficacy as an oral vaccine against GCRV, cholera toxin B subunit (CTB) of Vibrio cholerae fused to VP7 (CTB-VP7) was transformed into BL21(DE3) for expression. SDS-PAGE and Western blotting showed that the purified CTB-VP7 fusion protein (rCTB-VP7) was approximately 49.0 kDa. Meanwhile, CTB-VP7 was transformed into rice callus cells by Agrobacterium tumefaciens-mediated gene transformation. CTB-VP7 was integrated into the nuclear genome by PCR, and mRNA transcripts of CTB-VP7 were detected. ELISA and Western blot analyses revealed that the CTB-VP7 fusion protein (CTB-VP7) could be expressed in rice callus lines. The level of expression was determined to be 1.54% ± 0.43 of the total soluble protein. CTB-VP7 showed a binding affinity for monosialoganglioside(GM1), a receptor for CTB. CTB-VP7 showed a higher affinity towards GM1 compared to rCTB-VP7. CTB-VP7 bonded to GM1 with different affinities under different temperatures. Maximum binding of CTB-VP7 to GM1 was reported to occur within 2 h at 37 °C, and approximately half of the binding affinity remained at 25 °C. Our results suggest that CTB-VP7 could be produced in rice calli, increasing the possibility that edible plants can be employed in mucosal vaccines for protection against GCRV in aquaculture., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Grass carp (Ctenopharyngodon idellus) hemorrhagic disease (GCHD), caused by grass carp reovirus (GCRV), has given rise to an enormous loss in grass carp industry during the past years. Up to date, vaccination remained to be the most effective way to protect grass carp from GCHD. Oral vaccination is of major interest due to its advantages of noninvasive, time-saving, and easily-operated. The introduction of oral vaccination has profound impact on aquaculture industry because of its feasibility of extensive application for fish in various size and age. However, the main challenge in developing oral vaccine is that antigens are easily degraded and are easy to induce tolerance. Bacillus subtilis (B. subtilis) spores would be an ideal oral vaccine delivery system for their robust specialty, gene operability, safety and adjuvant property. VP4 protein is the major outer capsid protein encoded by GCRV segment 6 (S6), which plays an important role in viral invasion and replication. In this study, we used B. subtilis spores as the oral delivery system and successfully constructed the B. subtilis CotC-VP4 recombinant spores (CotC-VP4 spores) to evaluate its protective efficacy in grass carp. Grass carp orally immunized with CotC-VP4 spores showed a survival rate of 57% and the relative percent survival (RPS) of 47% after the viral challenge. Further, the specific IgM levels in serum and the specific IgZ levels in intestinal mucus were significantly higher in the CotC-VP4 group than those in the Naive group. The immune-related genes including three innate immune-related genes (IL-4/13A, IL-4/13B, CSF1R), four adaptive immune-related genes (BAFF, CD4L, MHC-II, CD8), three inflammation-related genes (IL-1β, TNF-α, TGF-β) and interferon type I (IFN-I) related signaling pathway genes were significantly up-regulated in the CotC-VP4 group. The study demonstrated that the CotC-VP4 spores produced protection in grass carp against GCRV infection, and triggered both innate and adaptive immunity post oral immunization. This work highlighted that Bacillus subtilis spores were powerful platforms for oral vaccine delivery, and the combination of Bacillus subtilis spores with GCRV VP4 protein was a promising oral vaccine., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Although some commercial vaccines against grass carp reovirus (GCRV) are available, given the many varieties of GCRV and limited types of vaccines, the disease caused by GCRV remains a major problem, which leads to economic losses in grass carp aquaculture. A reovirus strain (GCRV-HN14) was recently isolated from local diseased fish in our laboratory. The S11 segment of GCRV-HN14 was speculated to encode the virus capsid protein VP35. In our study, the S11 segment was cloned into the eukaryotic expression vector pcDNA3.1(+) to construct the recombinant plasmid pcDNA3.1-s11, which was then transfected into CIK cells, and the VP35 protein was successfully expressed. Grass carp was immunized with pcDNA3.1-s11, and the in vivo distribution and expression of the pcDNA3.1-s11 plasmids were analyzed by PCR and Western blot. Recombinant plasmids were detected in the blood, liver, spleen, kidney, and muscle. However, protein expression could only be detected in the muscle. The immune protection of the pcDNA3.1-s11 plasmid in grass carp was evaluated using a series of experiments. Results showed that the population of white blood cells significantly increased at 1, 7, and 14 days post-immunization (dpi) and reached a peak with (9.58 ± 0.72) × 10 7 /ml at 7 dpi (P < 0.01 or P < 0.05). The percentage of neutrophils reached a peak with (24.13 ± 2.38)% at 7 dpi (P < 0.01), whereas the lymphocytes peaked with (93.30 ± 4.71)% at 14 dpi (P < 0.05). Serum antibody levels were significantly enhanced in immunized fish at 14, 21, and 28 dpi (P < 0.01). The mRNA expression levels of type I interferon, immunoglobulin M, Toll-like receptor 22, and major histocompatibility complex class I were significantly up-regulated in the head kidney and spleen of immunized fish (P < 0.05). Grass carp immunized with pcDNA3.1-s11 exhibited a higher survival percentage (70.4%-73.3%) than the controls (5%-13%). Overall, as a DNA vaccine, the pcDNA3.1-s11 plasmid could induce immune protection against GCRV., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Grass carp reovirus (GCRV) is the causative agent of a hemorrhagic disease that causes severe economic losses in the grass carp (Ctenopharyngodon idella) farming industry in China. Discrimination between wild-type field and vaccine strains of GCRV is crucial for meaningful disease diagnosis and epidemiological investigation, yet current detection methods do not discriminate between these different viruses. This study exploited sequence differences between vaccine viruses and the virulent and field strains in the S6 gene that is present in all GCRV strains to develop a high resolution melting curve assay to differentiate between virus strains. The high resolution melting curve analysis was as analytically sensitive as real-time quantitative-Polymerase Chain Reaction (qPCR) detection and at least 10 times sensitive than the conventional PCR. This one-step assay will facilitate grass carp hemorrhagic disease outbreak responses and control., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Grass carp reovirus II (GCRV II) causes severe hemorrhagic disease with high mortality in grass carp, Cyenopharyngodon idellus. DNA vaccination has been proven to be a very effective method in conferring protection against fish viruses. However, DNA vaccines for GCRV II have not yet been conducted on grass carp. In the current work, we vaccinated grass carp with a DNA vaccine consisting of the segment 6 (pC-S6; encoding VP4) or 10 (pC-S10; encoding NS38) of GCRV II and comparatively analyzed the immune responses induced by these two vaccines. The protective efficacy of pC-S6 and pC-S10, in terms of relative percentage survival (RPS), was 59.9% and 23.1% respectively. This suggests that pC-S6 and pC-S10 DNA vaccines could increase the survival rate of grass carp against GCRV, albeit with variations in immunoprotective effect. Immunological analyses indicated the following. First, post-vaccination (pv), both pC-S6 and pC-S10 up-regulated the expression of interferon (IFN-1), Mx1, IL-1β, and TNF-α. However, CD4 and CD8α were up-regulated in the case of pC-S6 but not pC-S10. Second, comparing non-vaccinated and pC-S10-vaccinated fish, the T cell response related genes, such as CD4, CD8α, and GATA3, were elevated in pC-S6-vaccinated fish at 48 h post-challenge (pc). Third, pC-S6 and pC-S10 induced similar patterns of specific antibody response pv. However, only anti-VP4 IgM in the sera of surviving fish infected with GCRV was significantly increased pc compared with that pre-challenge. Taken together, these results indicate that pC-S6 promotes both innate (IFN-1 and Mx1 induction) and adaptive (T cell and specific antibody response) immunity pv and that the induction of a memory state promptly primes the immune response upon later encounters with the virus, whereas pC-S10 only induces the type I IFN-related response pv and a lower inflammatory response pc., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The grass carp (Ctenopharyngodon idella) hemorrhagic disease, caused by grass carp reovirus (GCRV), is one of the most severe infectious diseases in aquaculture. Given that antiviral therapies are currently limitedly available, vaccination remains the most effective means for the prevention of viral diseases, such as GCRV. A reovirus strain, which was temporarily named GCRV-HN14, was recently isolated from grass carp in Henan province, China. The S11 gene fragment of GCRV-HN14 was speculated to encode viral structural protein VP35, which has no equivalent gene in other aquareviruses but has antigenic epitopes. In this study, the recombinant plasmid pET-32a-vp35 was constructed to express recombinant VP35 proteins in prokaryotic cells, which was used to create a novel subunit vaccine. The immune protection of recombinant VP35 protein was evaluated by a series of experiments in grass carp. Results showed that the number of white blood cells (WBC) in the peripheral blood increased significantly to 7.92 ± 0.72 × 10 7 /ml 5 days after vaccination (P < 0.05). The number of neutrophils and monocytes in WBC were significantly higher than those of the control 3 days after vaccination (P < 0.05) and maximally got to 12.22 ± 1.28% and 18.70 ± 1.78%, respectively. Owing to the significant increase in the number of lymphocytes (92.37 ± 2.10%; P < 0.01), the percentages of neutrophils and monocytes declined significantly (14 dpi; P < 0.01). Serum antibody levels induced by recombinant VP35 protein significantly increased 7 days post immunization and continued to increase until 5 weeks post vaccination. The mRNA expression levels of type I interferon (designated as IFN1), immunoglobulin M, Toll-like receptor 22 and major histocompatibility complex class I were up-regulated significantly in the head kidneys and spleens of immunized fish (P < 0.01). Grass carp immunized by recombinant VP35 protein showed that the relative percentage of survival was about 60% after it was challenged with GCRV. Overall, the results suggested that recombinant VP35 protein can induce immunity and protect grass carp against GCRV infection. Thus, it can be used as a subunit vaccine., (Copyright © 2018. Published by Elsevier Ltd.)

Infection with Grass carp reovirus (GCRV) is becoming unprecedentedly widespread in grass carp (Ctenopharyngodon idella) aquaculture industry, yet the management of GCRV infection still remains a challenge. Therefore, it is of importance to develop effective means against GCRV. As a delivery system of viral antigens, surface displaying of heterologous proteins on bacteria using anchoring motifs has successfully been implemented in human and veterinary vaccines research. In this study, a novel vaccine (BL21/InpN/vp7) was developed based on surface displaying a major capsid protein (vp7) of GCRV using the anchoring motif of N-terminal unique domain of ice-nucleation protein (InpN) on Escherichia coli BL21 (DE3) vaccine. Then the grass carp were immunized by surface displaying BL21/InpN/vp7 vaccine against GCRV using both intraperitoneal injection and bath immunization and their immune responses were tested. The results revealed that some non-specific immune parameters (acid phosphatase (ACP), alkaline phosphatase (AKP) and total antioxidant capacity (T-AOC)) were strongly increased in grass carp post injection inoculation (vp7 dose ranged from 10 to 20 μg). The specific antibody levels against GCRV and the transcriptional of immune-related genes (TNF-α, IL-1β, MHCI and IgM) were also significantly enhanced in grass carp by injection inoculation (vp7 dose ranged from 5 to 20 μg). On the other hand, only the highest dose of bath vaccination significantly induced the production of specific antibody and up-regulated transcriptions of several immune-related genes (IgM and MHCI) in grass carp. The lower cumulative mortality of grass carp in vaccinated groups after GCRV challenge clearly demonstrated that surface displayed vp7 vaccine could protect fish against GCRV infection. The relative percentage survival (RPS) value in injection vaccinated group (88.89%) was much higher compared to bath group (18.89%), which was in consistent with the production of specific serum antibodies, non-specific immune response and immune related genes expression. To sum up, our results indicated the surface display of heterologous antigenic proteins on E. coli BL21 (DE3) using the anchoring motif of ice-nucleation protein may provide a promising approach to the vaccine development of aquatic animals and suggested its potential to be used as vaccine to fight against GCRV infection., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Grass carp reovirus (GCRV) is one of the most pathogenic aquareovirus and can cause lethal hemorrhagic disease in grass carp (Ctenopharyngodon idella). However, management of GCRV infection remains a challenge. Therefore, it is necessary to find effective means for the control of its infection. The uses of bacterial ghost (BG, non-living bacteria) as carriers for DNA delivery have received considerable attentions in veterinary and human vaccines studies. Nevertheless, there is still no report about intramuscular administration of bacterial ghost-based DNA vaccines in fish. In the current study, a novel vaccine based on Escherichia coli DH5α bacterial ghost (DH5α-BG), delivering a major capsid protein gene (vp7) of grass carp reovirus encoded DNA vaccine was developed to enhance the efficacy of a vp7 DNA vaccine against GCRV in grass carp. The grass carp was injected intramuscularly by different treatments -i) naked pcDNA-vp7 (containing plasmid 1, 2.5 and 5 μg, respectively), ii) DH5α-BG/pcDNA-vp7 (containing plasmid 1, 2.5 and 5 μg, respectively) and iii) naked pcDNA, DH5α-BG or phosphate buffered saline. The immune responses and disease resistance of grass carp were assessed in different groups, and results indicated that the antibody levels, serum total antioxidant capacity (T-AOC), superoxide dismutase (SOD) activity, acid phosphatase (ACP) activity and alkaline phosphatase (AKP) activity and immune-related genes were significantly enhanced in fish immunized with DH5α-BG/pcDNA-vp7 vaccine (DNA dose ranged from 2.5 to 5 μg). In addition, the relative percentage survival were significantly enhanced in fish immunized with DH5α-BG/pcDNA-vp7 vaccine and the relative percentage survival reached to 90% in DH5α-BG/pcDNA-vp7 group than that of naked pcDNA-vp7 (42.22%) at the highest DNA dose (5 μg) after 14 days of post infection. Moreover, the level of pcDNA-vp7 plasmid was higher in DH5α-BG/pcDNA-vp7 groups than naked pcDNA-vp7 groups in muscle and kidneys tissues after 21 days. Overall, those results suggested that DH5α bacterial ghost based DNA vaccine might be used as a promising vaccine for aquatic animals to fight against GCRV infection., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

A vertically integrated monitoring program was set up for breeders hatched in 2013 and their offspring to detect differences in performance related to the reovirus vaccination schedule. Within the same organization in Belgium, 17 breeder flocks were vaccinated with one dose of live and one dose of inactivated reovirus vaccine, while 14 flocks received two doses of inactivated vaccine without live priming. The hatchability of the eggs produced by these birds was examined. Further, the daily growth, feed conversion, mortality, slaughterhouse condemnation, production index, and antibiotic use were monitored in 110 broiler flocks derived from the breeders. All gathered data were examined statistically. In eggs obtained from breeders vaccinated twice with inactivated reovirus vaccine, a significant 2.88% higher hatchability rate was observed. The progeny broiler flocks of these breeders showed a significant 18.2% lower mortality during the fattening period. Although not statistically significant, the slaughterhouse condemnation rate was 10.1% lower as well. The results may indicate that-under the epidemiologic conditions of this study-double administration of inactivated reovirus vaccine in broiler breeders can at least contribute to higher hatchability of breeder eggs and lower broiler mortality.

Grass carp (Ctenopharyngodon idella) hemorrhagic disease, caused by grass carp reovirus (GCRV), is emerging as a serious problem in grass carp aquaculture. There is no available antiviral therapy and vaccination is the primary method of disease control. In the present study, the immunological effects and protective efficacy of an inactivated HuNan1307 vaccine in grass carp were evaluated. The GCRV isolate HuNan1307 was produced by replication onto the grass carp PSF cell line, and inactivated with 1% β-propiolactone for 60 h at 4 °C. Grass carp were injected with inactivated GCRV vaccine, followed by challenge with the isolate HuNan1307. The results showed that the minimum dosage of the inactivated vaccine was 10(5.5) TCID50/0.2 mL to induce immune protection. All grass carp immunized with the inactivated vaccine produced a high titer of serum antibodies and GCRV-specific neutralizing antibody. Moreover, the inactivated vaccine injection increased the expression of 6 immune-related genes in the spleen and head kidney, which indicated that a immune response was induced by the HuNan1307 vaccine. In addition, grass carp immunized with the inactivated vaccine showed a survival rate above 80% after the viral challenge, equal to that of grass carp immunized with a commercial attenuated vaccine, and the protection lasted at least for one year. The data in this study suggested that the inactivated HuNan1307 vaccine may represent an efficient method to induce immunity against GCRV infection and the induced disease in grass carp., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

VP7, an outer capsid protein of grass carp reovirus (GCRV), was expressed and displayed on the surface of Saccharomyces cerevisiae for developing an efficient vaccine against hemorrhagic disease of grass carp. The result of flow cytometry analysis indicated that protein VP7 could be displayed on the surface of yeast cells after inducing with galactose. The expression of VP7 was confirmed by western blot analysis and further visualized with confocal microscopy. The specific antibodies against VP7 generated from mice were detectable from all immune groups except the control group, which was immunized with untransformed yeast cells. The displaying VP7 on glycosylation-deficient strain EBYΔMnn9 was detected to induce a relatively low level of specific antibody amongst the three strains. However, the antiserum of EBYΔM9-VP7 showed relative high capacity to neutralize GCRV. Further neutralization testing assays indicated that the neutralizing ability of antiserum of the EBYΔM9-VP7 group appeared concentration dependent, and could be up to 66.7% when the antiserum was diluted to 1:50. This result indicates that appropriate gene modification of glycosylation in a yeast strain has essential effect on the immunogenicity of a yeast-based vaccine.

To reduce the lethal hemorrhagic disease caused by grass carp reovirus (GCRV) and improve the production of grass carp, efficient and economic prophylactic measure against GCRV is the most pressing desired for the grass carp farming industry. In this work, a novel SWCNTs-pEGFP-vp5 DNA vaccine linked vp5 recombinant in the form of plasmid pEGFP-vp5 and ammonium-functionalized SWCNTs by a chemical modification method was prepared to enhance the efficacy of a vp5 DNA vaccine against GCRV in juvenile grass carp. After intramuscular injection (1, 2.5 and 5 μg) and bath administration (1, 10, and 20 mg/L), the ability of the different immune treatments to induce transgene expression was analyzed. The results showed that higher levels of transcription and expression of vp5 gene could be detected in muscle tissues of grass carp in SWCNTs-pEGFP-vp5 treatment groups compare with naked pEGFP-vp5 treatment groups. Moreover, antibody levels, immune-related genes, and relative percentage survival were significantly enhanced in fish immunized with SWCNTs-pEGFP-vp5 vaccine. In addition, we found that a good immune protective effect was observed in bath immunization group; which at a concentration of 20 mg/L could reach the similar relative percentage survival (approximately 100%) in injection group at a dose of 5 μg. All these results indicated that ammonium-functionalized SWCNTs could provide extensive application prospect to aquatic vaccine and might be used to vaccinate fish by intramuscular injection or bath administration method., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

We developed adenovirus serotype 5 (Ad5) vectors displaying the sigma 1 protein from reovirus as mucosal vaccines. Ad5-sigma retargets to JAM-1 and sialic acid, but has 40-fold reduced gene delivery when compared to Ad5. While weaker at transduction, Ad5-sigma generates stronger T cell responses than Ad5 when used for mucosal immunization. In this work, new Ad5-fiber-sigma vectors were generated by varying the number of fiber β-spiral shaft repeats (R) between the fiber tail and sigma. Increasing chimera length led to decreasing insertion of these proteinsAd5 virions. Ad-R3 and R14 vectors effectively targeted JAM-1 in vitro while R20 did not. When wereused to immunize mice by the intranasal route, Ad5-R3-sigma produced higher serum and vaginal antibody responses than Ad5. These data suggest optimized Ad-sigma vectors may be useful vectors for mucosal vaccination., (Copyright © 2015 Elsevier Inc. All rights reserved.)

A suicidal DNA vaccine based on a Semliki Forest virus (SFV) replicon was evaluated for the development of a vaccine against novel duck reovirus (NDRV). The sigma C gene of NDRV was cloned and inserted into pSCA1, an SFV DNA-based replicon vector. After transfected into BHK-21 cells, the antigenicity of the sigma C protein was confirmed using an indirect immunofluorescence and Western blot assay. Immunogenicity was studied in ducklings. The results showed that NDRV-specific antibodies, neutralizing antibodies, IFN-γ and IL-4 were well induced in ducklings. Furthermore, all of the ducklings were protected against challenge with wild-type NDRV., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The uses of single walled carbon nanotubes (SWCNTs) as carriers for DNA delivery have received considerable attention in cell studies. DNA vaccination of fish has been shown to elicit durable transgene expression, but no reports exist on intramuscular administration of SWCNTs-DNA vaccine electrostatic complexes which prepared through non-covalent conjugation. In this study, we injected grass carp intramuscularly with a plasmid vector containing a major capsid protein gene (vp7) of grass carp reovirus as a) naked pcDNA-vp7, b) SWCNTs-pcDNA-vp7, c) empty plasmid vector, or phosphate buffered saline. After intramuscular administration, the ability of the different immune treatments to induce transgene expression was analyzed. The results indicated that higher levels of transcription and expression of the vp7 gene could be detected in muscle tissues of grass carp 28 days intramuscular injection in SWCNTs-pcDNA-vp7 treatment groups compare with naked pcDNA-vp7 treatment groups. Moreover, the serum respiratory burst activity, complement activity, lysozyme activity, superoxide dismutase activity, immune-related genes, antibody levels and relative percentage survival were significantly enhanced in fish immunized with SWCNTs-pcDNA-vp7 vaccine. The data in this study suggested that SWCNTs were promising carriers for plasmid DNA vaccine and might be used to vaccinate fish by intramuscular approach., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Objective: To investigate the impact of the injection of inactivated vaccine for hemorrhage of Grass carp on the expressions of main immune-related genes in spleen including immunoglobulin M (IgM), major histocompatibility complexI(MHC I), interferon I(IFN1), complement 3 (C3), interleukin-1β (IL-1β) and intelectin genes., Methods: Ctenopharyngoden idellus kidney (CIK) cells were inoculated with Grass carp reovirus (GCRV). Then inactivated vaccine was prepared by inactivating virus suspension with formaldehyde. Vaccine was 50- and 100-times diluted with normal saline. The experiments included four groups: undiluted group, 50-times diluted group, 100-times diluted group and normal saline control group. Then healthy Grass carps with the average body mass (20 ± 5) g were intraperitoneally injected with vaccine or normal saline separately for each group. Spleens were collected at different time points after injection (0, 6, 12, 24, 72 hours) and total RNA extraction was performed immediately. Real-time quantitative PCR (qRT-PCR) was used to detect mRNA expressions of the six immune-related genes., Results: Compared with normal saline control group, inactivated vaccine injection increased mRNA expressions of the six immune-related genes in the spleen of Grass carp, indicating that the vaccine stimulated the Grass carp to generate non-specific and specific immune responses. In the immunized groups, relative expressions of the genes intelectin, MHCI, IL-1β, C3 were all up-regulated at first and then declined as time went on. Relative expression of IFNIgene reached the peak at 6 hours post-injection and then decreased gradually to the normal level. Differently, relative expression of IgM gene increased continuously within 0-72 hours post-injection., Conclusion: The expressions of the six major immune-related genes were all up-regulated in the spleen of Grass carp injected with different doses of inactivated vaccine for hemorrhage of Grass carp.

Grass carp reovirus (GCRV), the most pathogenic aquareovirus, can cause fatal hemorrhagic disease in fingerling and yearling grass carp. Vaccination by injection is by far the most effective method of combating disease. However it is labor intensive, costly and not feasible to vaccinate large numbers of the fish. Thus, an efficient and economic strategy for the prevention of GCRV infection becomes urgent. Here, functionalized single-walled carbon nanotubes (SWCNTs) as carrier were used to manufacture SWCNTs-VP7 subunit vaccine with chemical modification. Different developmental stages of grass carps were immunized by VP7/SWCNTs-VP7 subunit vaccine against GCRV by intramuscular injection and bath immunization. The results indicate that better immune responses of grass carp immunized with the SWCNTs-VP7 subunit vaccine were induced in comparison with VP7 subunit vaccine alone. Immunization doses/concentrations are significantly reduced (about 5-8 times) to prevent GCRV infection in different developmental stages of grass carp with injection or bath treatment when SWCNTs carrier was used. A good immune protective effect (relative percentage survival greater than 95%) is observed in smaller size fish (0.2 g) with SWCNTs-VP7 bath immunization. In addition, serum respiratory burst activity, complement activity, lysozyme activity, superoxide dismutase activity, alkaline phosphatase activity, immune-related genes and antibody levels were significantly enhanced in fish immunized with vaccine. This study suggested that functionalized SWCNTs was the promising carrier for recombinant subunit vaccine and might be used to vaccinate fish by bath approach., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Grass carp reovirus Guangdong 108 strain (GCRV-GD108) was recently isolated in Guangdong province, China. M6 gene of GCRV-GD108 was speculated encoding virus major outer capsid protein VP4. Blast analysis showed that the amino acid sequence of GCRV-GD108 VP4 was homologous to the structural protein VP4 of known Aquareoviruses (27.3-32.9%). Immunogenicity prediction by DNAStar software revealed there were multiple B cell epitopes on GCRV-GD108 VP4. Prokaryotic expression vector pET32a was used to express VP4 recombinant protein (rVP4) in E. coli BL21(DE3) strain. As expected, the molecular weight of recombinant VP4 was about 87 kDa showed by SDS-PAGE result. Neutralization assay demonstrated that the rabbit polyclonal antibody of rVP4 could prevent virus infection efficiently. After 14 days immunization with the rVP4, grass carps were challenged with GCRV-GD108, the results showed that different doses of rVP4 (1 μg/g, 3 μg/g and 5 μg/g) all provided protection against virus infection (47-82%). The relative percent survival reached 82% in the group immunized with 3 μg/g of rVP4. ELISA revealed rVP4 induced high antibody titer in immunized fish. IgM expression levels in head kidney of grass carp were detected by RT-PCR, and the results showed that IgM expressed at a significantly higher level in immunization groups than in blank control, indicating the rVP4 can induce strong immune response. In conclusion, rVP4 is a candidate vaccine against GCRV-GD108., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: Grass carp reovirus (GCRV) is the causative pathogen of grass carp hemorrhagic disease, one of the major diseases damaging grass carp Ctenopharyngon idellus breeding industry in China. Prevention and control of the disease is impeded largely due to the lack of research in economic subunit vaccine development. This study aimed to evaluate the potential of viral outer shell protein VP5 as subunit vaccine., Methods: The vp5 gene was isolated from the viral genome through RT-PCR and genetically engineered to express the recombinant VP5 protein in E coli. The viral origin of the recombinant protein was confirmed by Western blot analysis with a monoclonal antibody against viral VP5 protein. Polyclonal antibody against the recombinant VP5 protein was prepared from mice. A microneutralization assay was developed to test its neutralizing ability against GCRV infection in cell culture., Results: The GST-VP5 fusion protein (rVP5) was produced from E. Coli with expected molecular weight of 90 kDa. The protein was purified and employed to prepare anti-VP5 polyclonal antibody from mice. The anti-VP5 antibody was found to neutralize GCRV through in vitro microneutralization assay and viral progeny quantification analysis., Conclusions: The present study showed that the viral VP5 protein was involved in viral infection and bacterially-expressed VP5 could be suitable for developing subunit vaccine for the control of GCRV infection.

Transgenic lines of Arabidopsis thaliana producing recombinant TC protein were developed. The S1 gene encoding sigmaC protein of an avian reovirus (ARV) was amplified by reverse-transcription PCR (RT-PCR). The amplified product was cloned into a plant-expression vector, pE1857, with a strong promoter for expression. The resulting construct with the BAR gene cassette for bialaphos selection was designated rpE-sigmaC and was introduced into Agrobacterium tumefaciens by electroporation. Agrobacterium containing the rpE-sigmaC constructs that transform A. thaliana and transgenic plants were selected using bialaphos selection. The presence of S1 transcript in plants and their relative level of expression were determined by real time RT-PCR. Western blot analysis further confirmed the presence of sigmaC protein in the plants. Transgenic lines with high levels of sigmaC protein were selected for immunization experiments using specific-pathogen free chickens. Recombinant sigmaC protein produced in plants induced a variety of immunoglobulin G (IgG) antibody responses in chickens. Recombinant protein administered either subcutaneously or orally in birds showed significant protection against challenge. Results suggested that the recombinant sigmaC protein produced in plants has the potential for large-scale vaccination against ARV in commercial poultry.

Mammalian orthoreoviruses (reoviruses) are highly tractable experimental models for studies of double-stranded (ds) RNA virus replication and pathogenesis. Reoviruses infect respiratory and intestinal epithelium and disseminate systemically in newborn animals. Until now, a strategy to rescue infectious virus from cloned cDNA has not been available for any member of the Reoviridae family of dsRNA viruses. We report the generation of viable reovirus following plasmid transfection of murine L929 (L) cells using a strategy free of helper virus and independent of selection. We used the reovirus reverse genetics system to introduce mutations into viral capsid proteins sigma1 and sigma3 and to rescue a virus that expresses a green fluorescent protein (GFP) transgene, thus demonstrating the tractability of this technology. The plasmid-based reverse genetics approach described here can be exploited for studies of reovirus replication and pathogenesis and used to develop reovirus as a vaccine vector.

A commercial reovirus vaccine alone or experimental reovirus vaccine plus antibody complex were inoculated into 18-day-old specific pathogen free (SPF) broiler embryos at 0.1 of the recommended chick dose. The following groups were used: group 1A was not vaccinated or challenged; group 1B was not vaccinated, but was challenged with virulent reovirus; group 2 received the vaccine complexed with 1/4 dilution of antiserum; group 3 received the vaccine with 1/8 dilution of antiserum; group 4 received the vaccine with 1/16 dilution of antiserum, and group 5 received vaccine alone. At 1, 3, 6, 9, and 12 days of age, serum was collected and antibody against avian reovirus was analyzed by enzyme-linked immunosorbent assay (ELISA). At the same times, spleens were collected and vaccine virus detected by inoculating chicken embryo fibroblasts (CEFs) and examining for cytopathic effect. At 15 days of age, chickens in groups 2-5 were challenged with reovirus. At 22 days of age, birds were euthanatized and weighed. Efficacy of the vaccines was based on safety, percent protection, and antibody response. In ovo vaccination with the commercial or experimental vaccines did not adversely affect hatchability of SPF chickens. The vaccine complexed with antibody resulted in significantly less posthatch mortality (3.7%) when compared to mortality of chickens that received vaccine alone (17%). Both vaccine virus recovery and antibody response were delayed at least 3 days in birds receiving the experimental vaccines. In evo administration of reovirus antibody complex vaccines provided at least 70% protection. The experimental reovirus-antibody complex vaccines were safe and efficacious when given in ovo to SPF broiler embryos.

Objective: Rotavirus gastroenteritis causes substantial morbidity, including hospital admission, in young children. In the context of recent vaccine developments, this study aimed to estimate the cost-effectiveness of a rotavirus vaccination program in Australia., Method: Standard methods of health economic evaluation were used to assess the total cost of rotavirus immunisation (as the difference between estimated vaccination program costs and the cost of disease that would be avoided by immunisation) and relate this to the number of cases of disease that would be prevented. Estimates were made from both societal and health care systems perspectives., Results: Based on Australian data on disease incidence and cost of hospitalisation, the current annual cost of rotavirus disease is about $26.0 million. Using conservative vaccine efficacy estimates, current immunization uptake rates and a cost of $30 per dose of vaccine, rotavirus immunisation would incur a net societal cost of $2.9 million ($11 per child), at a gross program cost of $21.6 million. These estimates are sensitive to two sources of uncertainty in the estimation of program delivery costs: vaccine price and whether separate immunization visits would be required., Conclusion: A rotavirus immunisation program would be cost-neutral to Australian society at a vaccine price of $26 per dose (or $19 when health care system costs only are considered)., Implications: Rotavirus immunization may be cost-effective in Australia, but considerable uncertainty remains. Policy decisions will depend heavily on pricing of the vaccine and may also need to consider intangible costs not accounted for in this analysis.

Coarse-spray (CS) administration of a commercial S1133 reovirus vaccine was evaluated in 1-day-old specific-pathogen-free broilers for prevention of clinical infection induced by intratracheal challenge with two enteric reovirus isolates. In Expt. 1, chickens were challenged at 4 days of age with either the 2408 or CO8 isolate. In Expt. 2, chickens were challenged at 7 days of age with either isolate. In Expt. 3, chickens were challenged at 3, 5, or 7 days of age with the 2408 isolate. In Expt. 1, vaccinated birds showed significant protection against challenge with either isolate at 4 days of age as measured by morbidity, mortality, gross lesions, and body weight. In Expt. 2, vaccinated birds showed greater protection against challenge at 7 days of age. In Expt. 3, resistance in vaccinated birds increased with time between vaccination and challenge. Vaccinated birds challenged at 3 days of age showed no significant protection, whereas vaccinated birds challenged at 5 or 7 days of age had increased resistance. This vaccine did not induce a drop in weight gain, morbidity, mortality, or microscopic lesions in the tendons.

A live commercial reovirus vaccine, Enterovax, was administered to adult broiler breeder hens via the drinking water to determine its efficacy in stimulation of circulating antibody. This vaccine was compared with a commercial inactivated reovirus vaccine. Only the inactivated product resulted in increased antibody as measured by a commercial enzyme-linked immunosorbent assay. However, the live reovirus vaccine caused diarrhea in the hens and decreased eggshell quality, fertility, and hatchability. In addition, the live vaccine virus was vertically transmitted from hens to their progeny, resulting in increased embryonic mortality and viral tenosynovitis.

Two commercially available live reovirus vaccines, alone or in combination with two infectious bursal disease virus (IBDV) vaccines, were evaluated for safety and efficacy in specific-pathogen-free leghorn chicks. Four trials were conducted to evaluate the vaccine combinations. At periodic intervals during the trials, tissues were collected and assayed for residual reovirus and examined for histological changes. Six weeks following reovirus vaccination, all treatment groups were challenged with a virulent field isolate of reovirus and sampled 1 week later for the final time. The two reovirus vaccines were safe and effective if given at 1 week of age, regardless of whether the vaccinates had been exposed to IBDV at 1 day. However, both reovirus vaccines persisted in the tendons of 1-day-old vaccinates. The effects of IBDV vaccines were generally minor and reflected by increases in the number of pre-challenge or post-challenge virus recoveries from some of the treatment groups receiving both type vaccines.

Coarse-spray (CS) administration of a commercial S1133 reovirus vaccine in chickens for prevention of clinical viral tenosynovitis (VT) infection was evaluated. In Expt. 1, one-day-old specific-pathogen-free (SPF) white leghorns were vaccinated with a combination of reovirus, Newcastle disease (ND), and infectious bronchitis (IB) vaccines by CS and infectious bursal disease vaccine by the subcutaneous (SQ) route. In Expt. 2, one-day-old commercial broilers were vaccinated by CS with reovirus vaccine and Marek's disease (MD) vaccine by SQ. In Expt. 3, one-day-old commercial broilers received reovirus vaccine in combination with ND-IB vaccines at 1 day of age by CS and MD vaccine by SQ. Some birds received an initial or second vaccination at 7 days of age by CS or the drinking-water (DW) route. Birds vaccinated by CS at 1 day of age with reovirus vaccine did not produce circulating virus-neutralizing antibody against reovirus, although they had resistance to VT infection. In contrast, initial or booster vaccination at 7 days of age by CS or DW resulted in an antibody response and greater resistance to challenge than did CS vaccination at 1 day of age. There was no difference in efficacy between CS and DW routes at 7 days of age. The reovirus vaccine did not interfere with other vaccines as measured by serologic (ND-IB-IBD) or challenge (MD) studies.

Day-old chicks with maternally derived infectious bursal disease (IBD) antibodies were inoculated with IBD oil emulsion vaccine. The vaccine protected at least 85 per cent of the chicks when they were challenged at four weeks old with virulent IBD virus and at least 90 per cent of those challenged at seven weeks of age. There was no benefit in using a combination of the oil emulsion vaccine and a live IBD vaccine.

Bluetongue virus proteins derived from baculovirus expression vectors have been administered in different combinations to sheep, a vertebrate host susceptible to bluetongue virus, and the neutralizing antibody responses were measured. Vaccinated sheep were subsequently challenged, and the indices of clinical reaction were calculated. The results indicated that the outer capsid protein VP2 alone in doses of greater than 50 micrograms per sheep elicited protection. A dose of ca. 50 micrograms of VP2 protected some but not all sheep. However, when used in combination with ca. 20 micrograms of the other outer capsid protein, VP5, 50-micrograms quantities of VP2 not only protected all the vaccinated sheep but also elicited a higher neutralizing-antibody response. The addition of viral core proteins VP1, VP3, VP6, and VP7, the nonstructural proteins NS1, NS2, and NS3, and the outer capsid proteins VP2 and VP5 did not enhance this neutralizing-antibody response.

This study was conducted to test the pathogenicity and immunosuppressive effects of seven commercially available infectious bursal disease (IBD) vaccines. These vaccine strains are intermediate in their pathogenicity in susceptible specific-pathogen-free (SPF) chickens. One-day-old and 3-week-old SPF chickens were vaccinated with these vaccines. Two weeks after IBD vaccination, they were vaccinated with Newcastle disease virus (NDV). The pathogenic and immunosuppressive effects of the IBD vaccines were evaluated by the antibody response to NDV vaccination, the bursa: body weight index, and histopathological lesions of the bursa. It was found that these strains were highly variable in their virulence and immunosuppressive properties. Three of the strains tested were found to be highly virulent and immunosuppressive; two others were moderate; and two could be classified as mild.

Experiments determined the efficacy of live vaccines in specific-pathogen-free broilers against serologic subtypes of infectious bursal disease virus (IBDV). Challenge isolates were the Delmarva variant E, a standard serotype I (APHIS) and a variant isolate from Mississippi. The vaccines were a cloned standard (CS) vaccine (Clone Vac-D78), a cloned variant (CV) vaccine (Bursa Vac IV), and an uncloned standard (UCS) vaccine (Bursine II). The severity of microscopic lesions was correlated with bursal atrophy as measured by bursa-weight-to-body-weight ratios. All vaccines provided adequate protection against the APHIS challenge. The three vaccines averaged 77% protection against APHIS in the first experiment and 78% in the second. Protection against the variant E and Miss isolates was considerably less for all vaccines. The three vaccines produced an average 70% protection against the Miss isolate in the first experiment and 69% in the second experiment. Against the variant E virus, the three vaccines averaged 67% protection in the first experiment and 65% in the second. There were significant differences in protection for each vaccine against individual IBDV subtypes. Results showed that no vaccine provided good protection (at least 80%) against all three subtypes of IBDV.

Convalescent serum given to 1-day-old poults delayed clinical signs of turkey coryza by several days and reduced mortality on infected farms. Turkey breeders immunized with cell-culture-adapted infectious bursal disease virus (IBDV) or turkey infectious bursal disease virus (TIBDV) had a marked increase in virus-neutralization (VN) antibody titers. The VN antibody titer was significantly higher in progeny poults than in poults from unimmunized breeders. Clinical turkey coryza and mortality was considerably less in poults from IBD- or TIBD-vaccinated breeders than in control poults. They also responded more favorably to hemorrhagic enteritis and fowl cholera vaccination.

A mixed breed flock of lambs, consisting of Suffolks, Hampshires, Columbias and Finnish breeds, were vaccinated with binary ethylenimine inactivated blue-tongue virus (BTV) serotypes 11, 17 and a mixture of 11 and 17 in aluminum hydroxide. Agar gel precipitin antibodies were used as an indicator of immunity. Sero-conversion of Hampshires and Suffolk lambs was poor at 43% as compared to 84% in the Columbia and Finn lambs. These results indicate a breed difference in immunological response to inactivated BTV vaccine.

A computer-assisted single-serum-dilution indirect kinetic-based enzyme-linked immunosorbent assay (KELISA) was used for quantitating the natural decay rate of infectious bursal disease virus (IBDV) maternal antibody in progeny obtained from white leghorn breeders. The KELISA results were compared with those of a standard virus-neutralization (VN) test. Pullets were subjected to two IBDV immunization regimens. Group 1 was vaccinated at weeks 0, 2, and 10 with two live vaccines in drinking water and at week 20 with an oil-emulsified (SC) IBDV vaccine, group 2 received only the first and last immunizations, and group 3 served as the unvaccinated control. Pullets were artificially inseminated at 28 weeks of age. Progeny chicks from each group were bled every other day for 47 days. Both KELISA and VN test detected linear relationship in the decay of maternal antibodies. The VN test detected no significant differences (P greater than 0.05) in the IBDV maternal antibody titers at day 1 or in the rate of decay between the progeny from groups 1 and 2. The VN maternal antibody titers decreased at a rate of 0.16 log2 titer per day. In contrast, KELISA revealed higher IBDV maternal antibody titers in day-old progeny from pullets vaccinated 4 times (log2 = 14.3). However, KELISA titers of progeny from this group decreased at a faster rate than titers of progeny from pullets vaccinated twice (0.20 vs. 0.13 log2 titer per day).

Inactivated Newcastle disease virus (NDV) and infectious bursal disease virus (IBDV) were incorporated into water-in-oil emulsion vaccines alone or as a bivalent vaccine. Twenty-week-old broiler breeder chickens that had received previous live virus vaccination with NDV and IBDV were injected intramuscularly with the monovalent or bivalent vaccine. The antibody titers to either the monovalent vaccine or bivalent vaccine increased rapidly and then remained at high levels for the duration of the 40-week trial. There were no practical differences in amplitude or duration of the antibody response to either antigen used alone compared to that of the bivalent combination. Progeny hatched from the vaccinated breeders possessed maternal antibody levels at one day of age comparable to those of the hens at the time the eggs were laid. The maternal antibody titers declined at a steady rate until they reached negligibly detectable levels at approximately 3 weeks of age. This trend held true without regard to the initial antibody titer.

Four different oil emulsion infectious bursal disease virus (IBDV) vaccines were inoculated into four-week-old specific pathogen-free chickens. At weekly intervals for five weeks, sera were obtained from the vaccinated birds and from uninoculated control birds and examined for antibodies against IBDV by enzyme-linked immunosorbent assay (ELISA), the quantitative agar gel precipitin (QAGP) test and the virus neutralisation (VN) test. There was a highly significant correlation between the mean responses to all tests; the highest correlation (0.818) was between VN and QAGP and the lowest (0.573) between QAGP and ELISA. Generally the ELISA detected positive sera earlier than the VN test which in turn was more sensitive than the QAGP test. The ELISA and QAGP test were less variable, more reproducible and easier to perform than the VN test.

The serological response of chicks to Brucella abortus strain 19 was monitored over a period of seven weeks to assess the degree of immunosuppression caused by vaccination at one day of age with two infectious bursal disease vaccines. One of the vacy. This vaccine caused severe immunosuppression judged by the minimal serological response following B abortus inoculation. The test also detected a significant delay caused by the other vaccine in the development of the serological response but the maximum titre was not significantly different from that in chicks which had received no infectious bursal disease vaccine.

The experiment involved 392,955 broilers from breeders vaccinated with a commercially available killed infectious bursal disease (KIBD) virus vaccine (Treatments 1 and 2) and 373,371 broilers from breeders not vaccinated with a KIBD virus vaccine (Treatment 2). The broilers from the KIBD-vaccinated breeders were divided into two treatment groups: Treatment 1 consisted of 209,457 broilers and Treatment 2 consisted of 183,498 broilers. Treatment 2 broilers were vaccinated at 10 days of age intraocularly with a commercially available modified liver infectious bursal disease virus (IBD-MLV) vaccine. Broilers in all three treatments were raised on commercially contracted farms serviced by the same company representative. Broiler performance was based on mortality, body weight, feed efficiency, condemnations, and how each farm ranked with respect to contract settlements. Compared with birds in Treatments 2 and 3 respectively, Treatment 1 broilers had 1.65% and 0.89% less mortality; 0.22 lbs. and 0.09 lbs. increased weight per bird; 0.03 and 0.03 increased feed efficiency; 0.09% and 0.32% fewer air sacculitis condemnations; and 0.57% and 0.83% fewer total condemnations. Treatment 1 broilers ranked in the top 37%; Treatment 2 ranked in the bottom 36%; and Treatment 3 ranked in the lower 57% of contract settlements for all broilers processed. The results clearly showed that Treatment 1 broilers outperformed Treatment 2 amd 3 birds and that the IBD-MLV vaccine had a detrimental effect on Treatment 2 broilers.

A comparison was made between four inactivated infectious bursal disease (IBD) oil emulsion vaccines. Vaccine A, which was prepared from antigen derived from bursae of Fabricius, stimulated higher levels of IBD gel diffusion titres than vaccines B, C and D, which were prepared from antigens derived from embryo fluids. The progeny of parents vaccinated with vaccine A resisted IBD challenge for eight days longer than the progeny of any of the other vaccinated parents.

A 14-year-old male drank two glasses of milk from a gallon inoculated with 21 vials of live virus vaccine intended to immunize 1000 baby chicks against Newcastle Disease. The patient was managed with catharsis and careful observation, and remained completely asymptomatic in the following 28 days. Newcastle Disease virus (NDV) may cause paralytic neurotropic, viscerotropic, or pneumonotropic disease in chickens, but has limited effects on humans. Systemic exposure to this vaccine has not previously been reported in humans, although an aerosol NDV vaccine has caused self-limited conjunctivitis following ocular exposure. The human effects of veterinary biologicals remain poorly defined. The limited literature existing in regards to the human experience with common veterinary vaccines is reviewed.