Influence of Temperature on the Efficacy of Homologous and Heterologous DNA Vaccines against Viral Hemorrhagic Septicemia in Pacific Herring

Abstract

Homologous and heterologous (genogroup Ia) DNA vaccines against viral hemorrhagic septicemia virus (genogroup IVa) conferred partial protection in Pacific Herring Clupea pallasii. Early protection at 2 weeks postvaccination (PV) was low and occurred only at an elevated temperature (12.6°C, 189 degree days), where the relative percent survival following viral exposure was similar for the two vaccines (IVa and Ia) and higher than that of negative controls at the same temperature. Late protection at 10 weeks PV was induced by both vaccines but was higher with the homologous vaccine at both 9.0°C and 12.6°C. Virus neutralization titers were detected among 55% of all vaccinated fish at 10 weeks PV. The results suggest that the immune response profile triggered by DNA vaccination of herring was similar to that reported for Rainbow Trout Oncorhynchus mykiss by Lorenzen and LaPatra in 2005, who found interferon responses in the early days PV and the transition to adaptive response later. However, the protective effect was far less prominent in herring, possibly reflecting different physiologies or adaptations of the two fish species.

Received August 1, 2016; accepted March 10, 2017 Published online July 11, 2017     

Effect of booster shot and investigation of vaccination efficacy period against herpesviral haematopoietic necrosis (HVHN) in goldfish Carassius auratus

In this study, the efficacy period of an intraperitoneal vaccination and effect of a booster shot of vaccine against herpesviral haematopoietic necrosis (HVHN) in goldfish Carassius auratus were investigated. Cell culture supernatant of cyprinid herpesvirus 2 (CyHV-2), causative agent of HVHN, propagated in goldfish fin (GFF) cells was inactivated with formalin (0.1%, v/v) for 2 days at 4 °C. Three groups of the variety Ryukin were individually intraperitoneally injected with the vaccine and each group was separately maintained in replicate tanks. After 4 weeks (Vaccinated-4w-1 and 2) and 8 weeks (Vaccinated-8w-1 and 2) from the first vaccination, the fish were CyHV-2-challenged by the immersion route (10 TCID₅₀ l⁻¹). In addition, the other vaccinated group of fish were injected with a booster vaccine 4 weeks after the first vaccination as the Vaccinated-booster groups, then the fish of these groups were CyHV-2-challenged by the immersion route (10 TCID₅₀ l⁻¹) after 8 weeks from the first vaccination. The mean of the relative percentage survival (RPS) values of the Vaccinated-4w and 8w groups showed 42.5% and 57.6%, respectively. In addition, the mean RPS value of Vaccinated-booster groups showed 63.6%. Statistical analysis showed significantly higher survival rates in all the vaccinated groups than those of the respective negative control groups using Fisher's exact test. Moreover, the survival rates of vaccinated-booster groups were significantly higher (p = 0.036) compared with the respective control groups by Student's t test. The present study shows the efficacy period of the vaccine is at least 8 weeks and a booster shot showed a tendency to enhance the protection against HVHN in goldfish.     

DNA Vaccination of Rainbow Trout against Viral Hemorrhagic Septicemia Virus: A Dose–Response and Time–Course Study

Abstract

Viral hemorrhagic septicemia (VHS) in rainbow trout Oncorhynchus mykiss is caused by VHS virus (VHSV), which belongs to the rhabdovirus family. Among the different strategies for immunizing fish with a recombinant vaccine, genetic immunization has recently proven to be highly effective. To further investigate the potential for protecting fish against VHS by DNA vaccination, experiments were conducted to determine the amount of plasmid DNA needed for induction of protective immunity. The time to onset of immunity and the duration of protection following administration of a protective vaccine dose were also analyzed. The dose–response analysis revealed that significant protection of rainbow trout fingerlings was obtained following intramuscular injection of only 0.01 μg of plasmid DNA encoding the VHSV glycoprotein gene. In addition, higher doses of DNA induced immunity to a virus isolate serologically different from the isolate used for vaccine development. Following administration of 1 μg of a DNA vaccine, significant protection against VHS was observed in the fish as early as 8 d postvaccination. At 168 d postvaccination, the fish had increased in size by a factor of 10 and protection against a lethal dose of VHSV was still evident. The results confirm the great potential for DNA vaccination in inducing efficient immunoprophylaxis against viral diseases in aquacultured fish.     

Communications: Protective Immunity Induced by a Commercial Vibrio Vaccine in Hybrid Striped Bass

Abstract

Commercial Vibrio anguiliarum-V. ordalii bacterin was used to vaccinate hybrid striped bass (Morone saxatilis ♀ × M. chrysops ♂) to test the vaccine efficacy against vibriosis. Vaccination by direct immersion of fish in diluted Vibrio vaccine for 20 s resulted in increased protective immunity. The relative percent survival of hybrid striped bass challenged 35 d after vaccination was 66.7% for those challenged by 1-h immersion exposure to 7.03 × 10⁷ V. anguillarum cells/mL, 75.0% for those challenged by injection with 3.51 × 10⁵ cells/fish, and 86.7% for those challenged by injection with 3.51 × 10⁴ cells/fish.     

Prevention of Ulcer Disease in Goldfish by Means of Vaccination

Abstract

A vaccine comprising cells of Aeromonas bestiarum grown in tryptic soy broth and atypical A. salmonicida cells produced in iron-limited and iron-supplemented media protected goldfish Carassius auratus when administered by immersion (dosage ≈ 5 × 10⁷ cells/mL for 60 s) followed after 28 d by an oral booster (dosage = 5 × 10⁷ cells/g of feed), which was fed for 7 d so that each fish received about 1 g of vaccine-containing feed. After challenge by intramuscular injection of a virulent culture of atypical A. salmonicida, the relative percent survival (RPS) was more than 90%. The approach was more successful than using a commercial furunculosis vaccine with or without supplementation with A. bestiarum or atypical A. salmonicida cells. Moreover, a smooth derivative of the virulent rough culture of atypical A. salmonicida was less effective as a vaccine candidate, yielding an RPS of only 65%. Low antibody titers of 1:39–1:396 were found in the vaccinated fish. The vaccinated fish had a significantly higher proportion of dead head kidney macrophages (10.9 ± 3.5%; P = 0.0149) than did the controls (6.8 ± 3.1%). However, differences in the number of erythrocytes and leukocytes, the level of phagocytic and lysozyme activities, and the proportion of lymphocytes, monocytes, and polymorphonuclear cells were not statistically significant between the two groups.     

Comparative protection of two different commercial vaccines against Yersinia ruckeri serotype O1 and biotype 2 in rainbow trout (Oncorhynchus mykiss)

Differentially extended specific protection by two commercial vaccines against Yersinia ruckeri serotype O1 biotype 2 was studied following 30 s immersion exposure. Rainbow trout were challenged intra-peritoneally (i.p.) with Y. ruckeri serotype O1, biotype 2 (≈10⁶ to 10⁷ CFU/fish) at 4, 6 and 8 months after vaccination with vaccines containing either biotype 1 (AquaVac^® ERM) or both biotypes 1 and 2 (AquaVac^® RELERA?). The specific pattern of vaccine-mediated protection was evaluated by relative percentage survival (RPS) analysis at 4 and 6 months post-vaccination and by obtaining gross pathological observations at 4 and 8 months respectively. We determined specific significant and superior protection in terms of increased survivability in AquaVac^® RELERA? vaccinated fish and observed correspondingly fewer pathological changes. The challenge trials indicated a longer protection for at least 6 months without any booster vaccination. A specific and adaptive response induced by AquaVac^® RELERA? vaccine against Y. ruckeri biotype 2 was clearly indicated. In addition, some degree of cross protection rendered by AquaVac^® ERM containing biotype 1 during infection with Y. ruckeri biotype 2 was also noted.     

Conjunctival vaccination of the brushtail possum (Trichosurus vulpecula) with bacille Calmette-Guérin

AIM: To determine the efficacy of conjunctival vaccination of captive brushtail possums (Trichosurus vulpecula) with bacille Calmette-Guérin (BCG), as measured by immunological responses to vaccination and response to intratracheal challenge with Mycobacterium bovis.

METHODS: Nine adult male brushtail possums were vaccinated by the instillation of a suspension of BCG strain Pasteur 1173P2 into the conjunctival sac of each eye. Each drop contained approximately 2.5 × 10⁵ colony forming units (cfu). At 8 weeks post-vaccination (pv) the vaccinated possums and 10 unvaccinated possums were challenged by intratracheal instillation of approximately 100 cfu of M. bovis. Cellular immune responses to bovine purified protein derivative (PPD) antigen were measured using the lymphocyte proliferation assay (LPA). Possums surviving to 50–51 days after challenge were euth anised and subjected to detailed post-mortem examination, including histopathology, to assess protection against tuberculosis. Sections of lung and spleen were cultured for M. bovis.

RESULTS: No conjunctival inflammation or other adverse reactions to the administration of the vaccine were evident macroscopically. The vaccinated group showed a systemic cellular immune response to bovine PPD antigen at 4 and 8 weeks pv, and the response at 8 weeks was significantly greater than at 4 weeks (p<0.05). Conjunctival vaccination induced significant levels of protective immunity, measured as less mass of tuberculous lesions in lung (p<0.05) and less dissemination of disease in vaccinated compared with unvaccinated possums (p<0.05).

CONCLUSIONS: Conjunctival vaccination with BCG induced a significant level of protective immunity against M. bovis infection in possums. This route of vaccination, together with intranasal aerosol vaccination, could be utilised in the delivery of an aerosolised vaccine using a device that sprays the vaccine suspension into the eyes and nose of possums.     

Oral Vaccination of Channel Catfish against Enteric Septicemia of Catfish Using a Live Attenuated Edwardsiella ictaluri Isolate

Enteric septicemia of catfish (ESC), caused by Edwardsiella ictaluri, is the most problematic bacterial disease affecting catfish aquaculture in the southeastern United States. Efforts to develop an effective ESC vaccine have had limited industrial success. In commercial settings, ESC vaccines are typically administered by immersion when fry are transferred from the hatchery to rearing ponds. While this approach is a practical method of mass delivery, this strategy administers vaccines to very young fish, which lack a fully developed immune system. To circumvent this limitation, an oral vaccination strategy was evaluated as a means of immunizing catfish at the fingerling stage of production, when fish possess a more complete immune arsenal. A virulent E. ictaluri isolate (S97-773) was attenuated by successive passage on media containing increasing concentrations of rifamycin. In laboratory trials, cultured vaccine was diluted and mixed with feed (100 mL diluted vaccine/454 g feed). This mixture was then fed to Channel Catfish Ictalurus punctatus fingerlings. Two separate dilutions of cultured vaccine (1:10 and 1:100) were used to create the vaccine–feed mixture, equating to estimated doses of 5 × 10⁷ and 5 × 10⁶ CFU/g of feed, respectively. After 30 d, catfish were exposed by immersion (1 × 10⁶ CFU/mL) to the virulent parental strain of E. ictaluri. The target dose (1:100 dilution, ～5 × 10⁶ CFU/g of feed) offered exceptional protection (relative percent survival = 82.6–100%). In addition, negligible deaths occurred in fish vaccinated at 10 times the target dose (1:10 dilution, ～5 × 10⁷ CFU/g of feed). In pond trials, antibody production increased 18-fold in orally vaccinated fish. When compared with nonvaccinated controls, vaccination significantly improved survival, feed fed, feed conversion, biomass produced, and total harvest. This research demonstrates Channel Catfish can be successfully immunized in a commercial setting against E. ictaluri with a single dose of an orally delivered, live attenuated, E. ictaluri vaccine.

Received July 31, 2014; accepted March 2, 2015     

Efficacy of an inactivated recombinant vaccine encoding a fimbrial protein of Pasteurella multocida B:2 against hemorrhagic septicemia in goats

This study was carried out to determine the antibody responses and protective capacity of an inactivated recombinant vaccine expressing the fimbrial protein of Pasteurella multocida B:2 following intranasal vaccination against hemorrhagic septicemia in goats. Goats were vaccinated intranasal with 10⁶ CFU/mL of the recombinant vaccine (vaccinated group) and 10⁶ CFU/mL of pET32/LIC vector without fimbrial protein (control group). All three groups were kept separated before all goats in the three groups were challenged with 10⁹ CFU/mL of live pathogenic P. multocida B:2. During the course of study, both serum and lung lavage fluid were collected to evaluate the antibody levels via enzyme-linked immunosorbent assay. It was found that goats immunized with the inactivated recombinant vaccine developed a strong and significantly (p < 0.05) higher specific IgA and IgG responses in both serum and lung lavage fluid samples compared to the control and unvaccinated groups. Following intratracheal challenge, the rate of isolation was 17% for the vaccinated group, 67% for the control group and 100% for the unvaccinated group. However, none of the goat from the vaccinated group had P. multocida B:2 in the liver, tonsil and heart. Therefore, the study revealed that an inactivated recombinant vaccine significantly provides significant protection against high dose challenge and enhances the stimulation of the local and systemic immunities.     

Improvement of trivalent leptospira vaccine by removal of anaphylactic agents

The anaphylactic reactions in cattle following leptospira vaccination mostly booster dose in different parts of Iran have been reported. The serum proteins as allergic substances are components of liquid phase of the vaccine. Therefore, the vaccine was modified by washing the whole cultures by centrifugations. The modified vaccine was safe in laboratory animals and cattle as well as under field conditions. Microagglutination test revealed a similar pattern of antibody response to the three Leptospira interrogans serovars (Canicola, Grippotyphosa, and Sejro hardjo) in all vaccinated cattle groups while was higher than the response of control animals. The results of the present investigation revealed that we can minimize postvaccination shock in vaccinated cattle populations with removing the shock proteins.     

Efficacy of lipopolysaccharide antigen of Yersinia ruckeri in rainbow trout by intraperitoneal and bath immersion administration

In this study, Intraperitoneal (IP) and bath immersion (BI) vaccine trials were conducted in fish with a mean weight of 6.3 g. Rainbow trout vaccinated with lipopolysaccharide (LPS) was 50 mg/L protein concentration and challenged by IP injection with 9.8 × 10⁶ cell/ml of Yersinia ruckeri at 45 days post-immunization had a relative percent survival (RPS). To obtain an effective bath immersion vaccine against yersiniosis, LPS preparation was obtained from the Y. ruckeri and with the LPS antigen. After 28 and 60 days vaccinated fish with first and second immunizations by LPS were challenged via intraperitoneal injection with 9.8 × 10⁶ cell/ml of Y. ruckeri for evaluating the mortality rates and calculating the relative percentage of survival (RPS). RPS value of experimental groups, which was significantly (P < 0.05) larger than that of the control group.     

VACCINATION OF HEIFERS WITH A REDUCED DOSE OF BRUCELLA ABORTUS STRAIN 19 VACCINE BEFORE FIRST MATING

Ten Jersey heifers aged 14 to 23 months were vaccinated with 2.25 times 10⁸ cells of living Brucella abortus strain 19 vaccine. They and 10 similar non-vaccinated heifers were subsequently mated and when about 6 months pregnant were challenged by the conjunctival application of a virulent culture of B. abortus. The serological response to vaccination was much less than is usually seen following vaccination with the normal dose of strain 19, especially when the indirect haemolysis test was used. A persistent vaccinal reaction was observed in one heifer. Significant resistance to infection was demonstrated which was greater than that previously observed in calfhood vaccinates given the full dose but less than that shown by cows given the smaller dose in early pregnancy. The effectiveness of strain 19 vaccination appears to be related to the age of the animal at vaccination.     

Lymphocyte activation as cytokine gene expression and secretion is related to the porcine reproductive and respiratory syndrome virus (PRRSV) isolate after in vitro homologous and heterologous recall of peripheral blood mononuclear cells (PBMC) from pigs vaccinated and exposed to natural infection

The present study evaluated the lymphocyte activation in PRRSV-vaccinated pigs subsequently exposed to natural infection by in vitro stimulation of peripheral blood mononuclear cells (PBMC) with homologous vaccine and two heterologous PRRSV isolates. The responsiveness was assessed by determining IFN-γ secreting cells by ELISpot assay, lymphocyte CD8 phenotype by intracellular staining/flow cytometry, cytokine gene expression by real-time quantitative PCR and cytokine secretion by ELISA. Conventional pigs were weaned at 28 days of age and inoculated intramuscularly (IM) or needle-less intradermally (ID) with a modified-live PRRSV vaccine suspended in adjuvant, while control pigs were injected with adjuvant alone (ADJ). Blood samples were collected at vaccination, 35 days post-vaccination and after 35 days post-exposure to natural infection by a heterologous field strain. Thirty-five days post-vaccination, PRRSV vaccine induced a low but significant virus-specific IFN-γ secreting cell response upon stimulation with both the vaccine strain and the two isolates in vaccinated pigs. Conversely, after 35 days post-exposure, only the vaccine strain and the BS/114/S isolate triggered this response. Intracellular staining showed that PRRSV-specific immune cells reacting upon vaccine strain and BS/114/S stimulation were mostly CD8⁺ IFN-γ producing cells whereas the stimulation with BS/55 isolate induced an IFN-γ production associated to the CD8^?IFN-γ⁺ phenotype. At 35 days post-vaccination, PBMC from vaccinated pigs showed lower IL-10 expression and release, and higher TNF-α gene expression upon stimulation with both the vaccine and viral isolates. After infection, both cytokines were not differently modulated in different groups. Immune parameters give evidence that IFN-γ secreting cells in the peripheral blood can be elicited upon PRRSV infection although vaccination itself does not stimulate high levels of these reactive cells. Moreover, the cross-reactivity against divergent PRRS viruses can show a different intensity and be differently associated with cytotoxic CD8⁺IFN-γ⁺ as well as CD8^?IFN-γ⁺ cells. Overall, the obtained data confirmed that the immune activation against PRRSV is not dependent on the genetic divergence of the virus. Especially after infection, a different immune reactivity was evident upon stimulation with the different isolates in terms of frequency and CD8 phenotype of PRRSV-specific IFN-γ producing cells. The modulation of cytokines in vaccinated pigs appeared to be more dependent on vaccination or infection conditions than on stimulation by different isolates, and the changes of IL-10 more relevant than those of TNF-α at gene and protein levels. Moreover, under the conditions of this study, the PRRSV vaccine administered via the intradermal route by a needle-less device was confirmed to induce an immune response comparable or in some cases higher than the intramuscular route.     

Yeast-expressed classical swine fever virus glycoprotein E2 induces a protective immune response

Classical swine fever (CSF) is an economically important swine disease worldwide. The glycoprotein E2 of classical swine fever virus (CSFV) is a viral antigen that can induce a protective immune response against CSF. A recombinant E2 protein was constructed using the yeast Pichia pastoris expression system and evaluated for its vaccine efficacy. The yeast-expressed E2 (yE2) was shown to have N-linked glycosylation and to form homodimer molecules. Four 6-week-old specified-pathogen-free (SPF) piglets were intramuscularly immunized with yE2 twice at 3-week intervals. All yE2-vaccinated pigs could mount an anamnestic response after booster vaccination with neutralizing antibody titers ranging from 1:96 to 1:768. Neutralizing antibody titers at 10 weeks post booster vaccination ranged from 1:16 to 1:64. At this time, the pigs were subjected to challenge infection with a dose of 1 × 10⁵ TCID₅₀ (50% tissue culture infective dose) virulent CSFV strain. At 1 week post challenge infection, all of the yE2-immunized pigs were alive and without symptoms or signs of CSF. Neutralizing antibody titers at this time ranged from 1:4,800 to 1:12,800 and even to 1:51,200 one week later. In contrast, the control pigs continuously exhibited signs of CSF and had to be euthanized because of severe clinical symptoms at 6 days post challenge infection. All of the yE2-vaccinated pigs were E^rns antibody negative and had seroconverted against E^rns by post challenge day 11, suggesting that yE2 is a potential DIVA (differentiating infected from vaccinated animals) vaccine. The yeast-expressed E2 protein retains correct immunogenicity and is able to induce a protective immune response against CSFV infection.     

Evaluation of the immune response induced by intradermal vaccination by using a needle-less system in comparison with the intramuscular route in conventional pigs

The immune response induced by intradermal vaccination using a needle-less device was evaluated in conventional pigs in comparison with the more conventional intramuscular vaccination; to this purpose, vaccination against Aujeszky’s Disease (AD) was used as a model of antiviral immunity. Two groups of pigs (n = 10 each) were vaccinated 4 weeks apart respectively by the intramuscular (IM group) and intradermal route (ID group; needle-less I.D.A.L.® vaccinator) with an AD modified live virus. Ten pigs injected with the vaccine adjuvant only were kept as sham-vaccinated controls (C group).On blood samples collected at 0, 2, 4, 5, 6 and 7 weeks post-vaccination (PV) ADV-specific virus neutralizing (VN) antibodies, IFN-γ secreting cells (SC), lymphocyte subsets and IFN-γ gene expression in PBMC were evaluated.VN antibodies increased after the 1st vaccination and peaked after the 2nd vaccination in both vaccinated groups. Also IFN-γ SC reached maximum levels in both groups after administration of the booster dose. Pigs in the control group remained negative for both parameters throughout the study. Flow cytometry showed persistently higher levels of CD3−CD8α+ Natural Killer cells in both vaccinated pigs. The ID group showed an earlier and regulated activation characterized by an increase of cytotoxic CD8β+ T lymphocytes and CD25+ cells after the boosting dose. No statistically significant differences between treated and control groups were detected for memory CD4+CD8α+^low T cells. Upregulation of IFN-γ gene expression in PBMC was detected in ID and IM pigs after both vaccine administrations, although at a different extent. Overall, the results showed that the intradermal vaccine delivery by a needle-less device can prime a strong humoral and cellular immune response comparable to that obtained by the intramuscular vaccination.     

Vaccination of Rainbow Trout against Infectious Hematopoietic Necrosis (IHN) by Using Attenuated Mutants Selected by Neutralizing Monoclonal Antibodies

Abstract

A neutralizing monoclonal antibody against infectious hematopoietic necrosis virus (IHNV) was used to select neutralization-resistant mutants from isolates of virus obtained from adult steelhead Oncorhynchus mykiss returning to the Round Butte Hatchery (RB mutants) on the Deschutes River in Oregon, USA, and from rainbow trout (nonanadromous O. mykiss) at a commercial hatchery in the Hagerman Valley of Idaho, USA (193-110 mutants). Two of the mutants, RB-1 and 193-110-4, were significantly (P < 0.001) attenuated compared with parental strains. Vaccination of rainbow trout by waterborne exposure to the mutants conferred solid protection against challenge with wild-type virus. In some trials, fish vaccinated with the RB-1 mutant at 50% tissue culture infectious doses (TCID50) of 1 × 10⁴–1 × 10⁵ TCID50/mL or with the 193-110-4 mutant at 1 × 10²–1 × 10³ TCID50/mL, held for 14 d, then challenged with the homologous wild-type strain at 1 × 10⁵ TCID50/mL showed relative percent survival of 95–100% (P < 0.005). There was no significant difference (P > 0.05) in protection among fish exposed to the RB-1 vaccine strain at a dose of 1 × 10⁵ TCID50/mL for periods of either 1, 12, or 24 h, held for 14 d, and then challenged with the wild-type RB isolate, although the 1-h exposure seemed to be somewhat less effective. Fish were vaccinated with the RB-1 strain at 1 × 10³–1 × 10⁵ TCID50/mL for 24 h then challenged after 1, 7, 14, or 21 d with the wild-type RB isolate. No significant (P > 0.1) protection was observed at 1 d postvaccination, but the relative percent survival increased progressively at each subsequent challenge period, becoming statistically significant by day 7 (P < 0.001) and beyond. These results suggested that resistance to challenge with wild-type virus resulted from development of IHNV-specific immunity and not from viral interference or interferon induction, and they reinforce the potential of an attenuated vaccine to control this important disease.     

Effects of infectious bursal disease on Marek's disease vaccination: suppression of antiviral immune response

Studies were made to determine whether infectious bursal disease virus (IBDV) infection would affect the response of chickens to turkey herpesvirus (HVT) vaccination in the development and level of HVT viremia and virus-neutralizing (VN) antibodies to HVT. The HVT viremia in the vaccinated chickens was not affected by IBDV, whether IBDV was inoculated simultaneously with HVT vaccination at one day of age or whether it was inoculated 3 weeks postvaccination with HVT. However, VN antibody response to HVT was significantly suppressed (P less than 0.001) when vaccinated chickens were exposed to IBDV either at the time of vaccination or at 3 weeks postvaccination. Such immunosuppression by IBDV of VN antibody response to HVT vaccination may result in a reduced antiviral immunity against Marek's disease virus.