Dexamethasone reduces infectious bursal disease mortality in chickens.

Very virulent infectious bursal disease virus (vvIBDV) causes high mortality in chickens but measures to reduce the mortality have not been explored. Chickens (8-9 weeks) were treated with 3 agents before and during vvIBDV inoculation. Dexamethasone treatment reduced the mortality of infected chickens (40.7% vs. 3.7%; p < 0.001), but treatment with aspirin or vitamin E plus selenium did not affect the mortality. The bursa of Fabricius appeared to have shrunk in both dead and surviving chickens (p < 0.01). The results indicate that dexamethasone can reduce mortality in vvIBDV-infected chickens and may provide therapeutic clues for saving individual birds infected by the virus.

Experimental co-infection of variant infectious bursal disease virus and fowl adenovirus serotype 4 increases mortality and reduces immune response in chickens.

Infectious bursal disease virus (IBDV) and fowl adenovirus serotype 4 (FAdV-4) cause infectious bursal disease (IBD) and hydropericardium-hepatitis syndrome, respectively. Recently, studies have reported co-infections of poultry with IBDV and FAdV-4, which is an important problem in the poultry industry. Here, the variant IBDV strain ZD-2018-1 and FAdV-4 isolate HB1501 were used to assess the pathogenicity of co-infection in 1-day-old specific pathogen-free (SPF) chickens. Compared with chickens infected with only FAdV-4, those coinfected with IBDV and FAdV-4 showed enhanced clinical symptoms, higher mortality, more severe tissue lesions, and higher biochemical index levels. Furthermore, the expression of interleukin (IL)-6, IL-1ß, and interferon-γ mRNAs in the IBDV-FAdV-4 coinfected chickens was delayed, and the antibody response levels were significantly lower in those birds compared with the FAdV-4-infected chickens. These results indicate that co-infection with variant IBDV ZD-2018-1 and FAdV-4 HB1501 could significantly promote the pathogenicity of FAdV-4 and reduce the immune response in chickens. This study provides the foundation for further investigation of the interaction mechanism in IBDV and FAdV-4 co-infection.

Bursa atrophy at 28 days old caused by variant infectious bursal disease virus has a negative economic impact on broiler farms in Japan.

Infectious bursal disease (IBD), caused by IBD virus (IBDV), is highly contagious, immunosuppressive and causes a negative economic impact on poultry industry. IBDV-vaccinated broiler farms at south Kyushu, Japan had a bursa-to-bodyweight ratio (BB ratio) reduction at 28 days (d) old, followed by high mortality 30 d later. We analysed the influence of the IBDV on atrophy of the bursa of fabricius (BF) and the subsequent mortality after 30 d. Ten broilers were sampled at each timepoint from the farm with high mortality at 21, 25, 28 and 35 d. A second flock from the same farm was sampled at 14, 21, 25, 28, 35 and 42 d. IBDV was detected in BF samples at 25, 28 and 35 d and at 21, 25, 28 and 35 d in the first and second flocks, respectively, using immunohistochemical staining and RT-PCR. IBDV isolates from both flocks were closely related to the China KM523643 strain. Histopathology and TUNEL assay indicated apoptosis, severe lymphoid depletion, vacuoles within follicles, lymphoid follicle atrophy and fibrosis in the BF. We observed 75% of the polyserositis and 10% of the airsacculitis at 30 D in dead broilers. The antigenic variant IBDV infection was appeared to be the main influencing factor on BF atrophy and BB ratio reduction in the broilers. High mortality in the broilers after 30 d could be due to secondary infection. The disease caused by IBDV had a negative economic impact in the farm. RESEARCH HIGHLIGHTS New variant IBDV caused bursa atrophy and reduced BB ratio in 28-day-old broilers. After vIBDV had infected broilers, at 21 days old they became immunosuppressed. High mortality at 30 days old in broilers was due to secondary infection. New vIBDV has a negative economic impact on broiler farms in Japan.

Comparison of mortality and viral load in rainbow trout (Oncorhynchus mykiss) infected with infectious pancreatic necrosis virus (IPNV) genogroups 1 and 5.

Infectious pancreatic necrosis virus (IPNV) is the aetiological agent of a highly contagious disease that affects farmed salmonids. IPNV isolates have been phylogenetically classified into eight genogroups, of which two are present in Chile, genogroups 1 and 5. Here, we compare the mortality rate caused by isolates from both genogroups in rainbow trout (Oncorhynchus mykiss) fry to determine if there is an association between host susceptibility and phylogenetic characterization of IPNV. Fish were challenged by immersion with one of four isolates (two for each genogroup), and mortality curves were assessed after 30 days. Viral load was measured in all mortalities and in live fish sampled at 1, 7 and 20 days post-infection. Although mortality was low throughout the challenge, differences were found between fish infected with different isolates. Both isolates from genogroup 1 caused greater cumulative mortalities than either of the isolates from genogroup 5. When combined, the overall mortality rate of fish challenged with genogroup 1 isolates was significantly higher than those infected with genogroup 5. However, viral load was lower on trout infected with genogroup 1 isolates. These results suggest that rainbow trout are more susceptible to IPNV isolates from genogroup 1 than genogroup 5.

Genome-Wide Association Analysis for Resistance to Infectious Pancreatic Necrosis Virus Identifies Candidate Genes Involved in Viral Replication and Immune Response in Rainbow Trout (Oncorhynchus mykiss).

Infectious pancreatic necrosis (IPN) is a viral disease with considerable negative impact on the rainbow trout (Oncorhynchus mykiss) aquaculture industry. The aim of the present work was to detect genomic regions that explain resistance to infectious pancreatic necrosis virus (IPNV) in rainbow trout. A total of 2,278 fish from 58 full-sib families were challenged with IPNV and 768 individuals were genotyped (488 resistant and 280 susceptible), using a 57K SNP panel Axiom, Affymetrix. A genome-wide association study (GWAS) was performed using the phenotypes time to death (TD) and binary survival (BS), along with the genotypes of the challenged fish using a Bayesian model (Bayes C). Heritabilities for resistance to IPNV estimated using genomic information, were 0.53 and 0.82 for TD and BS, respectively. The Bayesian GWAS detected a SNP located on chromosome 5 explaining 19% of the genetic variance for TD. The proximity of Sentrin-specific protease 5 (SENP5) to this SNP makes it a candidate gene for resistance against IPNV. In case of BS, a SNP located on chromosome 23 was detected explaining 9% of the genetic variance. However, the moderate-low proportion of variance explained by the detected marker leads to the conclusion that the incorporation of all genomic information, through genomic selection, would be the most appropriate approach to accelerate genetic progress for the improvement of resistance against IPNV in rainbow trout.

Effect of infectious bursal disease (IBD) vaccine on Salmonella Enteritidis infected chickens.

BACKGROUND: Chickens infected with both infectious bursal disease virus (IBDV) and Salmonella had higher mortality. In this work, we investigated the effect of IBDV vaccine (modified live-virus bursal disease vaccine, Nobilis strain 228E®) on experimentally infected chickens with Salmonella Enteritidis (SE). METHODS: Four experimental groups were included in this study, negative control group, 228E®group, 228E®+SE infected group, and SE infected group. Chickens were ocularly administrated 228E® at 12days of age and orally infected with S. Enteritidis at 13days of age. Sera, intestinal fluid, blood, cloacal swabs and tissue samples were collected at 1, 2 and 3weeks post vaccination (PV). RESULTS: The recorded mortalities were higher in the 228E®+SE infected group, compared to the SE infected group. The anti-S. Enteritidis serum antibody titer and the intestinal mucosal IgA level were higher in the SE infected group at 2 and 3weeks PV, compared to 228E®+SE infected group. S. Enteritidis fecal shedding and organ colonization were significantly higher in the 228E®+SE infected group than the SE infected group at 2 and 3weeks PV. The 228E®+SE group had significantly lower bursa to body weight ratios at 2 and 3weeks PV, as well as had higher bursal lesion scores than the SE infected group. IBDV vaccine depressed the specific-SE systemic and mucosal antibody responses, but did not affect the specific-SE cellular immune responses. CONCLUSION: Chickens administrated IBDV vaccine, followed by S. Enteritidis infection, could cause a significant effect on the bursa of Fabricius, resulting in failure of systemic and mucosal antibody responses to the S. Enteritidis and reduce the elimination and the clearance of S. Enteritidis.

In vivo study of viral haemorrhagic septicaemia virus and infectious pancreatic necrosis virus coexistence in Senegalese sole (Solea senegalensis).

The effect of IPNV-VHSV coinfection and superinfection on the mortality caused by both viruses in Senegalese sole has been analysed. No effect was observed after coinfection. However, a clear viral interference was recorded between a primary IPNV and a subsequent VHSV infection, which led to a survival increase in the infected sole of 50% points when compared with fish infected only with VHSV. The significantly higher Mx transcriptional values in the fish pre-exposed to IPNV (at least at first days after superinfection) and the increased daily mortality when low Mx transcriptional levels were recorded suggest that Mx may be involved in the protective effect against VHSV infection. However, in fish subjected to VHSV primary/IPNV secondary infection, no interference was observed.

Prophylactic potential of resiquimod against very virulent infectious bursal disease virus (vvIBDV) challenge in the chicken.

The study evaluated the prophylactic potential of resiquimod (R-848), a synthetic TLR7 agonist, against very virulent infectious bursal disease virus (vvIBDV) infection in chicken. Specific pathogen free White Leghorn chicks of three week age were treated with R-848 (50µg/bird, intramuscular) or PBS (n=26/group). Twenty four hour later, half of the birds from each group were challenged with 10(5) ELD50 of vvIBDV and observed for 10days. To understand the effect of R-848, immune response genes such as interferon (IFN)-ß, IFN-γ, IL-1ß, IL-4, iNOS and TLR7 were analyzed at 24 and 48h post-challenge in PBMCs ex vivo by real-time PCR (n=6/group). On day 4 post-challenge, representative birds (n=3/group) were sacrificed to study the bursal damage and IBDV antigen clearance. Immunosuppression was assessed by antibody response against live Newcastle disease virus (NDV) vaccine, which was administered on day 10 post-challenge. R-848 pre-treatment significantly upregulated the transcripts of each immune response gene studied (P<0.05). There was 50% mortality on vvIBDV challenge in control birds, while it was only 20% with R-848 group. R-848 pre-treatment reduced the bursal damage as indicated by lower bursal lesion score in histopathology, reduced IBDV antigen signal in immunohistochemistry and improved antigen clearance in agar gel immunodiffusion test. Further, it protected significantly against vvIBDV induced immunosuppression as indicated by HI antibody titre. It is concluded that pre-treatment of R-848 conferred partial protection from mortality and bursal damage while complete protection against immunosuppression in chicken when challenged with vvIBDV, which could be due to the upregulation of immune response genes.

Persistent infections with infectious pancreatic necrosis virus (IPNV) of different virulence in Atlantic salmon, Salmo salar L.

Infectious pancreatic necrosis virus (IPNV) is a prevalent pathogen in fish worldwide. The virus causes substantial mortality in Atlantic salmon juveniles and smolts when transferred to sea water and persistent infection in surviving fish after disease outbreaks. Here, we have investigated the occurrence of the virus as well as the innate immune marker Mx in the head kidney (HK) of Atlantic salmon throughout an experimental challenge covering both a fresh and a seawater phase. The fish were challenged with a high (HV) and low virulence (LV) IPNV. Both isolates caused mortality due to reactivation of the virus after transfer to sea water. In the freshwater phase, higher levels of virus transcripts were detected in the HK of fish infected with LV IPNV compared to HV, suggesting that the HV isolate is able to limit its own replication to a level where the innate immune system is not alerted. Further, ex vivoHK leucocytes derived from fish infected with the two isolates were stimulated with CpG DNA. Significantly, higher IFN levels were found in the LV compared to the HV group in the freshwater phase. This suggests that the viruses attenuate the antiviral host immune response at different levels which may contribute to the observed differences in disease outcome.

DNA vaccination with VP2 gene fragment confers protection against Infectious Bursal Disease Virus in chickens.

Infectious Bursal Disease Virus (IBDV) causes immunosuppression in young chickens by destruction of antibody producing B cells in the Bursa of Fabricius and poses a potential threat to the poultry industry. We have examined the protective efficacy of a subunit DNA vaccine against IBDV infection in chickens in this study. An immunodominant VP2 gene fragment (VP252-417) was cloned into CMV promoter based DNA vaccine vector pVAX1 and in vitro expression of the DNA encoded antigens was confirmed by transfection of CHO cells with vaccine constructs followed by RT-PCR and western blot analysis using IBDV-antiserum. Two weeks old chickens were immunized intramuscularly with pVAXVP252-417 and the in vivo transcription of the plasmid DNA was confirmed by RT-PCR analysis of DNA injected muscle tissue at different intervals of post immunization. Tissue distribution analysis revealed that the plasmid DNA was extensively distributed in muscle, spleen, kidney, liver, and bursa tissues. Chickens immunized with pVAXVP252-417 developed high titer (1:12,000) of anti-VP252-417 antibodies. Further, chicken splenocytes from pVAXVP252-417 immunized group showed a significantly high proliferation to the whole viral and recombinant antigen (P<0.01) compared to control groups, which implies that pVAXVP252-417 codes for immunogenic fragment which has epitopes capable of eliciting both B and T cell responses. This is evident by the fact that, pVAXVP252-417 immunized chicken conferred 75% protection against virulent IBDV (vIBDV) challenge compared to the control group. Thus, the present study confirms that the immunodominant VP2 fragment can be used as a potential DNA vaccine against IBDV infection in chickens.

Food pellets as an effective delivery method for a DNA vaccine against infectious pancreatic necrosis virus in rainbow trout (Oncorhynchus mykiss, Walbaum).

A DNA vaccine based on the VP2 gene of infectious pancreatic necrosis virus (IPNV) was incorporated into feed to evaluate the effectiveness of this oral delivery method in rainbow trout. Lyophilized alginate-plasmid complexes were added to feed dissolved in water and the mixture was then lyophilized again. We compared rainbow trout that were fed for 3 consecutive days with vaccine pellets with fish that received the empty plasmid or a commercial pellet. VP2 gene expression could be detected in tissues of different organs in the rainbow trout that received the pcDNA-VP2 coated feed (kidney, spleen, gut and gill) throughout the 15 day time-course of the experiments. This pcDNA-VP2 vaccine clearly induced an innate and specific immune-response, significantly up-regulating IFN-1, IFN-γ, Mx-1, IL8, IL12, IgM and IgT expression. Strong protection, with relative survival rates of 78%-85.9% were recorded in the vaccinated trout, which produced detectable levels of anti-IPNV neutralizing antibodies during 90 days at least. Indeed, IPNV replication was significantly down-regulated in the vaccinated fish 45 days pi.

Codon optimization and woodchuck hepatitis virus posttranscriptional regulatory element enhance the immune responses of DNA vaccines against infectious bursal disease virus in chickens.

The present study was undertaken to evaluate the protective efficacy of DNA vaccines against infectious bursal disease virus (IBDV) in chickens and to determine whether codon optimization and the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) could improve the immunogenicity of the DNA vaccines. The VP2, VP243 and codon-optimized VP243 genes of IBDV were cloned into pCAGGS vector, and designated as pCAGVP2, pCAGVP243 and pCAGoptiVP243, respectively. Plasmids pCAGWVP243 and pCAGWoptiVP243 carrying the WPRE elements were also constructed as DNA vaccines. To evaluate vaccine efficacy, 2-week-old chickens were injected intramuscularly with the constructed plasmids twice at 2-week intervals and challenged with very virulent IBDV 2 weeks post-boost. Plasmid pCAGVP243 induced better immune responses than pCAGVP2. Chickens immunized with pCAGoptiVP243 and pCAGWVP243 had higher levels of antibody titers, lymphoproliferation responses and cytokine production compared with pCAGVP243. Furthermore, plasmid pCAGWoptiVP243 induced the highest levels of immune responses among the groups. After challenged, DNA vaccines pCAGVP2, pCAGVP243, pCAGoptiVP243, pCAGWVP243 and pCAGWoptiVP243 conferred protection for 33%, 60%, 80%, 87% and 100% of chickens, respectively, as evidenced by the absence of clinical signs, mortality, and bursal atrophy. These results indicate that codon optimization and WPRE could enhance the protective efficacy of DNA vaccines against IBDV and these two approaches could work together synergistically in a single DNA vaccine.

Factors affecting variation in mortality of marine Atlantic salmon Salmo salar in Scotland.

Databases of site production have an important role to play in the investigation and understanding of diseases, since they store valuable amounts of disease and management data. Diseases pose an important constraint to economic expansion of aquaculture. They are dependent on the complex interacting factors of pathogen, environment, and host, and the causes of death can be related to nutritional, environmental, and genetic factors of the host or infectious agents. We examined the drivers of mortality from a single site-production database, which represented one-third of Scottish farmed salmon Salmo salar L. production in 2005, to determine whether mortality 'benchmarking' data could be generalised across sites and production cycles. We show that farm mortality records play an important role in studying mortality losses and identifying of management problems in production. We found that mortalities varied across the months of the year and with the time of year of initial stocking. Production cycles that started in the third quarter of the year had the highest mortality overall. Furthermore, we found site-to-site variation in mortality that may have been caused by either random occurrence of epidemics and environmental events or other local effects.

Survival, growth and sexual maturation in Atlantic salmon exposed to infectious pancreatic necrosis: a multi-variate mixture model approach.

BACKGROUND: Outbreaks of infectious pancreatic necrosis (IPN) in Atlantic salmon can result in reduced growth rates in a fraction of the surviving fish (runts). Genetic and environmental variation also affects growth rates within different categories of healthy animals and runts, which complicates identification of runts. Mixture models are commonly used to identify the underlying structures in such data, and the aim of this study was to develop Bayesian mixture models for the genetic analysis of health status (runt/healthy) of surviving fish from an IPN outbreak. METHODS: Five statistical models were tested on data consisting of 10 972 fish that died and 3959 survivors with recorded growth data. The most complex models (4 and 5) were multivariate normal-binary mixture models including growth, sexual maturity and field survival traits. Growth rate and liability of sexual maturation were treated as two-component normal mixtures, assuming phenotypes originated from two potentially overlapping distributions, (runt/normal). Runt status was an unobserved binary trait. These models were compared to mixture models with fewer traits (Models 2 and 3) and a classical linear animal model for growth (Model 1). RESULTS: Assuming growth as a mixture trait improved the predictive ability of the statistical model considerably (Model 2 vs. 1). The final models (4 and 5) yielded the following results: estimated (underlying) heritabilities were moderate for growth in healthy fish (0.32 ± 0.04 and 0.35 ± 0.05), runt status (0.39 ± 0.07 and 0.36 ± 0.08) and sexual maturation (0.33 ± 0.05), and high for field survival (0.47 ± 0.03 and 0.48 ± 0.03). Growth in healthy animals, runt status and survival showed consistent favourable genetic associations. Sexual maturation showed an unfavourable non-significant genetic correlation with runt status, but favourable genetic correlations with other traits. The estimated fraction of healthy fish was 81-85%. The estimated breeding values for runt status and (normal) growth were consistent for the most complex models (4 and 5), but showed imperfect correlations with estimated breeding values from the simpler models. CONCLUSIONS: Modelling growth in IPN survivors as a mixture trait improved the predictive ability of the model compared with a classical linear model. The results indicated considerable genetic variation in health status among survivors. Mixture modelling may be useful for the genetic analysis of diseases detected mainly through indicator traits.

Stress-induced reversion to virulence of infectious pancreatic necrosis virus in naïve fry of Atlantic salmon (Salmo salar L.).

We have studied stress-induced reversion to virulence of infectious pancreatic necrosis virus (IPNV) in persistently infected Atlantic salmon (Salmo salar L.) fry. Naïve fry were persistently infected with a virulent strain (T(217)A(221) of major structural virus protein 2, VP2) or a low virulent (T(217)T(221)) variant of IPNV. The fry were infected prior to immunocompetence as documented by lack of recombination activating gene-1, T-cell receptor and B-cell receptor mRNA expression at time of challenge. The fish were followed over 6 months and monitored monthly for presence of virus and viral genome mutations. No mutation was identified in the TA or TT group over the 6 months period post infection. Six months post infection TA and TT infected groups were subject to daily stress for 7 days and then sampled weekly for an additional period of 28 days post stress. Stress-responses were documented by down-regulation of mRNA expression of IFN-α1 and concomitant increase of replication levels of T(217)T(221) infected fish at day 1 post stress. By 28 days post stress a T221A reversion was found in 3 of 6 fish in the T(217)T(221) infected group. Sequencing of reverted isolates showed single nucleotide peaks on chromatograms for residue 221 for all three isolates and no mix of TA and TT strains. Replication fitness of reverted (TA) and non-reverted (TT) variants was studied in vitro under an antiviral state induced by recombinant IFN-α1. The T(217)A(221) reverted variant replicated to levels 23-fold higher than the T(217)T(221) strain in IFN-α1 treated cells. Finally, reverted TA strains were virulent when tested in an in vivo trial in susceptible salmon fry. In conclusion, these results indicate that stress plays a key role in viral replication in vivo and can facilitate conditions that will allow reversion from attenuated virus variants of IPNV.

Both genome segments contribute to the pathogenicity of very virulent infectious bursal disease virus.

Infectious bursal disease virus (IBDV) causes an economically significant disease of chickens worldwide. Very virulent IBDV (vvIBDV) strains have emerged and induce as much as 60% mortality. The molecular basis for vvIBDV pathogenicity is not understood, and the relative contributions of the two genome segments, A and B, to this phenomenon are not known. Isolate 94432 has been shown previously to be genetically related to vvIBDVs but exhibits atypical antigenicity and does not cause mortality. Here the full-length genome of 94432 was determined, and a reverse genetics system was established. The molecular clone was rescued and exhibited the same antigenicity and reduced pathogenicity as isolate 94432. Genetically modified viruses derived from 94432, whose vvIBDV consensus nucleotide sequence was restored in segment A and/or B, were produced, and their pathogenicity was assessed in specific-pathogen-free chickens. We found that a valine (position 321) that modifies the most exposed part of the capsid protein VP2 critically modified the antigenicity and partially reduced the pathogenicity of 94432. However, a threonine (position 276) located in the finger domain of the virus polymerase (VP1) contributed even more significantly to attenuation. This threonine is partially exposed in a hydrophobic groove on the VP1 surface, suggesting possible interactions between VP1 and another, as yet unidentified molecule at this amino acid position. The restored vvIBDV-like pathogenicity was associated with increased replication and lesions in the thymus and spleen. These results demonstrate that both genome segments influence vvIBDV pathogenicity and may provide new targets for the attenuation of vvIBDVs.

Study of virulence in field isolates of infectious pancreatic necrosis virus obtained from the northern part of Norway.

In order to study the variety of infectious pancreatic necrosis virus (IPNV) strains involved in outbreaks of infectious pancreatic necrosis (IPN) in Atlantic salmon fish farms, samples were collected from 19 different outbreaks of IPN in the northern part of Norway. The main objective of this study was to examine whether IPNV isolates of different virulence were involved in the outbreaks and could explain the variable IPN protection observed in vaccinated post-smolts in the field. Both the molecular basis of virulence of all field isolates and virulence expressed by mortality after bath challenge of unvaccinated post-smolts with eight of the isolates were studied. Very little variation among the field isolates was detected when the 578-bp variable region encoding the VP2 protein known to be involved in virulence was sequenced. The cumulative mortality after experimental challenge with field isolates genetically characterized as highly virulent was always high (40-56%), while the cumulative mortality of the same strains in vaccinated post-smolts during the field outbreaks varied from 1 to 50%. Although the tested samples came from fish vaccinated with the same vaccine product, the protection against IPN varied. These results demonstrate that differences in virulence of the isolates were not the main reason for the variation in mortality in the field outbreaks. Most of the field isolates were of high virulence, which is shown in experimental challenges to be important for mortality, but clearly other factors that might affect the susceptibility of IPN also play an important role in the outcome of an IPNV infection.

Complete genome sequence analysis of a natural reassortant infectious bursal disease virus in China.

A novel isolate of infectious bursal disease virus (IBDV) was designated GX-NN-L. The GX-NN-L IBDV was a very virulent infectious bursal disease virus (vvIBDV) isolated from broiler flocks in Guangxi province, China, in 2011. The GX-NN-L IBDV caused high mortality, immunosuppression, low weight gain, and bursal atrophy in commercial broilers. Here, we report the complete genome sequence of the GX-NN-L IBDV, a reassortment strain with segments A and B derived from very virulent strains and attenuated IBDV, respectively. These findings from this study provide additional insights into the genetic exchange between attenuated and very virulent strains of IBDV and continuous monitoring of the spread of the virus in chicken.