Pathogenicity associated with coinfection with very virulent infectious bursal disease and Infectious bursal disease virus strains endemic in the United States.

The pathogenicity induced by co-challenge with the rB strain of very virulent Infectious bursal disease virus (vvIBDV) and IBDV pathotypes endemic in the United States was evaluated in specific pathogen-free chickens. Four- and 6-week-old birds were simultaneously challenged with a 10(5) 50% egg infectious dose (EID50) of rB mixed with a 10(5) EID50 of one of the following viruses: standard classic (STC), subclinical variant (Del-E), subclinical variant (T1), or avirulent serotype 2 (OH). Each challenge group consisted of 5 chickens. The severity of disease was assessed by comparing the 5-day mortality rates, bursal lesions (mean bursal lesion scores), and mean bursal-to-body weight ratios in each of the challenged groups. A mortality of 100% (10/10 and 5/5) was observed in birds inoculated with only the vvIBDV (rB) strain at 4 weeks and 6 weeks of age, respectively. Although the sample sizes were low, a significant reduction in mortality and severity of disease, based on mean bursal lesion scores, was observed in groups co-challenged with rB and the less virulent pathotypes Del-E, T1, or OH at 4 weeks of age. Co-challenge with rB and the antigenically similar STC strain did not result in a significant decrease in mortality compared to challenge with the pathogenic rB strain at 4 weeks of age, but a significant reduction in the mean bursa lesion score was observed. At 6 weeks of age, a significant decrease in mortality and mean bursa lesion score was observed in the rB groups co-challenged with STC, Del-E, or T1 but not OH.

Diversity of genome segment B from infectious bursal disease viruses in the United States.

Several phylogenetic lineages of the infectious bursal disease virus (IBDV) genome segment B have been identified. Although this genome segment has been shown to contribute to virulence, little is known about the genetic lineages that exist in the United States. The nucleotide genome segment B sequences of 67 IBDV strains collected from 2002 to 2011 in the United States were examined. Although they were from nine different states, a majority (47) of these viruses were from California. A 722-base pair region near the 5' end of genome segment B, starting at nucleotide 168 and ending at 889, was examined and compared to sequences available in GenBank. The nucleotide sequence alignment revealed that mutations were frequently observed and that they were uniformly spaced throughout the region. When the predicted amino acids were aligned, amino acids at positions 145, 146, and 147 were found to change frequently. Six different amino acid triplets were observed and the very virulent (vv) IBDV strains (based on presence of vvIBDV genome segment A sequence) all had the triplet T145, D146, and N147. None of the non-vvIBDV strains had this TDN triplet. Phylogenetic analysis of the 67 nucleotide sequences revealed four significant genome segment B lineages among the U.S. viruses. One of these included the genome segment B typically found in vvIBDV and three contained non-vvIBDV genome segment B sequences. When the available sequences in GenBank were added to the analysis, two additional lineages were observed that did not contain U.S. viruses; one included viruses from China and the other contained viruses from the Ivory Coast. Although the samples tested do not represent all poultry producing regions in the United States, serotype 1 viruses from states outside California all belonged to one genome segment B lineage. The other three lineages observed in the United States were populated with viruses exclusively found in California, except the serotype 2 lineage, where the type strain was a serotype 2 virus from Ohio. The data provide further evidence for the importance of genome segment B identification during routine molecular diagnosis of all IBDV strains.

Identification and pathogenicity of a natural reassortant between a very virulent serotype 1 infectious bursal disease virus (IBDV) and a serotype 2 IBDV.

Infectious bursal disease virus (IBDV) causes an economically important, immunosuppressive disease in chickens. There are two serotypes of the virus that contain a bi-segmented double-stranded RNA genome. In December 2008, the first very virulent (vv)IBDV was identified in California, USA and in 2009 we isolated reassortant viruses in two different locations. Genome segment A of these reassortants was typical of vvIBDV serotype 1 but genome segment B was most similar to IBDV serotype 2. The CA-K785 reassortant caused 20% mortality in chickens but no morbidity or mortality in commercial turkey poults despite being infectious. There have been previous reports of natural reassortants between vvIBDV and other serotype 1 strains, but a natural reassortant between IBDV serotypes 1 and 2 has not been described. The apparent reassorting of California vvIBDV with an endemic serotype 2 virus indicates a common host and suggests vvIBDV may have entered California earlier than originally thought.

Amino acids contributing to antigenic drift in the infectious bursal disease Birnavirus (IBDV).

We examined the effect of amino acids 222 and 254 on antigenicity of the variant Del-E strain of infectious bursal disease virus (IBDV). Using molecular epidemiology, we identified a virus designated as Del-E-222 that was identical to Del-E except for alanine at position 222. A second virus was generated using reverse genetics of the Del-E backbone to create Del-E-254 that contained an asparagine at amino acid 254. The Del-E-222 and Del-E-254 viruses were tested for their ability to escape neutralizing immunity provided by parenteral vaccination. The bursas from birds vaccinated with parental Del-E and challenged with Del-E-222 or Del-E-254 had macroscopic lesions typical of an IBDV infection, and their B-BW ratios were significantly smaller than the controls. Microscopic lesions included lymphocyte depletion and confirmed the ability of Del-E-222 and Del-E-254 to break through the immunity induced by the parental Del-E virus vaccination. Both mutations appear to be contributing to antigenic drift.

Detection and characterization of infectious bursal disease viruses in broilers at processing.

The presence of infectious bursal disease virus (IBDV) in broilers entering processing plants was examined. The dissemination of IBDV and the introduction of non-native strains for example very virulent (vv) IBDV have had a negative economic impact on poultry production in many countries. Restrictions have been placed on the import and export of poultry products by some countries. There is a perceived risk that IBDV can be spread through transportation and contamination of processing equipment, poultry protein products and processing plant personnel. This risk, however, is fundamentally unknown because scientific studies have not been conducted on the presence of IBDV in birds entering processing plants or the variables that may affect this risk during and post-harvest. The goal of this study was to determine if infectious IBDV was present in broilers entering processing plants. A total of 47 pooled bursa samples from 26 processing plants in the Eastern U.S. were examined. Molecular testing indicated that an IBDV specific RT-PCR was positive in 12 (25.5%) of the samples from 11 different processing plants. Nucleotide sequence analysis was conducted on the 12 RT-PCR positive samples and indicated the IBDV was not commercially available attenuated vaccine strains. Most of the sequences were unique and a phylogenic analysis indicated they were most closely related to variant strains of IBDV. Five RT-PCR positive samples were selected at random for testing in specific-pathogen-free chickens. All five samples contained infectious IBDV as evidenced by macroscopic lesions and bursa/body weight ratios that were significantly lower in infected birds than in the non-inoculated controls. The five viruses were re-identified in bursa tissue from chickens in their respective groups at necropsy using RT-PCR and nucleotide sequencing. The results indicate that infectious and pathogenic IBDV are entering processing plants in this geographic region of the U.S. Additional studies are needed on post-harvest variables that could increase or decrease the risk that these viruses are being disseminated during this process.

The diagnosis of very virulent infectious bursal disease in California pullets.

This report documents the occurrence of a very virulent infectious bursal disease virus (vvIBDV) in Northern California commercial brown pullets. Diagnosis was made from multiple accessions from two neighboring and epidemiologically related ranches submitted to the California Animal Health and Food Safety (CAHFS) laboratory. Pullets, 11 and 14 wk of age from ranch A (rA) and ranch B (rB) respectively, were submitted from infectious bursal disease virus vaccinated flocks experiencing a drastic increase in mortality. The December 2008 outbreak resulted in 26% and 34% mortality on rA and rB respectively. Gross and histologic lesions characteristic of acute vvIBDV were observed. Gross lesions included edematous bursas, hemorrhages at the junction of the proventriculus and gizzard as well as hemorrhages on skeletal muscles. Microscopic lesions included severe lymphoid necrosis and inflammation in edematous bursas, lymphoid necrosis in thymus, spleen, Peyer's patches and cecal tonsils. Diagnosis of vvIBDV was confirmed by molecular characterization of the IBDV from bursas as well as viral pathogenicity in specific-pathogen-free birds. RT-PCR and nucleotide sequencing of the hypervariable region of the VP2 (vVP2) gene segment of the IBDV genome was performed on rA, rB and embryo passaged rA virions.The amino acids compatible with vvIBDV isolates: 222(Ala), 242(Ile), 256(Ile), 294(Ile) and 299(Ser) were reported from both ranches. In addition, nucleotide sequencing of a fragment of the VP1 gene demonstrated the viruses have the segment B genotype associated with highly pathogenic vvIBDV. Inocula of 10(5.5) 50% egg infective dose of vvIBDV virus from rA and rB were introduced orally into two groups (g1 and g2 respectively) of 4 wk 2-day-old SPF leghorns. At 4 days postinoculation, there was 100% (22/22) morbidity in g1 and g2; 91% (20/22) mortality in g1; 100% (22/22) mortality for g2; 0% (0/20) morbidity and 0% (0/ 20) mortality was reported in the control group. This is the first occurrence of vvIBDV reported from birds in the United States.

Characteristics of a very virulent infectious bursal disease virus from California.

An outbreak of infectious bursal disease (IBD) in two California layer flocks resulted in the isolation of two infectious bursal disease viruses designated rA and rB. Increased mortality plus gross and histopathology in the layer flocks suggested rA and rB could be very virulent infectious bursal disease virus (vvIBDV). Preliminary studies indicated that high mortality resulted when bursa homogenates from the layer farms were used to inoculate specific-pathogen-free (SPF) chicks. In addition, rA and rB contained VP2 amino acid sequences typically seen in vvIBDV. Molecular and in vivo studies were conducted to more thoroughly identify and characterize the rA and rB viruses. Nucleotide sequence analysis demonstrated that rA and rB had identical sequences across the hypervariable VP2 (hvVP2) and segment B regions examined, and their amino acid sequences in the hvVP2 region were identical to the vvwIBDV type strains UK 661, OKYM, and Harbin. Furthermore, the genome segment B nucleotide sequences examined for rA and rB were a 98.1% match with vvIBDV and only an 88.0% match with classic IBDV strains. Phylogenetic analysis placed the rA and rB viruses with other vvIBDV and confirmed these viruses were close genetic descendants of vvIBDV seen around the world. Pathogenicity studies in 4-wk-old SPF chicks demonstrated that at a high dose (105.5 50% egg infective dose [EID50]) and a low dose (102.0 EID50) of rA and rB, mortality ranged from 91% to 100%. A pathogenic classic virus, standard challenge (STC), at similar doses did not cause mortality in the SPF chicks. In addition, mortality occurred in three out of four SPF birds exposed by direct contact to rA and rB inoculated chicks. Serum from convalescent birds inoculated with rA had high titers to IBDV but were negative for antibodies to infectious bronchitis virus, avian influenza virus, chicken anemia virus, Newcastle disease virus, Mycoplasma gallisepticum, and Mycoplasma synoviae. Virus isolation attempts on the rA and rB bursa homogenate inocula also indicated that no contaminating microorganisms contributed to the high mortality and pathology observed in the SPF chicks. In one experiment, broilers with maternal immunity to IBDV were protected from infection and disease when they were challenged with 10(2) EID50 and 10(5) EID50 of the STC virus. When challenged with 10(2) EID50 of the rA virus, the maternally immune broilers were protected from disease but not infection as evidenced by a positive reverse transcription-polymerase chain reaction (RT-PCR) assay for the virus. When the broilers were challenged with 10(5) EID50 of the rA virus, they had typical gross and histopathologic signs of IBD but no mortality by 7 days postinoculation. It was concluded that the rA and rB viruses meet the genotypic and phenotypic characteristics of a vvIBDV.

Genetic characteristics of infectious bursal disease viruses from four continents.

Following the initial discovery of very virulent infectious bursal disease virus (vvIBDV) strains in Europe, these viruses spread to many parts of the world. In this study, we examined the phylogenetic relationship of never-before-published IBDV from 18 countries on four continents. All the samples were collected between 1997 and 2005 and were reported to be from broiler flocks experiencing higher than expected mortality which is often associated with acute very virulent infectious bursal disease. A total of 113 samples were imported into the U.S. and viral genetic material was used to determine the nucleotide sequence of the VP2 gene hypervariable region. Although all the samples were reported to be associated clinically with high mortality, genetic analysis suggests that some were not vvIBDV strains. Two viruses from South Africa were genetically similar to U.S. variant viruses. A majority (71/113) of the viruses examined had the amino acid Alanine at position 222 and sixty-seven of these suspect vvIBDV also had amino acids I242, I256, I294 and S299 which are highly conserved among vvIBDV strains. Phylogenetic analysis placed putative vvIBDV strains from many different countries and geographic regions in a single clade with some minor non-significant branching.