An unusual case of haemorrhagic bursa in a pullet flock associated with a genogroup 2 infectious bursal disease virus.

An unusual case of infectious bursal disease (IBD) was observed in eight-week-old commercial caged pullets. This flock (House 1) exhibited a one-day spike in mortality. On gross necropsy examination, enlarged, diffusely haemorrhagic bursas were observed. This lesion has been frequently described in cases of very virulent infectious bursal disease virus (vvIBDV). A five-week-old caged pullet flock (House 2) in an adjacent building did not display haemorrhagic bursa lesions. Microscopic examination of bursas from the eight-week-old pullets in House 1 showed marked diffuse haemorrhages and extensive lymphoid necrosis. Histopathology of bursas from the five-week-old pullets in House 2 showed severe, diffuse lymphoid depletion without haemorrhages. IBD ELISA results from birds in House 1 at 9 weeks had a GMT of 6395 and birds in House 2 had a GMT of 82 in the same timeframe. Diagnostic testing for avian influenza virus, Mycoplasma gallisepticum, Mycoplasma synoviae, virulent Newcastle disease virus, infectious bronchitis virus, infectious laryngotracheitis virus, chicken anaemia virus and fowl pox virus were negative. The predicted amino acid sequence of the hypervariable region of VP2 indicated the IBDV observed in both flocks (1/chicken/USA/1300OH/19 from House 1 and 1/chicken/USA/1301OH/19 from House 2) was identical and was not a vvIBDV. Their sequences were similar to a genogroup 2 IBDV from Ontario, Canada (EF138967). No mortality was observed when the 1/chicken/USA/1300OH/19 virus was inoculated into specific-pathogen-free (SPF), four-week-old pullets. Gross and microscopic lesions were observed in bursa tissue, but the bursal haemorrhages observed in the original field case were not reproduced in challenged SPF pullets.

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.

New introduction of a very virulent infectious bursal disease virus in New York, USA.

Bursa tissue samples from a pullet flock in New York State that was experiencing immune suppression related disease were sent to our laboratory in 2018. A very virulent infectious bursal disease virus (vvIBDV) was identified in those samples through molecular and pathogenicity studies and designated 1/chicken/USA/1054NY/18. Phylogenetic analyses of the hypervariable VP2 nucleotide sequence region indicated that this strain belonged to genogroup 3 which comprises the vvIBDV. Partial sequence data of the VP1 gene indicated this virus also had a VP1 typical of vvIBDV. While vvIBDV have previously been identified in the United States in California and Washington State, the 1054NY vvIBDV was most closely related to isolates from Ethiopia, suggesting it is a new introduction into the U.S. The 1054NY vvIBDV was used to challenge four-week old specific-pathogen-free (SPF) layer chicks where it caused 100% morbidity and 68.7% mortality within 4 days. Upon necropsy, gross pathological findings in infected SPF birds included small yellowish coloured bursas, some with haemorrhages on the serosal and mucosal surfaces. Microscopic lesions included inflammation, severe lymphocyte necrosis, atrophy of the follicles and follicular depletion of lymphocytes. RESEARCH HIGHLIGHTS A very virulent infectious bursal disease virus (vvIBDV) was detected in a pullet flock in New York state, USA. Nucleotide sequence analysis of the vvIBDV VP2 gene indicates it is not related to previous US vvIBDV isolates and appears to be a new introduction into the US. The New York vvIBDV caused 100% morbidity and 68.7% mortality in four-week-old specific-pathogen-free chicks.

A proposed nomenclature for infectious bursal disease virus isolates.

Infectious bursal disease virus (IBDV) was initially identified in the USA. For decades, these viruses were not categorized using a typing system because they were considered to be antigenically and pathogenically similar. In the 1980s, a second major serotype, serotype 2, was found in turkeys. Classification of IBDV became more complex with the discovery of antigenic variant strains called "variants" in the United States and a highly virulent strain known as "very virulent" or vvIBDV identified in Europe. To distinguish the IBDV strains identified prior to this time from the antigenic variant viruses, the term "classic viruses" was adopted. Studies over the next three decades produced a wealth of information on the antigenicity, pathogenicity and molecular structure of IBDV isolates. These data made it clear that the descriptive nomenclature used for IBDV was inadequate. For example, not all viruses identified as vvIBDV by genotyping are highly pathogenic; some have reassorted genome segments that result in lower virulence. Furthermore, variant viruses are not an antigenically homogeneous group and the term "classic virus" has been used interchangeably to describe antigenic and pathogenic types of IBDV. These and other issues make the current naming system for strains of IBDV archaic. The lack of uniform testing and standards for antigenicity and pathogenicity makes it difficult to categorize IBDV strains on a global basis. A new nomenclature that includes a genotyping system that can easily be applied worldwide is proposed and serves as a platform to begin discussions on its value to the scientific community.

Classification of infectious bursal disease virus into genogroups.

Infectious bursal disease virus (IBDV) causes infectious bursal disease (IBD), an immunosuppressive disease of poultry. The current classification scheme of IBDV is confusing because it is based on antigenic types (variant and classical) as well as pathotypes. Many of the amino acid changes differentiating these various classifications are found in a hypervariable region of the capsid protein VP2 (hvVP2), the major host protective antigen. Data from this study were used to propose a new classification scheme for IBDV based solely on genogroups identified from phylogenetic analysis of the hvVP2 of strains worldwide. Seven major genogroups were identified, some of which are geographically restricted and others that have global dispersion, such as genogroup 1. Genogroup 2 viruses are predominately distributed in North America, while genogroup 3 viruses are most often identified on other continents. Additionally, we have identified a population of genogroup 3 vvIBDV isolates that have an amino acid change from alanine to threonine at position 222 while maintaining other residues conserved in this genogroup (I242, I256 and I294). A222T is an important mutation because amino acid 222 is located in the first of four surface loops of hvVP2. A similar shift from proline to threonine at 222 is believed to play a role in the significant antigenic change of the genogroup 2 IBDV strains, suggesting that antigenic drift may be occurring in genogroup 3, possibly in response to antigenic pressure from vaccination.

Development of a porcine reproductive and respiratory syndrome virus-like-particle-based vaccine and evaluation of its immunogenicity in pigs.

Porcine reproductive and respiratory syndrome (PRRS) is a leading cause of economic burden to the pork industry worldwide. The routinely used modified live PRRS virus vaccine (PRRS-MLV) induces clinical protection, but it has safety concerns. Therefore, in an attempt to develop a safe and protective inactivated PRRSV vaccine, we generated PRRS-virus-like-particles (PRRS-VLPs) containing the viral surface proteins GP5-GP4-GP3-GP2a-M or GP5-M using a novel baculovirus expression system. Our in vitro results indicated that the desired PRRSV proteins were incorporated in both the VLPs preparations based on their reactivity in immunogold electron microscopy and ELISA. To boost their immunogenicity in pigs, we entrapped the PRRS-VLPs in PLGA nanoparticles and coadministered them intranasally with a potent adjuvant. We then evaluated their efficacy in pigs against a viral challenge using a virulent heterologous field isolate. Our results indicated that PRRS-VLPs induced an anamnestic immune response, since we observed boosted IgG and IFN-γ production in vaccinated and virus-challenged animals, but not during the pre-challenge period. Importantly, a two-log reduction in the lung viral load was detected in PRRS-VLP-vaccinated animals. In conclusion, we generated PRRS-VLPs containing up to five viral surface proteins and demonstrated their immunogenicity in pigs, but further studies are required to improve its immunogenicity and efficacy as a vaccine candidate.

Identification and molecular analysis of infectious bursal disease in broiler farms in the Kurdistan Regional Government of Iraq.

The present study was undertaken to characterize field isolates of infectious bursal disease virus (IBDV). The identification was done using reverse transcription-polymerase chain reaction (RT-PCR) and partial sequencing of the VP2 gene. Pooled bursal samples were collected from commercial broiler farms located in the Kurdistan Regional Government (KRG) of Iraq. The genetic material of the IBDV was detected in 10 out of 29 field samples. Sequences of the hypervariable VP2 region were determined for 10 of these viruses. Molecular analysis of the VP2 gene of five IBDVs showed amino acid sequences consistent with the very virulent (vv) IBDV. Two samples were identified as classic vaccine viruses, and three samples were classic vaccine viruses that appear to have mutated during replication in the field. Phylogenetic analysis showed that all five field IBDV strains of the present study were closely related to each other. On the basis of nucleotide sequencing and phylogenetic analysis, it is very likely that IBD-causing viruses in this part of Iraq are of the very virulent type. These IBDVs appear to be evolving relative to their type strains.

Multivalent virus-like-particle vaccine protects against classic and variant infectious bursal disease viruses.

Nucleotide sequences that encode the pVP2 proteins from a variant infectious bursal disease virus (IBDV) strain designated USA08MD34p and a classic IBDV strain designated Mo195 were produced with the use of reverse-transcriptase-polymerase chain reaction (RT-PCR) and cloned into a pGEM-T Easy vector. A nucleotide sequence that encodes the VP3 protein was also produced from the USA08MD34p viral genome with the use of RT-PCR and cloned into a pGEM-T Easy vector. The VP3 and pVP2 clones were inserted into the pVL1393 baculovirus transfer vector and sequenced to confirm their orientation to the promoter and to ensure they contained uninterrupted open reading frames. Recombinant baculoviruses were constructed by transfection in Sf9 cells. Three recombinant baculoviruses were produced and contained the USA08MD34p-VP3, USA08MD34p-pVP2, or Mo195-pVP2 genomic sequences. Virus-like particles (VLPs) were observed with the use of transmission electron microscopy when the USA08MD34p-VP3 baculovirus was co-inoculated into Sf9 cells with either of the pVP2 constructs. VLPs were also observed when the USA08MD34p-pVP2 and Mo195-pVP2 were coexpressed with USA08MD34p-VP3. These multivalent VLPs contained both classic and variant pVP2 molecules. Stability tests demonstrated the VLPs were stable at 4 and 24 C for 8 wk. The USA08MD34p, Mo195, and multivalent VLPs were used to vaccinate chickens. They induced an IBDV-specific antibody response that was detected by enzyme-linked immunosorbent assay (ELISA), and virus-neutralizing antibodies were detected in vitro. Chickens vaccinated with the multivalent VLPs were protected from a virulent variant IBDV strain (V1) and a virulent classic IBDV strain (STC). The results indicate the multivalent VLPs maintained the antigenic integrity of the variant and classic viruses and have the potential to serve as a multivalent vaccine for use in breeder-flock vaccination programs.

Molecular evidence for a geographically restricted population of infectious bursal disease viruses.

A population of infectious bursal disease virus (IBDV) in northeast Ohio that appears to be geographically restricted was identified. Thirteen broiler farms containing a total of 36 houses were examined for the presence of IBDV. Twenty-four of the 36 houses were positive for IBDV, and of those viruses, 15 viruses from six different broiler farms formed a unique phylogenetic group. Nucleotide sequence analysis identified glutamic acid (E) at position 253 in all 15 viruses. Only one other virus in the GenBank database contained this mutation, and it was also from northeast Ohio. All 15 viruses from this study and the one identified in GenBank also had a unique VP1 sequence. The amino acids located at position 253 in VP2 are typically histidine (attenuated viruses) and glutamine (pathogenic viruses). Because amino acid 253 has been linked to pathogenicity in IBDV, two viruses from the E253 population were selected for pathogenicity studies. They were observed to be pathogenic in 4-wk-old specific-pathogen-free layer chicks. When these two viruses were used to challenge broilers from the parent flock that supplies the birds to all 13 broiler farms examined in this study, the viruses were able to break through the maternal immunity at 14 and 21 days of age but not at 7 days of age. A similar scenario was observed on the six broiler farms that had these viruses. The phylogeographic data suggest this population of IBDV has been restricted for more than 14 yr to northeast Ohio. Because commercially available classic and variant vaccines do not effectively control this population of IBDV, other alternatives are needed.

Historical, spatial, temporal, and time-space epidemiology of very virulent infectious bursal disease in California: a retrospective study 2008-2011.

In December of 2008 very virulent infectious bursal disease virus (vvIBDV) was identified in a commercial flock in northern California. Since then several other backyard and commercial facilities in California have had flocks affected by the same strain and other unique (previously unseen) strains of IBDV. Previous to this incident, very virulent infectious bursal disease (vvIBD) had never been identified in North America. Following the initial outbreak in 2008, California became the first state to undertake a voluntary surveillance effort to try to determine the geographical prevalence of vvIBD based on sequencing of a portion of the segment A region of the vvIBDV genome. To date we have complete geographical information on approximately 500 separate accessions representing approximately 1500 birds from over 200 commercial (-85% of the facilities) and backyard facilities (-15% of the facilities) throughout the state. Sequencing of targeted regions of both the segment A and segment B regions of the genome has revealed three distinct types of IBDV in California chickens. One type is genetically and in pathogenically consistent with vvIBDV. The second and third types only have a segment A region consistent with vvIBDV. Geographic information system mapping coupled with spatial-temporal cluster analysis identified significant spatial and time-space clustering; however, no temporal clustering was noted. The lack of temporal clustering coupled with negative vvIBDV results in tested avian wildlife implies that avian wildlife in California do not currently appear to play a significant role in vvIBDV transmission. In the voluntary surveillance that was done in the Central Valley of California, which has a high density of commercial poultry, no positive farms were found when 142 of 504 farms were sampled. Given this level of sampling, the confidence (probability) of detecting an affected commercial flock was calculated to be between 28% and 81% depending on whether one or five hypothetically affected farms were affected.

Molecular epidemiologic evidence of homologous recombination in infectious bursal disease viruses.

Nucleotide and predicted amino acid sequences of the infectious bursal disease virus (IBDV) surface protein VP2 have been used to identify strains of the virus and place them into phylogenetic groups. The amino acids across the hypervariable sequence region of VP2 (hvVP2) vary, but typically variant viruses have amino acids 222T, 249K, 286I, and 318D and classic viruses have 222P, 249Q, 286T, and 318G. A molecular epidemiologic study was conducted from 2001 to 2011 in commercial chickens (Gallus gallus) from Mexico, Colombia, and Venezuela. Although many IBDVs were identified, most had the typical variant or classic amino acid sequences across the hvVP2 region. Four viruses identified in 2004, one in 2006, and 10 in 2011 from Mexico had the amino acids 222T, 249Q, 286T, and 318D. Six samples from Venezuela in 2001, one sample from Colombia in 2001, two samples from Venezuela in 2004, and one sample from Venezuela in 2005 had the amino acids 222P, 249K, 286I, and 318G. These combinations of classic and variant amino acid sequence markers had not been identified previously in any IBDV strains. The VP2 amino acid sequences in the P(BC) and P(HI) loop structures of the Venezuela and Colombia viruses were similar to most classic viruses, whereas their minor P(DE) and P(FG) loop sequences were typical of Delaware variant strains. The Mexico viruses had VP2 P(BC) loop sequences that were typical of variant IBDV strains, but their minor PDE and PFG loop structures contained amino acids that were similar but not identical to classic strains. The P(HI) loop sequences of the Mexico viruses had 318D that is typical of a Delaware variant virus, but the other amino acids in this loop structure distinguished them from all other IBDV strains. The data suggest that one or more recombination events may have occurred to create this type of sequence diversity. Because of importation regulations, immunologic studies could not be conducted in the United States to determine the antigenicity of the viruses examined in this study. The amino acid sequence data, however, suggest they would contain antigenic epitopes of both variant and classic IBDVs.

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.

Viral competition and maternal immunity influence the clinical disease caused by very virulent infectious bursal disease virus.

The very virulent form of infectious bursal disease virus (vvIBDV) causes an immunosuppressive disease that is further characterized by the rapid onset of morbidity and high mortality in susceptible chickens. In 2009, vvIBDV was first reported in California, U. S. A., and since that time only a few cases of acute infectious bursal disease attributed to vvIBDV have been recognized in California. In other countries where vvIBDV has become established, it rapidly spreads to most poultry-producing regions. Two factors that may be involved in limiting the spread or reducing the severity of the clinical disease caused by vvIBDV in the U. S. A. are maternal immunity and competition with endemic variant strains of the virus. In this study, the ability of vvIBDV to infect and cause disease in maternally immune layer chickens was examined at weekly intervals over a 5-wk period during which their neutralizing maternal antibodies waned. Birds inoculated with vvIBDV at 2, 3, and 4 wk of age seemed healthy throughout the duration of the experiment, but macroscopic and microscopic lesions were observed in their bursa tissues. A real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay also confirmed the presence of vvIBDV RNA in their bursa tissues, indicating this virus was infecting the birds even at 2 wk of age when neutralizing maternal antibodies to infectious bursal disease virus were still relatively high (> 2000 geometric mean antibody titer). No mortality was observed in any birds when inoculated at 2, 3, or 4 wk of age; however, inoculation at 5 and 6 wk of age resulted in 10% and 20% mortality, respectively. Three experiments on the competition between vvIBDV and the two variant viruses T1 and FF6 were conducted. In all three experiments, specific-pathogen-free (SPF) birds that were inoculated with only the vvIBDV became acutely moribund, and except for Experiment 1 (62% mortality) all succumbed to the infection within 4 days of being exposed. When the variant viruses were inoculated into SPF chickens at 4 wk of age and the vvIBDV was administered 10 days later, a real-time reverse transcriptase (RT)-PCR assay was positive for the variant T1 and FF6 viral RNA but negative for vvIBDV RNA, indicating the vvIBDV isolate could not establish an infection in the bursa tissues of these birds. No clinical disease or mortality was observed in the birds, but macroscopic and microscopic lesions typical of a variant virus infection were observed. When the interval was shortened to 2 days, both variant viruses and vvIBDV were detected by RT-PCR, but the vvIBDV viral load in the bursa tissue was significantly lower compared with that of birds inoculated with vvIBDV alone. No clinical disease or mortality was observed, and macroscopic and microscopic lesions in the bursa of these birds were not typical of a vvIBDV infection but instead resembled a pathogenic classic virus infection. When the variant viruses and vvIBDV were inoculated simultaneously into 4-wk-old SPF chickens only the vvIBDV RNA was detected in the bursa tissues of the birds, and no reduction in viral load as measured by real-time RT-PCR was detected. Mortality (30%-40%) was reduced in these duel-infected birds compared with the 100% mortality observed in birds that received vvIBDV alone. The data suggest that maternal immunity and competition with variant viruses are acting to reduce the clinical signs and anticipated mortality after an vvIBDV infection, which may increase the chances that vvIBDV is not being recognized in commercial poultry operations.

Molecular Epidemiology of Infectious Zoonotic and Livestock Diseases.

Zoonotic and livestock diseases are very important globally both in terms of direct impact on human and animal health and in terms of their relationship to the livelihood of farming communities, as they affect income generation and food security and have other, indirect consequences on human lives. More than two-thirds of emerging infectious diseases in humans today are known to be of animal origin. Bacterial, viral, and parasitic infections that originate from animals, including hypervirulent and multidrug-resistant (MDR) bacterial pathogens, such as livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA), invasive nontyphoidal Salmonella of animal origin, hyperviruent Clostridium difficile, and others, are of major significance to public health. Understanding the origin, risk factors, transmission, prevention, and control of such strains has been a challenge for various reasons, particularly due to the transdisciplinary partnership between and among human, environment, and animal health sectors. MDR bacteria greatly complicate the clinical management of human infections. Food animal farms, pets in communities, and veterinary hospital environments are major sources of such infections. However, attributing such infections and pinpointing sources requires highly discriminatory molecular methods as outlined in other parts of this curated series. Genotyping methods, such as multilocus sequence typing, pulsed-field gel electrophoresis, restriction fragment length polymorphism, and several others, have been used to decipher sources of foodborne and other zoonotic infectious diseases. In recent years, whole-genome-sequence-based approaches have been increasingly used for molecular epidemiology of diseases at the interface of humans, animals, and the environment. This part of the series highlights the major zoonotic and foodborne disease issues. *This article is part of a curated collection.

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.

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.

Molecular epidemiology of endemic and very virulent infectious bursal disease virus genogroups in backyard chickens in California, 2009-2017.

Pathogenic strains of infectious bursal disease virus (IBDV) are associated with increased morbidity, mortality, and immunosuppression in susceptible chickens. Backyard poultry is increasing in popularity in the United States, but very little is known about the prevalence and molecular epidemiology of IBDV within these flocks. We performed a retrospective study and phylogenetic analyses of IBDV detected in backyard chickens (BYCs) submitted to the California Animal Health and Food Safety (CAHFS) diagnostic laboratory system in 2009-2017. There were 17 CAHFS autopsy cases of very virulent IBDV (vvIBDV) segment A detected by RT-rtPCR in BYC flocks from 7 counties in California from 2009-2017. During this same time period, non-vvIBDV genotypes were detected by RT-rtPCR in 16 autopsy cases originating from BYC premises in 10 counties in California. Subsequent RT-PCR and phylogenetic analysis of a segment of the hvVP2 and VP1 gene identified vvIBDV, interserotypic reassortant IBDV (vvIBDV segment A and serotype 2 segment B), and non-vvIBDV (variant/subclinical IBDV and classic IBDV) strains in BYC flocks in California.

Madin-Darby canine kidney cell sialic acid receptor modulation induced by culture medium conditions: Implications for the isolation of influenza A virus.

BACKGROUND: The influenza A virus (IAV) binds to α-2,3- and α-2,6-linked sialic acid (SA) receptors expressed by Madin-Darby canine kidney (MDCK) cells. The receptor distribution may therefore be important in regulating IAV propagation. Serum-free medium (SFM) avoids variability in conventional culture medium containing fetal bovine serum (FBS), which can have variable composition and may contain endotoxins. However, little is known about the distribution of SA receptors on cells maintained in SFM. OBJECTIVES: We assessed the influence of culture media on MDCK cell SA receptor distribution along with the effect of SA receptor distribution on IAV recovery. We hypothesized that SFM would increase the proportion of α-2,6-linked SA receptors present and alter isolate recovery. METHODS: Madin-Darby canine kidney cells were cultured in medium containing FBS and two SFMs. Cell surface distribution of α-2,6- and α-2,3-linked receptors was determined using flow cytometry. Recovery of swine- and avian-lineage IAVs from MDCK cells maintained in each medium was quantified as TCID50 . RESULTS: Madin-Darby canine kidney cells cultured in UltraMDCK SFM expressed both SA receptors and supported the growth of both swine- and avian-lineage IAVs. Cells maintained in other medium inconsistently expressed each receptor and the avian IAV grew to lower titers in cells cultured with FBS. CONCLUSIONS: Medium conditions altered the distribution of SA receptors present on MDCK cells and affected IAV recovery. Culture in UltraMDCK SFM resulted in cells expressing both receptors and IAVs grew to higher titers than in the other culture condition, indicating that this medium may be useful for culturing IAV from multiple species.

Advances in vaccine research against economically important viral diseases of food animals: Infectious bursal disease virus.

Numerous reviews have been published on infectious bursal disease (IBD) and infectious bursal disease virus (IBDV). Many high quality vaccines are commercially available for the control of IBD that, when used correctly, provide solid protection against infection and disease caused by IBDV. Viruses are not static however; they continue to evolve and vaccines need to keep pace with them. The evolution of IBDV has resulted in very virulent strains and new antigenic types of the virus. This review will discuss some of the limitations associated with existing vaccines, potential solutions to these problems and advances in new vaccines for the control of IBD.

Infectious bursal disease virus-induced immunosuppression exacerbates Campylobacter jejuni colonization and shedding in chickens.

Campylobacter jejuni is the leading cause of food-borne bacterial gastroenteritis in humans in the United States. Infectious bursal disease virus (IBDV) causes an immunosuppressive disease in young chickens. To analyze a possible role of IBDV-induced immunosuppression in colonization and shedding of C. jejuni, two experiments were conducted. In both experiments, group 1 consisted of noninoculated control chickens, groups 2 and 3 were inoculated with varying doses of C. jejuni, and groups 4 and 5 were inoculated initially with IBDV followed by doses of C. jejuni similar to groups 2 and 3. Campylobacter jejuni was recovered from the cloaca and cecum, but not the small intestines, from all chickens in groups 2 and 3. In groups 4 and 5, C. jejuni was recovered from the small intestines, cecum, and cloaca from all chickens. The amount (colony-forming units/sample) of C. jejuni recovered from chickens in groups 4 and 5 was significantly greater (P < 0.05) than the amount recovered from chickens in groups 2 and 3; and C. jejuni was also present sooner in these groups than in groups 2 and 3. Bursa samples from chickens in groups 4 and 5 were significantly smaller (P < 0.05) than in the other groups. Additionally, real-time polymerase chain reaction results for IBDV were positive in groups 4 and 5 and negative in all other groups. This study indicated that IBDV infection exacerbated colonization and shedding of C. jejuni, presumably through the immune suppression this virus causes in chickens. It highlights the need for further investigation into the role of immunosuppression in preharvest control strategies for food-borne disease-causing agents.