This study focuses on virus isolation of avian reoviruses from a tenosynovitis outbreak between September 2015 and June 2018, the molecular characterization of selected isolates based on partial S1 gene sequences, and the full genome characterization of seven isolates. A total of 265 reoviruses were detected and isolated, 83.3% from tendons and joints, 12.3% from the heart and 3.7% from intestines. Eighty five out of the 150 (56.6%) selected viruses for sequencing and characterization were successfully detected, amplified and sequenced. The characterized reoviruses grouped in six distinct genotypic clusters (GC1 to GC6). The most represented clusters were GC1 (51.8%) and GC6 (24.7%), followed by GC2 (12.9%) and GC4 (7.2%), and less frequent GC5 (2.4%) and GC3 (1.2%). A shift on cluster representation throughout time occurred. A reduction of GC1 and an increase of GC6 classified strains was noticed. The highest homologies to S1133 reovirus strain were detected in GC1 (~77%) while GC2 to GC6 homologies ranged between 58.5 and 54.1%. Over time these homologies have been maintained. Seven selected isolates were full genome sequenced. Results indicated that the L3, S1 and M2 genes, coding for proteins located in the virus capsid accounted for most of the variability of these viruses. The information generated in the present study helps the understanding of the epidemiology of reoviruses in California. In addition, provides insights on how other genes that are not commonly studied add variability to the reovirus genome.

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.

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.

In 2017, the Turlock branch of the California Animal Health & Food Safety laboratory system received a significant increase in infectious coryza (IC) necropsy cases, with a total of 54 submissions originating from commercial broilers (n = 40), commercial layers (n = 11), and backyard chickens (n = 3). Layer flocks positive for IC were distributed within the adjacent counties of Merced and Stanislaus, while broiler flocks were concentrated within Merced County. The backyard flocks were located in Alameda and Sacramento counties. The clinical and pathologic presentation was consistent with IC, although septicemic lesions were also noticed. Avibacterium paragallinarum was isolated and identified by PCR from the respiratory tract as well as from extrarespiratory sites. Polymicrobial infections involving other viral (infectious bronchitis virus, infectious bursal disease virus) and bacterial (Mycoplasma spp., Escherichia coli, Ornithobacterium rhinotracheale, Gallibacterium anatis biovar haemolytica) agents were commonly reported. Thirteen selected Av. paragallinarum isolates were successfully characterized as serovar C (Page scheme) and serovar C2 (Kume scheme). They shared a unique enterobacterial repetitive intergenic consensus (ERIC) PCR, differing from the four reference strains, and showed consistent high minimum inhibitory concentration values for tetracycline, suggesting a common origin from a single clone. Based on these results, high biosecurity standards and proper immunization of susceptible, multi-age flocks should always be implemented and adjusted as needed. The importance of backyard flocks should not be underestimated due to their unique epidemiologic role.

The reemergence of infectious coryza (IC) caused by Avibacterium paragallinarum (AP) as an acute and occasionally chronic respiratory disease in domestic poultry has caused severe losses in several U.S. states. The disease is also associated with decreased egg production in layers and increased condemnations from air sac infections in broilers. A series of applied experiments were performed to elucidate the persistence of AP in infected broiler flocks, to genotype AP strains isolated from field cases, and to evaluate commercial and autogenous vaccine protection in commercial and specific-pathogen-free (SPF) chickens. Experimental evaluation of environmental persistence suggests that AP did not persist more than 12 hr in a hypothetically contaminated environment. Additionally, other detected potential pathogens such as Gallibacterium anatis and infectious bronchitis virus caused mild respiratory signs in the exposed birds. The HMTp210 and HagA genes of four IC field strains were sequenced and compared with published sequences of HMTp210 and HagA. The HMTp210 phylogeny showed a marginally imperfect clustering of the sequences in genogroups A, B, and C. Although not definitive, this phylogeny provided evidence that the four field strains aligned with previously characterized serovar C strains. Moreover, the base pair homology of the four strains was 100% identical to serovar C reference strains (H-18 and Modesto). HagA phylogeny was unclear, but interestingly, the IC field strains were 100% homologous to C-1 strains reported from Mexico and Ecuador. Finally, vaccine protection studies in commercial hens indicate that clinical signs are induced by a combination of IC and other concomitant pathogens infecting commercial birds. Additionally, vaccine protection experiments performed in SPF hens indicated that protection provided by the two commercial vaccines tested provided a reduction in clinical signs and bacterial shedding after two applications.