Whole Genome Sequencing of Infectious Bursal Disease Viruses Isolated from a Californian Outbreak Unravels the Underlying Virulence Markers and Highlights Positive Selection Incidence.

Outbreaks of the immunosuppressive infectious bursal disease (IBD) are frequently reported worldwide, despite the vaccination regimes. A 2009 Californian IBD outbreak caused by rA and rB isolates was described as very virulent (vv) IBD virus (IBDV); however, molecular factors beyond this virulence were not fully uncovered. Therefore, segments of both isolates were amplified, successfully cloned, whole genome sequenced by Next Generation Sequencing, genotyped, and the leading virulence factors were entirely investigated in terms of phylogenetic and amino acid analysis and protein modeling for positive selection orientation and interaction analysis. rA and rB isolates displayed the highest amino acid identity (97.84-100%) with Genotype 3 strains. Interestingly, rA and rB contained all virulence hallmarks of hypervariable (HVR), including 222A, 242I, 249Q, 256I, 284A, 286T, 294I, 299S, and 318G, as well as the serine-rich heptapeptide sequence. Moreover, we pinpointed the A3B2 genotype of rA and rB, predominant in non-reassortants, and we highlighted the absence of recombination events. Furthermore, gene-wise phylogenetic analysis showed the entire genes of rA and rB clustered with the vvIBDVs and emphasized their share in IBDV virulence. VP5 showed a virulence marker, MLSL (amino acid sequence). VP2 encountered three significant novel mutations apart from the HVR, including G163E in rA and Y173C and V178A in rB, all residing within interacting motifs. VP4 contained 168Y, 173N, 203S, and 239D characteristic for the vv phenotype. A235V mutation was detected at the dsRNA binding domain of VP3. In VP1, the TDN triplet and the mutation (V4I) were detected, characteristic of hypervirulence occurring at the N-terminus responsible for protein priming. Although selection analysis revealed seven sites, codon 222 was the only statistically significant selection site. The VP2 modeling of rA and rB highlighted great structure fitness, with 96.14% Ramachandran favored positioning including the 222A, i.e., not influencing the structure stability. The 222A was found to be non-interface surface residue, associated with no interaction with the attachment-mediated ligand motif. Our findings provide pivotal insights into the evolution and underlying virulence factors and will assist in the development of control strategies via sequence-based continuous monitoring for the early detection of novel vv strains.

Thymoma in an aged backyard Leghorn chicken, with reviews of a database and literature.

A 7-y-old backyard Leghorn chicken (Gallus domesticus) was submitted to the California Animal Health and Food Safety Laboratory System (CAHFS)-Turlock branch for postmortem examination, with a history of unexpected death. At postmortem examination, a hemorrhagic soft tissue mass was observed in the cervical region. Microscopically, a densely cellular neoplasm of polygonal epithelial cells and small lymphocytes was observed. The microscopic features of the neoplasm in combination with positive immunohistochemistry for pancytokeratin and CD3 were used to classify the lesion as a thymoma. Thymoma was diagnosed in only 5 birds submitted to CAHFS from 1990 to 2019. Thymoma has been described only rarely in birds, and is an unusual diagnosis in backyard chickens.

Infectious Bronchitis Virus Associated with Nephropathy Lesions in Diagnostic Cases from Commercial Broiler Chickens in California.

In March 2019, the California Animal Health and Food Safety Laboratory (CAHFS), Turlock branch, received two submissions of broiler chickens from commercial flocks reporting increased mortality. Submissions consisted of either white or brown broilers. Submitted chickens appeared depressed with ruffled feathers. At necropsy, moderate to severely enlarged and pale kidneys were observed, with gross lesions indicative of dehydration. Microscopically, renal tubules were degenerated and distended with necrotic debris and tubular casts. The kidney parenchyma contained mononuclear inflammatory cell infiltrates and interstitial edema. Infectious bronchitis virus (IBV) was isolated and identified by reverse transcription quantitative PCR from kidney tissue pools and tracheal swab pools from both cases. Partial sequencing of the S1 hypervariable region was most similar to a local California variant, CA1737. The outbreak lasted roughly 1 wk in both flocks, with 2% total mortality in the brown broilers and 20% total mortality in the white broilers. Final proof of the IBV strains causing nephropathy will require fulfillment of Koch postulates. IBV associated with nephropathy has been sporadically reported in California chicken flocks and represents a significant pathogen due to its potential for inducing high flock mortality. The incidence of IBV associated with a nephropathy diagnosis in chicken necropsy submissions to the CAHFS system-wide from 1998 to 2019 is also reviewed.

Chlamydiosis in a Gouldian Finch (Erythrura gouldiae).

Avian chlamydiosis is an infection caused by obligate intracellular and Gram-negative bacteria belonging to the family Chlamydiaceae and has been reported in more than 450 avian species distributed in 30 orders. In particular, a high prevalence of infection has been demonstrated in wild passerine populations, including both asymptomatic and clinically ill individuals, suggesting a role of these avian species as important carriers. In May 2018, avian chlamydiosis was diagnosed in a 1-year-old male Gouldian finch (Erythrura gouldiae) at the Turlock Branch of the California Animal Health and Food Safety Laboratory System. The bird belonged to an outdoor aviary with mixed avian species, including Gouldian finches, doves (Geopelia cuneata and Spilopelia chinensis), and psittacines (Aratinga, Psittacula, Pyrrhura, and Trichoglossus sp.). Severe respiratory distress and mortality were noted among the finches. Gross and histopathologic lesions were concentrated in the liver and spleen, with a mild involvement of the upper respiratory tract. Chlamydia spp. were detected in the spleen and kidney by real-time PCR and were further confirmed by immunohistochemistry. Subsequently, Chlamydia psittaci was isolated from the liver and spleen and characterized as a CP3-like strain (genotype B). In addition, viral particles compatible with circovirus were identified in the liver by direct electron microscopy. To the authors' knowledge, this is the first report of avian chlamydiosis with hepatic viral particles consistent with circovirus infection in a Gouldian finch.

Reporte de caso- Clamidiosis en un pinzón diamante de Gould (Chloebia gouldiae). La clamidiosis aviar es una infección causada por bacterias intracelulares y Gramnegativas obligadas que pertenecen a la familia Chlamydiaceae y se ha reportado en más de 450 especies de aves distribuidas en 30 órdenes. En particular, se ha demostrado una alta prevalencia de infección en poblaciones de paseriformes silvestres, incluyendo individuos asintomáticos y clínicamente enfermos, lo que sugiere un papel de estas especies aviares como portadores importantes. En mayo del año 2018, se diagnosticó clamidiosis aviar en un pinzón diamante de Gould (Chloebia gouldiae) de un año de edad remitido a la sede en Turlock del Sistema de Laboratorios de Salud Animal y Seguridad Alimentaria del Estado de California. El ave pertenecía a un aviario al aire libre con especies mixtas de aves, incluyendo los diamantes de Gould, palomas (Geopelia cuneata y Spilopelia chinensis) y psitacinas (Aratinga, Psittacula, Pyrrhura y Trichoglossus sp.). Se observaron problemas respiratorios severos y mortalidad entre los pinzones. Las lesiones macroscópicas e histopatológicas se concentraron en el hígado y el bazo, con problemas leves en el tracto respiratorio superior. Se detectó Chlamydia spp. en el bazo por PCR en tiempo real y fueron confirmados por inmunohistoquímica. Posteriormente, se aisló Chlamydia psittaci del hígado y el bazo y se caracterizó como una cepa de tipo CP3 (genotipo B). Además, se identificaron partículas virales compatibles con circovirus en el hígado mediante microscopía electrónica directa. Según el conocimiento de los autores, este es el primer informe de clamidiosis aviar con partículas virales hepáticas compatibles con infección por circovirus en un pinzón diamante de Gould.

Retrospective Summary of Erysipelothrix rhusiopathiae Diagnosed in Avian Species in California (2000-19).

Erysipelas is a bacterial disease caused by Erysipelothrix rhusiopathiae that affects multiple mammalian and avian species. In poultry, the disease is of sporadic prevalence and more often observed in older birds, leading to decreased egg production and mortality. Among avian species, turkey breeders seem to be the most affected, but outbreaks have been reported in ducks, layer chickens, quails, geese, and various captive and free-range birds. Sixty-seven cases of erysipelas have been diagnosed in animals submitted for necropsy evaluation at the California Animal Health and Food Safety Laboratory System from January 2000 to December 2019. Of these, 38 cases (56.72%) were in avian species, and a retrospective analysis of these avian cases was performed. The majority of the avian cases were in turkeys (17/38, 44.74%). Most of the turkey breeder cases reported performing artificial insemination prior to the increase in mortality. In other birds, mortality was often observed without observing previous clinical signs. The majority of cases presented with coinfections with other pathogens (23/38, 60.53%), which might have affected the clinical outcome. Despite the occasional occurrence in avian species, erysipelas is an important pathogen in poultry and should be considered as a differential diagnosis in other avian species when acute septicemia is suspected as the cause of mortality.

Rotavirus A Associated with Clinical Disease and Hepatic Necrosis in California Pigeons (Columba livia domestica).

Retrospective analysis of pigeon necropsy submissions to the California Animal Health and Food Safety Laboratory System from 2000 to 2018 revealed 14 submissions diagnosed with rotavirus A hepatic necrosis or "reoviruslike" viral hepatitis. Nine of the 14 submissions (64%) occurred in 2018. Submissions were racing pigeons and squab breeders from flocks with increased mortality. Juvenile and adult pigeons were submitted with a history of depression, diarrhea, regurgitation, labored breathing, and weakness. Flock morbidity peaked at 80% and mortality at 28%. The most consistent findings on postmortem examination were variably congested, mottled, and enlarged livers and spleens. Microscopically, mild to severe hepatic necrosis was observed with variable bile duct hyperplasia, sinusoidal congestion, hemosiderosis, and portal lymphoplasmacytic inflammation. Rotavirus A was detected in hepatocytes and inflammatory cells by immunohistochemistry. Negative-stain electron microscopy identified viral particles consistent with a member of Reoviridae in all negatively stained liver homogenates. Eleven cases were analyzed by reverse transcriptase-PCR targeting rotavirus A viral protein (VP) 6 and VP7 genes. Subsequent phylogenetic analysis of the VP6 and VP7 sequences compared to published Chinese, Nigerian, and German rotavirus A VP6 and VP7 sequences demonstrated the formation of two and three distinct clades, respectively. To the authors' knowledge, rotavirus A hepatic necrosis in pigeons has not been previously reported in the United States and represents a significant emerging disease for the pigeon industry due to the potential for high flock mortality and lost production.

Rotavirus A asociado con enfermedad clínica y necrosis hepática en palomas de California (Columba livia domestica). El análisis retrospectivo de los casos de necropsias de palomas remitidos al Sistema de Laboratorio de Salud Animal y Seguridad Alimentaria del Estado de California entre los años 2000 a 2018 reveló 14 casos con diagnóstico de necrosis hepática por rotavirus A, o hepatitis viral ocasionada por "virus similares a reovirus". Nueve de los 14 casos (64%) ocurrieron en el año 2018. Los casos fueron de palomas de competencia y de criadores de pichones de parvadas con aumento en la mortalidad. Se presentaron palomas jóvenes y adultas con antecedentes de depresión, diarrea, regurgitación, dificultad para respirar y debilidad. La morbilidad mayor fue de un 80% como máximo y la mortalidad fue de un 28%. Los hallazgos más consistentes en el examen post mortem incluyeron hígados y bazos con congestión, apariencia moteada y aumento de tamaño de forma variable. Microscópicamente, se observó necrosis hepática de leve a severa con hiperplasia variable de los conductos biliares, congestión de sinusoides, hemosiderosis e inflamación linfoplasmocítica portal. Se detectó rotavirus A en hepatocitos y células inflamatorias por inmunohistoquímica. La microscopía electrónica de tinción negativa identificó partículas virales consistentes con virus posiblemente miembros de la familia Reoviridae en todos los homogenizados de hígado teñidos negativamente. Se analizaron once casos mediante transcripción reversa y PCR dirigida a los genes de la proteína viral (VP) 6 y VP7 del rotavirus A. El análisis filogenético posterior de las secuencias de los genes VP6 y VP7 cuando se compararon con secuencias de genes VP6 y VP7 de rotavirus A de China, Nigeria y de Alemania previamente publicadas demostró la formación de dos y tres clados distintos, respectivamente. De acuerdo con el conocimiento de los autores, la necrosis hepática por rotavirus A en palomas no se había reportado previamente en los Estados Unidos y representa una enfermedad emergente importante para la industria de las palomas debido a su potencial de alta mortalidad de la parvada y a las pérdidas en la producción.

Retrospective analysis of infectious laryngotracheitis in backyard chicken flocks in California, 2007-2017, and determination of strain origin by partial ICP4 sequencing.

Infectious laryngotracheitis (ILT) can cause severe losses in backyard flocks (BYFs) and commercial poultry. The prevalence of ILT, the circulating strains of ILT virus (ILTV) in BYFs, and the correlation of disease in BYF and commercial operations, is largely unknown. Of 8,656 BYF submissions, 88 cases of ILT were diagnosed at the California Animal Health and Food Safety Laboratory System in 2007-2017. ILT diagnosis by year varied from 0.19% to 1.7% of the total BYF submissions, with the highest number of cases submitted from Amador and Riverside counties. Moderate tracheitis, conjunctivitis, and occluded tracheal lumen were commonly reported gross anatomic lesions. Microscopically, inflammation and edema were observed in the trachea, lung, and conjunctiva; 62 (70%) cases had intranuclear inclusion bodies (INIBs), with 10 cases containing INIBs only in conjunctival sections. To analyze the circulating ILTV strains and to differentiate between field and vaccine strains of ILTV, real-time PCR and sequencing of 996 base pairs of the infected-cell polypeptide 4 ( ICP4) gene was performed on 15 ILTV-positive tracheal samples and compared to reference field and vaccine ILTV ICP4 sequences in GenBank. Fourteen strains were identical or closely related to the chicken embryo origin live virus vaccine strains, and one strain was closely related to a Chinese isolate, the USDA reference strain, and a vaccine strain. The presence of ILT in BYFs in counties with high commercial poultry concentrations demonstrates a risk for disease transmission and emphasizes the importance of continued surveillance and improved biosecurity in BYFs.

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.

Fowl cholera and acute heart rupture in a backyard turkey.

Pasteurella multocida is the causative agent of fowl cholera, an economically important disease of commercial and backyard poultry. Turkeys are particularly susceptible to fowl cholera; both backyard and commercial turkeys can succumb to disease. On April 10, 2018, a dead 9-mo-old male Bronze turkey was submitted to the California Animal Health and Food Safety Laboratory System (CAHFS)-Turlock branch for postmortem examination. History included previous housing and fighting with another male turkey, after which separation by a fence was instituted. Fighting continued, and depression and anorexia of 2 d duration was followed by acute collapse and death. At autopsy, blood clots markedly expanded the pericardium, and a tear was visible in the left ventricular free wall. Vegetative aortic valvular lesions were observed. Microscopically, infarcts were observed in kidney, liver, heart, spleen, and pancreas, with large numbers of gram-negative bacterial colonies present in most organs. P. multocida was isolated from multiple organs, and identified as serotype 2,5 and fingerprint 1604. Septic embolization from the vegetative valvular aortic lesions likely led to infarcts in multiple organs, including the left ventricular free wall, which ultimately caused weakening of the ventricular wall, ventricular rupture, and exsanguination into the pericardial space. Rupture of the left ventricular free wall has not been previously documented in turkeys with P. multocida infection, to our knowledge, and demonstrates an atypical presentation of fowl cholera in this backyard turkey.

Characterization of an Outbreak of Infectious Coryza (Avibacterium paragallinarum) in Commercial Chickens in Central California.

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.

Caracterización de un brote de coriza infecciosa (Avibacterium paragallinarum) en pollos comerciales en la parte central de California. En el año 2017, la sede en Turlock del Sistema de Laboratorios de Salud Animal y Seguridad Alimentaria de California recibió un aumento significativo en el número de casos de necropsia por coriza infecciosa, con un total de 54 casos, incluyendo casos provenientes de pollos de engorde comerciales (n = 40), gallinas de postura comerciales (n = 11) y aves de traspatio (n = 3). Las parvadas de gallinas de postura positivas para coriza infecciosa se distribuyeron en los condados adyacentes de Merced y Stanislaus, mientras que las parvadas de pollos de engorde se concentraron en el condado de Merced. Las parvadas de traspatio estaban ubicadas en los condados de Alameda y Sacramento. La presentación clínica y patológica fue consistente con coriza infecciosa, aunque también se observaron lesiones septicémicas. Se aisló Avibacterium paragallinarum y se identificó mediante PCR en el tracto respiratorio y también de sitios extrarespiratorios. Las infecciones polimicrobianas relacionadas con otros virus (virus de la bronquitis infecciosa, virus de la enfermedad infecciosa de la bolsa) y bacterias (Mycoplasma spp., Escherichia coli, Ornithobacterium rhinotracheale, Gallibacterium anatis biovar haemolytica) fueron reportadas comúnmente. Trece aislamientos seleccionados de A. paragalinarum se caracterizaron con éxito como serovar C (esquema de Page) y serovar C2 (esquema de Kume). Estos aislamientos Compartieron por PCR un consenso intergénico repetitivo enterobacterial (ERIC) único, que difiere de las cuatro cepas de referencia y mostraron valores constantes de concentración mínima inhibitoria alta para tetraciclina, lo que sugiere un origen común de un solo clon. Con base en estos resultados, siempre se deben implementar y ajustar estándares de bioseguridad altos y la inmunización adecuada de parvadas susceptibles de edades múltiples, según sea necesario. La importancia de las parvadas de traspatio no debe subestimarse debido a su función epidemiológica especial.

Liposarcoma in a Backyard Silkie and Retrospective Summary of Neoplasms Diagnosed in Backyard Chickens Submitted to the California Animal Health and Food Safety Laboratory System, 2008-2017.

Liposarcomas are a malignant neoplasm of adipocytes, and are rarely diagnosed in avian species. This case report describes the evidence supporting a diagnosis of metastatic liposarcoma in a backyard silkie chicken. On September 28, 2017, a dead 3-yr-old backyard silkie chicken, with a history of unknown skin lesions involving the entire body and severe weight loss, was submitted to California Animal Health and Food Safety Laboratory System-Turlock branch for necropsy. At necropsy, raised necrotic lesions involving the majority of the skin and multiple nodules in the liver, spleen, and bone marrow were noticed. Microscopically, stellate, spindle, and myxoid cells containing large vacuoles, which were confirmed as lipid droplets by Oil Red O, were observed infiltrating the dermis and underlying a necrotic epidermis, with metastasis to liver, spleen, bone marrow, and ovary being the most significant findings. PAS, Oil Red O, Ziehl-Neelsen, Congo red, Gram, and Von Kossa stains, along with immunohistochemistry for pan cytokeratin, vimentin, S100, CD3, pp38, and Meq were used to classify the lesions. Intensely positive vimentin immunohistochemistry, along with large quantities of Oil Red O-positive lipid droplets within the neoplastic cells, were supportive of our diagnosis of liposarcoma. The incidence of neoplastic diseases diagnosed in backyard flock submissions to CAHFS system wide from 2008 to 2017 was also reviewed.

Retrospective Study of Pasteurella multocida Diagnosed in Commercial Turkeys Submitted to California Animal Health and Food Safety Laboratory System; 1991-2017.

Fowl cholera is caused by the bacterium Pasteurella multocida and is known to cause significant economic losses in the commercial turkey industry. Four hundred and thirty cases of P. multocida in commercial turkeys, submitted to the California Animal Health and Food Safety Laboratory System (CAHFS) from January 1, 1991, to December 31, 2017, were analyzed. Records examined included CAHFS branch location, date of submission, clinical signs, company and premise of origin, age and sex of submitted turkeys, macroscopic findings, organs in which P. multocida was isolated, and serotype and fingerprint information. Increased mortality as high as 1200 birds per day was the most common complaint at submission, with acute septicemic lesions observed in the majority of cases. The mean age of turkeys diagnosed with fowl cholera was 14 wk, with a median age of 17 wk. Cases most frequently occurred from September to November, with 36% of cases occurring during this time period. Serotyping was performed in 350 cases, while fingerprinting was performed in 171 cases. Serotypes 3 and 3,4 were frequently identified in the 26-yr time period, while the fingerprints identified varied over time. Despite the decreasing population of commercial turkeys in California since the 1990s, fowl cholera continues to be an economically significant disease in this sector.

Estudio retrospectivo de Pasteurella multocida diagnosticado en pavos comerciales remitidos al Sistema de Laboratorios de Salud Animal y Seguridad Alimentaria del Estado de California; 1991­2017. La enfermedad de cólera aviar es causada por la bacteria Pasteurella multocida y se sabe que causa importantes pérdidas económicas en la industria comercial de pavos. Se analizaron cuatrocientos treinta casos de P. multocida en pavos comerciales, presentados al Sistema de Laboratorios de Salud Animal y Seguridad Alimentaria del Estado de California (CAHFS) desde el primero de enero del 1991 hasta el 31 de diciembre del 2017. Los registros examinados incluyeron la ubicación de la sede de CAHFS, la fecha de presentación, los signos clínicos, la compañía avícola y la granja de origen, la edad y el sexo de los pavos presentados, los hallazgos macroscópicos, los órganos en los que se aisló P. multocida y la información sobre serotipos y de genotipificación. El aumento de la mortalidad hasta 1200 aves por día fue la reclamación más común en el momento de la presentación, con lesiones septicémicas agudas observadas en la mayoría de los casos. La edad media de los pavos diagnosticados con cólera aviar fue de 14 semanas, con una edad media de 17 semanas. Los casos ocurrieron con mayor frecuencia de septiembre a noviembre, con el 36% de los casos ocurridos durante este período. La serotipificación se realizó en 350 casos, mientras que la genotificación se realizó en 171 casos. Los serotipos 3 y 3,4 se identificaron con frecuencia en el período de 26 años, mientras que los genotipos identificados variaron con el tiempo. A pesar de la disminución de la población de pavos comerciales en California desde la década de los 1990s, cólera aviar sigue siendo una enfermedad económicamente significativa en este sector.