Outbreak of densovirus with high mortality in a commercial mealworm (Tenebrio molitor) farm: A molecular, bright-field, and electron microscopic characterization.

Mealworms are one of the most economically important insects in large-scale production for human and animal nutrition. Densoviruses are highly pathogenic for invertebrates and exhibit an extraordinary level of diversity which rivals that of their hosts. Molecular, clinical, histological, and electron microscopic characterization of novel densovirus infections is of utmost economic and ecological importance. Here, we describe an outbreak of densovirus with high mortality in a commercial mealworm (Tenebrio molitor) farm. Clinical signs included inability to prehend food, asymmetric locomotion evolving to nonambulation, dehydration, dark discoloration, and death. Upon gross examination, infected mealworms displayed underdevelopment, dark discoloration, larvae body curvature, and organ/tissue softness. Histologically, there was massive epithelial cell death, and cytomegaly and karyomegaly with intranuclear inclusion (InI) bodies in the epidermis, pharynx, esophagus, rectum, tracheae, and tracheoles. Ultrastructurally, these InIs represented a densovirus replication and assembly complex composed of virus particles ranging from 23.79 to 26.99 nm in diameter, as detected on transmission electron microscopy. Whole-genome sequencing identified a 5579-nucleotide-long densovirus containing 5 open reading frames. A phylogenetic analysis of the mealworm densovirus showed it to be closely related to several bird- and bat-associated densoviruses, sharing 97% to 98% identity. Meanwhile, the nucleotide similarity to a mosquito, cockroach, and cricket densovirus was 55%, 52%, and 41%, respectively. As this is the first described whole-genome characterization of a mealworm densovirus, we propose the name Tenebrio molitor densovirus (TmDNV). In contrast to polytropic densoviruses, this TmDNV is epitheliotropic, primarily affecting cuticle-producing cells.

Greater Species Richness of Bacterial Skin Symbionts Better Suppresses the Amphibian Fungal Pathogen Batrachochytrium Dendrobatidis.

The symbiotic microbes that grow in and on many organisms can play important roles in protecting their hosts from pathogen infection. While species diversity has been shown to influence community function in many other natural systems, the question of how species diversity of host-associated symbiotic microbes contributes to pathogen resistance is just beginning to be explored. Understanding diversity effects on pathogen resistance could be particularly helpful in combating the fungal pathogen Batrachochytrium dendrobatidis (Bd) which has caused dramatic population declines in many amphibian species and is a major concern for amphibian conservation. Our study investigates the ability of host-associated bacteria to inhibit the proliferation of Bd when grown in experimentally assembled biofilm communities that differ in species number and composition. Six bacterial species isolated from the skin of Cascades frogs (Rana cascadae) were used to assemble bacterial biofilm communities containing 1, 2, 3, or all 6 bacterial species. Biofilm communities were grown with Bd for 7 days following inoculation. More speciose bacterial communities reduced Bd abundance more effectively. This relationship between bacterial species richness and Bd suppression appeared to be driven by dominance effects-the bacterial species that were most effective at inhibiting Bd dominated multi-species communities-and complementarity: multi-species communities inhibited Bd growth more than monocultures of constituent species. These results underscore the notion that pathogen resistance is an emergent property of microbial communities, a consideration that should be taken into account when designing probiotic treatments to reduce the impacts of infectious disease.

Evolutionary Dynamics and Global Diversity of Influenza A Virus.

UNLABELLED: The increasing number of zoonotic infections caused by influenza A virus (IAV) subtypes of avian origin (e.g., H5N1 and H7N9) in recent years underscores the need to better understand the factors driving IAV evolution and diversity. To evaluate the current feasibility of global analyses to contribute to this aim, we evaluated information in the public domain to explore IAV evolutionary dynamics, including nucleotide substitution rates and selection pressures, using 14 IAV subtypes in 32 different countries over a 12-year period (2000 to 2011). Using geospatial information from 39,785 IAV strains, we examined associations between subtype diversity and socioeconomic, biodiversity, and agricultural indices. Our analyses showed that nucleotide substitution rates for 11 of the 14 evaluated subtypes tended to be higher in Asian countries, particularly in East Asia, than in Canada and the United States. Similarly, at a regional level, subtypes H5N1, H5N2, and H6N2 exhibited significantly higher substitution rates in East Asia than in North America. In contrast, the selection pressures (measured as ratios of nonsynonymous to synonymous evolutionary changes [dN/dS ratios]) acting on individual subtypes showed little geographic variation. We found that the strongest predictors for the detected subtype diversity at the country level were reporting effort (i.e., total number of strains reported) and health care spending (an indicator of economic development). Our analyses also identified major global gaps in IAV reporting (including a lack of sequences submitted from large portions of Africa and South America and a lack of geolocation information) and in broad subtype testing which, until addressed, will continue to hinder efforts to track the evolution and diversity of IAV around the world. IMPORTANCE: In recent years, an increasing number of influenza A virus (IAV) subtypes, including H5N1, H7N9, and H10N8, have been detected in humans. High fatality rates have led to an increased urgency to better understand where and how novel pathogenic influenza virus strains emerge. Our findings showed that mutational rates of 11 commonly encountered subtypes were higher in East Asian countries than in North America, suggesting that there may be a greater risk for the emergence of novel pathogenic strains in East Asia. In assessing the potential drivers of IAV subtype diversity, our analyses confirmed that reporting effort and health care spending were the best predictors of the observed subtype diversity at the country level. These findings underscore the need to increase sampling and reporting efforts for all subtypes in many undersampled countries throughout the world.

California mussels (Mytilus californianus) as sentinels for marine contamination with Sarcocystis neurona.

Sarcocystis neurona is a terrestrial parasite that can cause fatal encephalitis in the endangered Southern sea otter (Enhydra lutris nereis). To date, neither risk factors associated with marine contamination nor the route of S. neurona infection to marine mammals has been described. This study evaluated coastal S. neurona contamination using California mussels (Mytilus californianus) as sentinels for pathogen pollution. A field investigation was designed to test the hypotheses that (1) mussels can serve as sentinels for S. neurona contamination, and (2) S. neurona contamination in mussels would be highest during the rainy season and in mussels collected near freshwater. Initial validation of molecular assays through sporocyst spiking experiments revealed the ITS-1500 assay to be most sensitive for detection of S. neurona, consistently yielding parasite amplification at concentrations ⩾5 sporocysts/1 mL mussel haemolymph. Assays were then applied on 959 wild-caught mussels, with detection of S. neurona confirmed using sequence analysis in three mussels. Validated molecular assays for S. neurona detection in mussels provide a novel toolset for investigating marine contamination with this parasite, while confirmation of S. neurona in wild mussels suggests that uptake by invertebrates may serve as a route of transmission to susceptible marine animals.

Detection of macropodid alphaherpesvirus 2 infection and lesions in sudden death of a captive Virginia opossum and a water opossum.

Macropodid alphaherpesvirus 2 (MaAHV2) is best described in macropods and has been implicated in outbreaks among captive marsupial populations in Australia. Natural disease caused by herpesviruses has not been reported previously in opossum species, to our knowledge. One Virginia opossum (Didelphis virginiana) and 1 water opossum (Chironectes minimus) were submitted for postmortem examination from a zoo that housed 6 opossums, all of which died within several weeks. Red kangaroos (Macropus rufus) and red-necked wallabies (Macropus rufogriseus) were also present at the facility. Liver samples from both opossums were submitted for transmission electron microscopy and whole-genome sequencing. Microscopically, both opossums had multifocal necrosis in the liver and lung, with intranuclear inclusion bodies within hepatocytes and pneumocytes. Another significant finding in the Virginia opossum was sepsis, with isolation of Streptococcus didelphis from various organs. Ultrastructural analysis of formalin-fixed liver tissue identified herpesviral replication complexes in both opossums; negative-stain electron microscopy of unfixed liver tissue repeatedly yielded a negative result. The herpesvirus had >99% nucleotide identity with MaAHV2. These 2 cases indicate that both opossum species are susceptible to MaAHV2 infection, and the outbreak has implications for mixed-species facilities that house macropods.

Natural Infection with H5N1 Highly Pathogenic Influenza (HPAI) Virus in 5- and 10-Day-Old Commercial Pekin Ducklings (Anas platyrhynchos domesticus).

Highly pathogenic avian influenza (HPAI) has resulted in catastrophic economic losses globally in poultry. This case report describes the diagnostic detection and pathology of HPAI H5N1 in 5-day-old commercial ducklings, which is an atypical age for detection of natural infection of HPAI in poultry. The pathology observed at 5 days of age was also compared to lesions observed in ducklings from the same flock evaluated at 10 days of age before depopulation. The California Animal Health and Food Safety (CAHFS) Laboratory, Tulare, received ten 5-day-old Pekin duckling (Anas platyrhynchos domesticus) carcasses for diagnostic evaluation due to mortality that started increasing at 3 days of age. The most common gross findings included bilateral pulmonary edema with congestion and enlarged, mottled livers and spleens. Microscopically, cerebral neuronophagia, pancreatic necrosis, and interstitial pneumonia with pulmonary edema were observed in the 5-day-old ducklings. Oropharyngeal and cloacal swabs were positive for avian influenza virus (AIV) by real-time reverse transcriptase PCR. The AIV was typed as HPAI, EA/AM 2.3.4.4b H5N1 goose/Guangdong clade lineage by the National Veterinary Services Laboratory. Ducks at the affected premises were depopulated 4 days after the 5-day-old ducklings were submitted to the CAHFS lab, at which time additional tissue samples were collected for comparison to 10-day-old ducklings on the same premises. Differences in microscopic lesions and AIV tissue distribution were observed between the 5-day and 10-day tissues collected. Notably, microscopic lesions were more severe in the brain and pancreas at 10 days of age. Findings in 10-day-old ducklings included cerebral lymphoplasmacytic perivascular cuffing, gliosis, neuronal degeneration, and pancreatic necrosis. AIV antigen distribution and intensity was greatest in the cerebral tissue of the brains at 10 days and in the lungs at 5 days of age. To the authors' knowledge, published studies are limited on AIV natural infection in domestic ducks less than 9 days of age.

Infección natural con el virus de la influenza altamente patógena (HPAI) H5N1 en patitos Pekín comerciales (Anas platyrhynchos domesticus) de 5 y 10 días de edad. La influenza aviar altamente patógena (HPAI) ha provocado pérdidas económicas catastróficas en todo el mundo entre las aves de corral. Este reporte de caso describe la detección diagnóstica y la patología de la infección por un virus de influenza aviar de alta patogenicidad H5N1 en patitos comerciales de 5 días de edad, que es una edad atípica para la detección de la infección natural del virus de la influenza aviar de alta patogenicidad en avicultura. La patología observada a los 5 días de edad también se comparó con las lesiones observadas en patitos de la misma parvada evaluados a los 10 días de edad, antes de la despoblación. El Laboratorio de Salud Animal y Seguridad Alimentaria de California (CAHFS), con sede Tulare, recibió 10 cadáveres de patito Pekín (Anas platyrhynchos domesticus) de 5 días de edad para su evaluación diagnóstica debido a que la mortalidad comenzó a aumentar a los 3 días de edad. Los hallazgos macroscópicos más comunes incluyeron edema pulmonar bilateral con congestión en hígado y bazos agrandados y moteados. Microscópicamente se observó neuronofagia cerebral, necrosis pancreática y neumonía intersticial con edema pulmonar en los patitos de 5 días de edad. Los hisopos orofaríngeos y cloacales fueron positivos para el virus de la influenza aviar (AIV) mediante transcripción reversa y PCR en tiempo real. El Laboratorio Nacional de Servicios Veterinarios clasificó al virus como de alta patogenicidad EA/AM 2.3.4.4b H5N1 clado de linaje de ganso/clado Guangdong. Los patos en las instalaciones afectadas fueron despoblados 4 días después de que los patitos de 5 días fueran enviados al laboratorio de CAHFS, momento en el cual se recolectaron muestras de tejido adicionales para compararlas con patitos de 10 días de las mismas instalaciones. Se observaron diferencias en las lesiones microscópicas y la distribución del tejido del AIV entre los tejidos recolectados de 5 y 10 días. En particular, las lesiones microscópicas fueron más severas en el cerebro y en el páncreas a los 10 días de edad. Los hallazgos en patitos de 10 días incluyeron infiltraciones linfoplasmocitarias perivasculares en el cerebro, gliosis, degeneración neuronal y necrosis pancreática. La distribución e intensidad del antígeno de influenza aviar fue mayor en el tejido cerebral de los cerebros a los 10 días y en los pulmones a los 5 días de edad. De acuerdo al conocimiento de los autores, los estudios publicados sobre la infección natural por el virus de la influenza aviar en patos domésticos de menos de 9 días de edad son limitados.

Evolution of antigen variation in the tick-borne pathogen Anaplasma phagocytophilum.

Anaplasma phagocytophilum is an obligately intracellular tick-transmitted bacterial pathogen of humans and other animals. During the course of infection, A. phagocytophilum utilizes gene conversion to shuffle ∼100 functional pseudogenes into a single expression cassette of the msp2(p44) gene, which codes for the major surface antigen and major surface protein 2 (MSP2). The role and extent of msp2(p44) recombination, particularly in hosts that only experience acute infections, is not clear. In the present study, we explored patterns of recombination and expression of the msp2(p44) gene of A. phagocytophilum in a serially infected mouse model. Even though the bacterium was passed rapidly among mice, minimizing the opportunities for the host to develop adaptive immunity, we detected the emergence of 34 unique msp2(p44) expression cassette variants. The expression of msp2(p44) pseudogenes did not follow a consistent pattern among different groups of mice, although some pseudogenes were expressed more frequently than others. In addition, among 263 expressed pseudogenes, 3 mosaic sequences each consisting of 2 different pseudogenes were identified. Population genetic analysis showed that genetic diversity and subpopulation differentiation tended to increase over time until stationarity was reached but that the variance that was observed in allele (expressed pseudogene) frequency could occur by drift alone only if a high variance in bacterial reproduction could be assumed. These findings suggest that evolutionary forces influencing antigen variation in A. phagocytophilum may comprise random genetic drift as well as some innate but apparently nonpurifying selection prior to the strong frequency-dependent selection that occurs cyclically after hosts develop strong adaptive immunity.

Infectious Bronchitis Virus California Variant CA1737 Isolated from a Commercial Layer Flock with Cystic Oviducts and Poor External Egg Quality.

False layer syndrome is a condition in which the reproductive tract of chicks is infected with infectious bronchitis virus (IBV) strains that cause permanent damage to the oviduct. These chickens subsequently develop cystic oviducts and do not lay eggs, and affected flocks fail to reach expected egg production peaks. The California Animal Health and Food Safety laboratory, Turlock Branch, received four separate case submissions from a 25-to-28-wk-old commercial ISA Brown layer flock. Birds were submitted for diagnostic evaluation due to suboptimal egg production and vent pecking. Submissions totaled 31 birds and consisted of live layers, recent mortality, and a flat of eggs. No clinical signs were observed in the submitted live birds. The most common gross findings included cystic left oviducts, signs of vent pecking, ovarian regression, and yolk coelomitis. The eggs were abnormally shaped with irregular, white, gritty deposits on the surface of the shell. Microscopically, there was atrophy of the oviducts, glandular hypoplasia, and lymphocytic salpingitis. In addition, lymphoplasmacytic tracheitis was observed, and renal tubules were dilated with multifocal areas of mineralization. IBV was identified by reverse transcription quantitative PCR from cecal tonsil tissue pools and tracheal swab pools. Sequencing of the S1 hypervariable region of IBV and whole-genome IBV sequencing were 97% homologous to the California variant CA1737/04. Definitive proof of the CA1737 strain's causing reproductive abnormalities will require challenge studies with fulfillment of Koch's postulates and evaluation of confounding and risk factors.

Reporte de caso- Virus de la bronquitis infecciosa Variante de California CA1737 aislada de una parvada comercial de ponedoras con oviductos quísticos y mala calidad externa del huevo. El síndrome de la falsa capa es una condición en la cual el tracto reproductivo de las gallinas está infectado con cepas del virus de la bronquitis infecciosa (IBV) que causan daño permanente al oviducto. Posteriormente, estas gallinas desarrollan oviductos quísticos y bajas en la postura de huevo, las parvadas afectadas no alcanzan los picos de producción de huevos esperados. El laboratorio de Salud Animal y Seguridad Alimentaria de California, con sede en Turlock, recibió cuatro casos separados de una parvada comercial de ponedoras ISA Brown de 25 a 28 semanas de edad. Las aves se enviaron para evaluación diagnóstica debido a una producción de huevos subóptima y por presencia de picoteo en las cloacas. Se recibieron un total de 31 aves y consistieron en aves de postura vivas, mortalidad reciente y además una charola de huevos. No se observaron signos clínicos en las aves vivas enviadas. Los hallazgos macroscópicos más comunes incluyeron oviductos izquierdos quísticos, signos de picoteo en las cloacas, regresión ovárica y celomitis de la yema. Los huevos tenían una forma anormal con depósitos irregulares, blancos y arenosos en la superficie de la cáscara. Microscópicamente, había atrofia de los oviductos, hipoplasia glandular y salpingitis linfocítica. Además, se observó traqueítis linfoplasmocítica y túbulos renales dilatados con áreas multifocales de mineralización. El virus de la bronquitis infecciosa se identificó mediante PCR cuantitativa de transcripción inversa a partir de grupos de tejidos de tonsilas cecales y muestras agrupadas de hisopos traqueales. La secuenciación de la región hipervariable S1 de IBV y la secuenciación de IBV del genoma completo fueron homólogas en un 97 % a la variante de California CA1737/04. La prueba definitiva de las anomalías reproductivas causantes de la cepa CA1737 requerirá estudios de desafío con el cumplimiento de los postulados de Koch y la evaluación de los factores de riesgo y de confusión.

Whole-genome sequence of a genotype VIII infectious bronchitis virus isolated from California layer chickens in 2021.

Herein, we report the complete genome for an avian infectious bronchitis virus isolated from cecal tonsils of California layers in 2021. This whole-genome sequence belongings to genotype GVIII, previously classified as a unique variant.

Antigen variability in Anaplasma phagocytophilum during chronic infection of a reservoir host.

Anaplasma phagocytophilum is an obligately intracellular, tick-transmitted, bacterial pathogen of humans and other animals. In order to evade host immunity during the course of infection, A. phagocytophilum utilizes gene conversion to shuffle approximately 100 functional pseudogenes into a single expression cassette of the msp2(p44) gene, which encodes the major surface antigen, major surface protein 2 (Msp2). The role and extent of msp2(p44) recombination in a reservoir host for A. phagocytophilum have not been evaluated. In the current study, we explored patterns of recombination and expression site variability of the msp2(p44) gene in three chronically infected woodrats, a reservoir for the disease in the Western USA. All three woodrats developed persistent infection of at least 6 months duration; two of them maintained active infection for at least 8 months. In total, we detected the emergence of 60 unique msp2(p44) expression site variants with no common temporal patterns of expression site recombination among the three A. phagocytophilum populations. Both the strength of infection (i.e. pathogen load) and the genetic diversity of pseudogenes detected at the msp2(p44) expression site fluctuated periodically during the course of infection. An analysis of the genomic pseudogene exhaustion rate showed that the repertoire of pseudogenes available to the A. phagocytophilum population could in theory become depleted within a year. However, the apparent emergence of variant pseudogenes suggests that the pathogen could potentially evade host immunity indefinitely. Our findings suggest a tightly co-evolved relationship between A. phagocytophilum and woodrats in which the pathogen perpetually evades host immunity yet causes no detectable disease.

Early circulation of rabbit haemorrhagic disease virus type 2 in domestic and wild lagomorphs in southern California, USA (2020-2021).

Rabbit haemorrhagic disease virus type 2 (RHDV2) causes a severe systemic disease with hepatic necrosis. Differently from classic RHDV, which affects only European rabbits (Oryctolagus cuniculus), RHDV2 can affect many leporid species, including hares (Lepus spp.) and cottontail rabbits (Sylvilagus spp.). RHDV2 emerged in Europe in 2010 and spread worldwide. During the last 5 years, there have been multiple outbreaks in North America since the first known event in 2016 in Quebec, Canada, including several detections in British Columbia, Canada, between 2018 and 2019, Washington State and Ohio, USA, in 2018 and 2019, and New York, USA, in 2020. However, the most widespread outbreak commenced in March 2020 in the southwestern USA and Mexico. In California, RHDV2 spread widely across several southern counties between 2020 and 2021, and the aim of this study was to report and characterize these early events of viral incursion and circulation within the state. Domestic and wild lagomorphs (n = 81) collected between August 2020 and February 2021 in California with a suspicion of RHDV2 infection were tested by reverse transcription quantitative real-time PCR on the liver, and histology and immunohistochemistry for pan-lagovirus were performed on liver sections. In addition, whole genome sequencing from 12 cases was performed. During this period, 33/81 lagomorphs including 24/59 domestic rabbits (O. cuniculus), 3/16 desert cottontail rabbits (Sylvilagus audubonii), and 6/6 black-tailed jackrabbits (Lepus californicus) tested positive. All RHDV2-positive animals had hepatic necrosis typical of pathogenic lagovirus infection, and the antigen was detected in sections from individuals of the three species. The 12 California sequences were closely related (98.9%-99.95%) to each other, and also very similar (99.0%-99.4%) to sequences obtained in other southwestern states during the 2020-2021 outbreak; however, they were less similar to strains obtained in New York in 2020 (96.7%-96.9%) and Quebec in 2016 (92.4%-92.6%), suggesting that those events could be related to different viral incursions. The California sequences were more similar (98.6%-98.7%) to a strain collected in British Columbia in 2018, which suggests that that event could have been related to the 2020 outbreak in the southwestern USA.

Emergence of fowl aviadenovirus C-4 in a backyard chicken flock in California.

Fowl aviadenovirus (FAdV) species D and E are associated with inclusion body hepatitis (IBH); species C, serotype 4 (hereafter, FAdV4) is associated with hepatitis-hydropericardium syndrome (HHS) in young chickens. Outbreaks of HHS have led to significant losses in the poultry industry in several countries, predominantly in China. In April 2020, FAdV4 was detected in a remote backyard flock in California. In a mixed flock of chickens of various breeds and ages (6 mo to 2 y old), 7 of 30 were found dead within a week without premonitory signs. One additional bird died after the flock was relocated to fresh pasture, bringing the total mortality to 8 of 30 (27%). Postmortem examination of 3 birds revealed good body condition scores and active laying. One chicken had subtle hemorrhages throughout the liver, and the other 2 had diffusely dark mahogany livers. On histopathology, 2 chickens had hepatic necrosis with hepatocytes containing large, mostly basophilic, intranuclear inclusion bodies, identified by electron microscopy as 82.2-nm diameter adenoviral particles. Virus isolation and genomic sequencing performed on a liver sample revealed strains with 99.9% homology to FAdV4 isolates reported from China. To our knowledge, FAdV4 has not been reported in the United States to date. Furthermore, the chickens affected here were all adults and exhibited a variation of serotype 4 disease in which IBH was present but not hydropericardium.

Sarcocystis neurona Transmission from Opossums to Marine Mammals in the Pacific Northwest.

Increasing reports of marine mammal deaths have been attributed to the parasite Sarcocystis neurona. Infected opossums, the only known definitive hosts, shed S. neurona sporocysts in their feces. Sporocysts can contaminate the marine environment via overland runoff, and subsequent ingestion by marine mammals can lead to fatal encephalitis. Our aim was to determine the prevalence of S. neurona in opossums from coastal areas of Washington State (USA) and to compare genetic markers between S. neurona in opossums and marine mammals. Thirty-two road-kill opossums and tissue samples from 30 stranded marine mammals meeting inclusion criteria were included in analyses. Three opossums (9.4%) and twelve marine mammals (40%) were confirmed positive for S. neurona via DNA amplification at the ITS1 locus. Genetic identity at microsatellites (sn3, sn7, sn9) and the snSAG3 gene of S. neurona was demonstrated among one harbor porpoise and two opossums. Watershed mapping further demonstrated plausible sporocyst transport pathways from one of these opossums to the location where an infected harbor porpoise carcass was recovered. Our results provide the first reported link between S. neurona genotypes on land and sea in the Pacific Northwest, and further demonstrate how terrestrial pathogen pollution can impact the health of marine wildlife.

Nanopore sequencing as a rapid tool for identification and pathotyping of avian influenza A viruses.

We report whole-genome sequencing of influenza A virus (IAV) with 100% diagnostic sensitivity and results available in <24-48 h using amplicon-based nanopore sequencing technology (MinION) on clinical material from wild waterfowl (n = 19), commercial poultry (n = 4), and swine (n = 3). All 8 gene segments of IAV including those from 14 of the 18 recognized hemagglutinin subtypes and 9 of the 11 neuraminidase subtypes were amplified in their entirety at >500× coverage from each of 16 reference virus isolates evaluated. Subgenomic viral sequences obtained in 3 cases using Sanger sequencing as the reference standard were identical to those obtained when sequenced using the MinION approach. An inter-laboratory comparison demonstrated reproducibility when comparing 2 independent laboratories at ≥99.8% across the entirety of the IAV genomes sequenced.

Infectious Bronchitis Virus Prevalence, Characterization, and Strain Identification in California Backyard Chickens.

Infectious bronchitis virus (IBV) causes significant losses in the poultry industry throughout the world. Here we characterize the lesions of infectious bronchitis (IB) and IBV prevalence and identify the circulating strains in small flocks in California. Backyard chickens (BYCs) submitted to the Davis (Northern California; NorCal) and San Bernardino (Southern California; SoCal) branches of the California Animal Health and Food Safety Laboratory System from January through March 2019 were included in the study. Trachea, kidney, and cecal tonsils were collected for real-time reverse transcriptase (qRT)-PCR, histology, immunohistochemistry (IHC), and sequence analysis. A total of 50 chickens out of 169 submissions tested positive for IBV by qRT-PCR. Of these, 16% (20/123) were from NorCal and 65% (30/46) from SoCal laboratory. The cecal tonsil was the most frequently positive tissue by qRT-PCR and IHC. Lymphoplasmacytic tracheitis was the most frequent histopathologic finding in 24 of 39 birds, while the kidney showed interstitial nephritis, tubular necrosis, tubular dilation, and/or gout in 14 of 43 chickens. Infectious bronchitis virus played a primary role or a synergistic effect in the mortality of chickens that succumbed to other infectious diseases. The sequences of IBV detected in 22 birds were analyzed, and 14 strains were most similar to CA1737. One strain each matched Conn46, Cal99, and ArkDPI, and the remaining five did not have a substantial match to any available reference strains. The findings in this study indicate that small flocks can be reservoirs of IBV and might facilitate evolution of new variants as well as reversion of attenuated strains to virulence.

Artículo regular­Prevalencia, caracterización e identificación de cepas del virus de la bronquitis infecciosa en pollos de traspatio de California. El virus de la bronquitis infecciosa (con las siglas en inglés IBV) causa pérdidas significativas en la industria avícola en todo el mundo. En este estudio se caracterizaron las lesiones de la bronquitis infecciosa (IB), la prevalencia del virus y se identificó a las cepas circulantes en pequeñas parvadas en California. Se incluyeron en el estudio pollos de traspatio (BYC) remitidos a las sedes en Davis (norte de California; NorCal) y San Bernardino (sur de California; SoCal) del Sistema de Laboratorios de Salud Animal y Seguridad Alimentaria de California de enero a marzo del 2019. Se recolectaron tráquea, riñón y tonsilas cecales para análisis cuantitativo en tiempo real (qRT)-PCR, histología, inmunohistoquímica (IHC) y análisis de secuencias. Un total de 50 pollos de 169 casos dieron positivo para la presencia del virus de bronquitis infecciosa por qRT-PCR. De estos, el 16% (20/123) provenían del norte de California y el 65% (30/46) del laboratorio del sur de California. Las tonsilas cecales fueron las muestras de tejidos positivos con mayor frecuencia por qRT-PCR e IHC. La traqueítis linfoplasmocítica fue el hallazgo histopatológico más frecuente en 24 de 39 aves, mientras que el riñón mostró nefritis intersticial, necrosis tubular, dilatación tubular y/o gota en 14 de 43 pollos. El virus de la bronquitis infecciosa jugó un papel principal o un efecto sinérgico en la mortalidad de los pollos que murieron por otras enfermedades infecciosas. Se analizaron las secuencias del virus de bronquitis detectadas en 22 aves y 14 cepas fueron muy similares al virus de bronquitis infecciosa CA1737. Tres virus coincidieron con Conn46, Cal99 y ArkDPI, y las cinco restantes no tenían una coincidencia sustancial con ninguna cepa de referencia disponible. Los hallazgos de este estudio indican que las pequeñas parvadas pueden ser reservorios del virus de la bronquitis infecciosa y podrían facilitar la evolución de nuevas variantes, así como la reversión de cepas atenuadas a formas virulentas.

Natural fatal Sarcocystis falcatula infections in free-ranging eagles in North America.

Three bald eagles (Haliaeetus leucocephalus) and 1 golden eagle (Aquila chrysaetos) were admitted to rehabilitation facilities with emaciation, lethargy, and an inability to fly. Intravascular schizonts and merozoites were present in 2 bald eagles, mainly in the lung tissue, whereas the third bald eagle and the golden eagle had lymphohistiocytic encephalitis with intralesional schizonts and merozoites. In all eagles, protozoal tissue cysts were present in skeletal musculature or heart. The protozoal organisms were morphologically compatible with a Sarcocystis sp. By immunohistochemistry, the protozoal merozoites were positive for Sarcocystis falcatula antigen in all cases when using polyclonal antisera. Furthermore, the protozoa were confirmed to be most similar to S. falcatula by polymerase chain reaction in 3 of the 4 cases. To the authors' knowledge, this report presents the first cases of natural infection in eagles with S. falcatula as a cause of mortality.

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.

Sarcocystis falcatula-associated encephalitis in a free-ranging great horned owl (Bubo virginianus).

A great horned owl (Bubo virginianus) was admitted to a rehabilitation clinic with severe neurologic signs that were unresponsive to supportive care. The animal was euthanatized because of a poor prognosis. Marked granulomatous encephalitis with focal brainstem malacia was detected microscopically. The brainstem was the most severely affected brain location and the only place in which schizonts and merozoites, morphologically compatible with Sarcocystis spp., were detected. Immunohistochemistry with the use of polyclonal antisera indicated the presence of Sarcocystis falcatula. The species identification of the protozoa as S. falcatula was confirmed by polymerase chain reaction. To the author's knowledge, this is the first report of spontaneous S. falcatula-associated encephalitis in a great horned owl.

Equine protozoal myeloencephalitis caused by Neospora hughesi in an adult horse in Saskatchewan.

A protozoal parasite identified as Neospora hughesi was found in inflammatory lesions in the central nervous system of a Canadian-born adult horse presented with neurological signs. This is believed to be the first case of equine protozoal myeloencephalitis (EPM) caused by Neospora hughesi in a horse outside of the United States.