Piscichuvirus-Associated Severe Meningoencephalomyelitis in Aquatic Turtles, United States, 2009-2021.

Viruses from a new species of piscichuvirus were strongly associated with severe lymphocytic meningoencephalomyelitis in several free-ranging aquatic turtles from 3 coastal US states during 2009-2021. Sequencing identified 2 variants (freshwater turtle neural virus 1 [FTuNV1] and sea turtle neural virus 1 [STuNV1]) of the new piscichuvirus species in 3 turtles of 3 species. In situ hybridization localized viral mRNA to the inflamed region of the central nervous system in all 3 sequenced isolates and in 2 of 3 additional nonsequenced isolates. All 3 sequenced isolates phylogenetically clustered with other vertebrate chuvirids within the genus Piscichuvirus. FTuNV1 and STuNV1 shared ≈92% pairwise amino acid identity of the large protein, which narrowly places them within the same novel species. The in situ association of the piscichuviruses in 5 of 6 turtles (representing 3 genera) with lymphocytic meningoencephalomyelitis suggests that piscichuviruses are a likely cause of lymphocytic meningoencephalomyelitis in freshwater and marine turtles.

A self-limited acute febrile sterile neutrophilic dermatosis (Sweet's-like syndrome) in a dog featuring target skin lesions with strong upregulation of interleukin-8 and T-helper 1 pathway.

In this report, we provide a case of self-limiting canine acute febrile sterile neutrophilic dermatosis in which the clinical signs featured typical target skin lesions with strong upregulation of T-helper 1 markers and interleukin-8, a potent neutrophil chemoattractant. Further, large case series are needed to characterize canine sterile neutrophilic dermatosis.

Dans cet article, nous présentons un cas de dermatose neutrophilique fébrile stérile aiguë canine spontanément résolutive dans laquelle les signes cliniques comportaient des lésions cutanées cibles typiques avec une forte régulation à la hausse des marqueurs T-helper 1 et de l'interleukine-8, un puissant chimioattractant des neutrophiles. D'autres grandes séries de cas sont nécessaires pour caractériser la dermatose neutrophile stérile canine.

En este artículo exponemos un caso de dermatosis neutrofílica estéril febril aguda canina autolimitante en la que los signos clínicos fueron lesiones cutáneas típicas en forma de diana con una intensa elevación de los marcadores T-helper 1 y de interleucina-8, un potente quimiotáctico de neutrófilos. Se necesitan series de casos más grandes para caracterizar la dermatosis neutrofílica estéril canina.

Neste relato, nós apresentamos um caso de dermatose neutrofílica estéril canina aguda febril em que os sinais clínicos típicos foram lesões cutâneas em alvo com forte ativação de marcadores T-helper 1 e interleucina-8, um potente quimioatrativo de neutrófilos. São necessários mais estudos com uma grande série de casos para caracterizar a dermatose neutrofílica estéril canina.

Putative Novel Avian Paramyxovirus (AMPV) and Reidentification of APMV-2 and APMV-6 to the Species Level Based on Wild Bird Surveillance (United States, 2016-2018).

Avian paramyxoviruses (APMVs) (subfamily Avulavirinae) have been isolated from over 200 species of wild and domestic birds around the world. The International Committee on Taxonomy of Viruses (ICTV) currently defines 22 different APMV species, with Avian orthoavulavirus 1 (whose viruses are designated APMV-1) being the most frequently studied due to its economic burden to the poultry industry. Less is known about other APMV species, including limited knowledge on the genetic diversity in wild birds, and there is a paucity of public whole-genome sequences for APMV-2 to -22. The goal of this study was to use MinION sequencing to genetically characterize APMVs isolated from wild bird swab samples collected during 2016 to 2018 in the United States. Multiplexed MinION libraries were prepared using a random strand-switching approach using 37 egg-cultured, influenza-negative, hemagglutination-positive samples. Forty-one APMVs were detected, with 37 APMVs having complete polymerase coding sequences allowing for species identification using ICTV's current Paramyxoviridae phylogenetic methodology. APMV-1, -4, -6, and -8 viruses were classified, one putative novel species (Avian orthoavulavirus 23) was identified from viruses isolated in this study, two putative new APMV species (Avian metaavulavirus 24 and 27) were identified from viruses isolated in this study and from retrospective GenBank sequences, and two putative new APMV species (Avian metaavulavirus 25 and 26) were identified solely from retrospective GenBank sequences. Furthermore, coinfections of APMVs were identified in four samples. The potential limitations of the branch length being the only species identification criterion and the potential benefit of a group pairwise distance analysis are discussed. IMPORTANCE Most species of APMVs are understudied and/or underreported, and many species were incidentally identified from asymptomatic wild birds; however, the disease significance of APMVs in wild birds is not fully determined. The rapid rise in high-throughput sequencing coupled with avian influenza surveillance programs have identified 12 different APMV species in the last decade and have challenged the resolution of classical serological methods to identify new viral species. Currently, ICTV's only criterion for Paramyxoviridae species classification is the requirement of a branch length of >0.03 using a phylogenetic tree constructed from polymerase (L) amino acid sequences. The results from this study identify one new APMV species, propose four additional new APMV species, and highlight that the criterion may have insufficient resolution for APMV species demarcation and that refinement or expansion of this criterion may need to be established for Paramyxoviridae species identification.

Runting and Stunting Syndrome in Broiler Chickens: Histopathology and Association With a Novel Picornavirus.

Runting stunting syndrome (RSS) in commercial chickens has been reported worldwide, and although several studies have attempted to clarify the cause and describe the lesions, there are gaps in knowledge of the epidemiology, pathogenesis, and etiology. The study objective was to use commercial chicks naturally affected by RSS to describe the histologic changes of RSS in all segments of the small intestine in chicks of different ages and to identify viral gene sequences in affected chicks and their association with histologic lesions. Chicks lacking clinical signs but from the same houses and from unaffected houses were used as controls. The average weight of affected chicks was significantly lower than expected for their flocks. Macroscopically, the small intestines had paler serosa, with watery, mucoid, or foamy contents and poorly digested food. Histologic lesions were characterized by necrotic crypts, crypt dilation, and flattening of the crypt epithelium. Histomorphometry of the intestines revealed villous atrophy especially in the jejunum and ileum. Histologic changes in other organs were not observed. Random next-generation sequencing of total RNA extracted from formalin-fixed paraffin-embedded tissues detected avian nephritis virus, avian rotavirus, and picornavirus in jejunal segments from 7-day-old chicks. No viruses were detected in the jejunum of 1-day-old chicks. Detection of picornaviral reads was significantly associated (P < .05) with histologic lesions of RSS. Sequence analysis of the picornavirus revealed genetic similarity with the genus Gallivirus. Using in situ hybridization for galliviral nucleic acid sequences, the signal was associated with crypt lesion severity, although signal was detected both in chicks with and without RSS.

Cladophialophora encephalitis in an alpaca.

A 4-year-old Huacaya hembra was evaluated for acute neurologic signs including recumbency and a left head tilt. Cranial nerve examination revealed a left ear droop, muzzle deviation to the right, mydriasis of the left eye, an absent menace response, bilateral absent pupillary light reflex when light was directed into the left eye, and bilateral horizontal nystagmus with fast phase to the right. Multifocal intracranial lesions were suspected. Computed tomography revealed an intracranial mass. Postmortem examination, histopathology, and sequencing of a polymerase chain reaction product confirmed a diagnosis of phaeohyphomycotic meningoencephalitis caused by Cladophialophora bantiana. Key clinical message: Advanced diagnostic imaging (computed tomography) was useful in achieving a diagnosis of an intracranial mass in an alpaca with acute neurological signs, later confirmed to be central nervous system (CNS) phaeohyphomycosis. Although uncommon, intracranial fungal infection should be considered as a differential diagnosis in camelid patients exhibiting CNS signs, particularly if they do not respond to initial antimicrobial and anthelmintic therapy.

Encéphalite à Cladophialophora chez un alpaga. Une femelle alpaga de race Huacaya âgée de 4 ans fut évaluée pour des signes neurologiques aigus incluant un décubitus et une inclinaison de la tête à gauche. L'examen des nerfs crâniens a révélé un affaissement de l'oreille gauche, une déviation vers la droite du museau, une mydriase de l'oeil gauche, une absence de réponse à la menace, l'absence bilatérale de réflexe pupillaire lorsqu'une lumière était pointée dans l'oeil gauche, et un nystagmus horizontal bilatéral avec phase rapide vers la droite. Des lésions intra-crâniales multifocales étaient suspectées. Un examen par tomodensitométrie révéla une masse intra-crâniale. L'examen post-mortem, l'histopathologie et le séquençage d'un produit de réaction d'amplification en chaîne par la polymérase confirmèrent un diagnostic de méningo-encéphalite phaeohyphomycotique causée par Cladophialophora bantiana.Message clinique clé :L'examen par imagerie diagnostique de pointe (tomodensitométrie) fut utile afin d'arriver à un diagnostic de masse intra-crâniale chez un alpaga avec des signes neurologiques aigus, plus tard confirmé par une phaeohyphomycose du système nerveux central (CNS). Bien que peu fréquente, une infection fongique intra-crâniale devrait être considérée comme un diagnostic différentiel chez des camélidés présentant des signes du CNS, particulièrement s'ils ne répondent pas à un traitement initial avec des antimicrobiens et des anthelmintiques.(Traduit par Dr Serge Messier).

Enhanced phylogenetic resolution of Newcastle disease outbreaks using complete viral genome sequences from formalin-fixed paraffin-embedded tissue samples.

Highly virulent Newcastle disease virus (NDV) causes Newcastle disease (ND), which is a threat to poultry production worldwide. Effective disease management requires approaches to accurately determine sources of infection, which involves tracking of closely related viruses. Next-generation sequencing (NGS) has emerged as a research tool for thorough genetic characterization of infectious organisms. Previously formalin-fixed paraffin-embedded (FFPE) tissues have been used to conduct retrospective epidemiological studies of related but genetically distinct viruses. However, this study extends the applicability of NGS for complete genome analysis of viruses from FFPE tissues to track the evolution of closely related viruses. Total RNA was obtained from FFPE spleens, lungs, brains, and small intestines of chickens in 11 poultry flocks during disease outbreaks in Pakistan. The RNA was randomly sequenced on an Illumina MiSeq instrument and the raw data were analyzed using a custom data analysis pipeline that includes de novo assembly. Genomes of virulent NDV were detected in 10/11 birds: eight nearly complete (> 95% coverage of concatenated coding sequence) and two partial genomes. Phylogeny of the NDV complete genome coding sequences was compared to current methods of analysis based on the full and partial fusion genes and determined that the approach provided a better phylogenetic resolution. Two distinct lineages of sub-genotype VIIi NDV were identified to be simultaneously circulating in Pakistani poultry. Non-targeted NGS of total RNA from FFPE tissues coupled with de novo assembly provided a reliable, safe, and affordable method to conduct epidemiological and evolutionary studies to facilitate management of ND in Pakistan.

Tropism of Newcastle disease virus strains for chicken neurons, astrocytes, oligodendrocytes, and microglia.

BACKGROUND: Newcastle disease (ND), which is caused by infections of poultry species with virulent strains of Avian orthoavulavirus-1, also known as avian paramyxovirus 1 (APMV-1), and formerly known as Newcastle disease virus (NDV), may cause neurological signs and encephalitis. Neurological signs are often the only clinical signs observed in birds infected with neurotropic strains of NDV. Experimental infections have shown that the replication of virulent NDV (vNDV) strains is in the brain parenchyma and is possibly confined to neurons and ependymal cells. However, little information is available on the ability of vNDV strains to infect subset of glial cells (astrocytes, oligodendrocytes, and microglia). The objective of this study was to evaluate the ability of NDV strains of different levels of virulence to infect a subset of glial cells both in vitro and in vivo. Thus, neurons, astrocytes and oligodendrocytes from the brains of day-old White Leghorn chickens were harvested, cultured, and infected with both non-virulent (LaSota) and virulent, neurotropic (TxGB) NDV strains. To confirm these findings in vivo, the tropism of three vNDV strains with varying pathotypes (SA60 [viscerotropic], TxGB [neurotropic], and Tx450 [mesogenic]) was assessed in archived formalin-fixed material from day-old chicks inoculated intracerebrally. RESULTS: Double immunofluorescence for NDV nucleoprotein and cellular markers showed that both strains infected at least 20% of each of the cell types (neurons, astrocytes, and oligodendrocytes). At 24 h post-inoculation, TxGB replicated significantly more than LaSota. Double immunofluorescence (DIFA) with markers for neurons, astrocytes, microglia, and NDV nucleoprotein detected the three strains in all three cell types at similar levels. CONCLUSION: These data indicate that similar to other paramyxoviruses, neurons and glial cells (astrocytes, oligodendrocytes, and microglia) are susceptible to vNDV infection, and suggest that factors other than cellular tropism are likely the major determinant of the neurotropic phenotype.

Rapid virulence prediction and identification of Newcastle disease virus genotypes using third-generation sequencing.

BACKGROUND: Newcastle disease (ND) outbreaks are global challenges to the poultry industry. Effective management requires rapid identification and virulence prediction of the circulating Newcastle disease viruses (NDV), the causative agent of ND. However, these diagnostics are hindered by the genetic diversity and rapid evolution of NDVs. METHODS: An amplicon sequencing (AmpSeq) workflow for virulence and genotype prediction of NDV samples using a third-generation, real-time DNA sequencing platform is described here. 1D MinION sequencing of barcoded NDV amplicons was performed using 33 egg-grown isolates, (15 NDV genotypes), and 15 clinical swab samples collected from field outbreaks. Assembly-based data analysis was performed in a customized, Galaxy-based AmpSeq workflow. MinION-based results were compared to previously published sequences and to sequences obtained using a previously published Illumina MiSeq workflow. RESULTS: For all egg-grown isolates, NDV was detected and virulence and genotype were accurately predicted. For clinical samples, NDV was detected in ten of eleven NDV samples. Six of the clinical samples contained two mixed genotypes as determined by MiSeq, of which the MinION method detected both genotypes in four samples. Additionally, testing a dilution series of one NDV isolate resulted in NDV detection in a dilution as low as 101 50% egg infectious dose per milliliter. This was accomplished in as little as 7 min of sequencing time, with a 98.37% sequence identity compared to the expected consensus obtained by MiSeq. CONCLUSION: The depth of sequencing, fast sequencing capabilities, accuracy of the consensus sequences, and the low cost of multiplexing allowed for effective virulence prediction and genotype identification of NDVs currently circulating worldwide. The sensitivity of this protocol was preliminary tested using only one genotype. After more extensive evaluation of the sensitivity and specificity, this protocol will likely be applicable to the detection and characterization of NDV.

Whole-genome sequencing of genotype VI Newcastle disease viruses from formalin-fixed paraffin-embedded tissues from wild pigeons reveals continuous evolution and previously unrecognized genetic diversity in the U.S.

BACKGROUND: Newcastle disease viruses (NDV) are highly contagious and cause disease in both wild birds and poultry. A pigeon-adapted variant of genotype VI NDV, often termed pigeon paramyxovirus 1, is commonly isolated from columbids in the United States and worldwide. Complete genomic characterization of these genotype VI viruses circulating in wild columbids in the United States is limited, and due to the genetic variability of the virus, failure of rapid diagnostic detection has been reported. Therefore, in this study, formalin-fixed paraffin-embedded (FFPE) samples were subjected to next-generation sequencing (NGS) to identify and characterize these circulating viruses, providing valuable genetic information. NGS enables multiple samples to be deep-sequenced in parallel. When used on FFPE samples, this methodology allows for retrospective studies of infectious organisms. METHODS: FFPE wild pigeon tissue samples (kidney, liver and spleen) from 10 mortality events in the U.S. between 2010 and 2016 were analyzed using NGS to detect and sequence NDV genomes from randomly amplified total RNA. Results were compared to the previously published immunohistochemistry (IHC) results conducted on the same samples. Additionally, phylogenetic analyses were conducted on the complete and partial fusion gene and complete genome coding sequences. RESULTS: Twenty-three out of 29 IHC-positive FFPE pigeon samples were identified as positive for NDV by NGS. Positive samples produced an average genome coverage of 99.6% and an average median depth of 199. A previously described sub-genotype (VIa) and a novel sub-genotype (VIn) of NDV were identified as the causative agent of 10 pigeon mortality events in the U.S. from 2010 to 2016. The distribution of these viruses from the North American lineages match the distribution of the Eurasian collared-doves and rock pigeons in the U.S. CONCLUSIONS: This work reports the first successful evolutionary study using deep sequencing of complete NDV genomes from FFPE samples of wild bird origin. There are at least two distinct U.S. lineages of genotype VI NDV maintained in wild pigeons that are continuously evolving independently from each other and have no evident epidemiological connections to viruses circulating abroad. These findings support the hypothesis that columbids are serving as reservoirs of virulent NDV in the U.S.

Early AMD-like defects in the RPE and retinal degeneration in aged mice with RPE-specific deletion of Atg5 or Atg7.

PURPOSE: To examine the effects of autophagy deficiency induced by RPE-specific deletion of Atg5 or Atg7 in mice as a function of age. METHODS: Conditional knockout mice with a floxed allele of Atg5 or Atg7 were crossed with inducible VMD2-rtTA/Cre transgenic mice. VMD2-directed RPE-specific Cre recombinase expression was induced with doxycycline feeding in the resulting mice. Cre-mediated deletion of floxed Atg5 or Atg7 resulted in RPE-specific inactivation of the Atg5 or Atg7 gene. Plastic and thin retinal sections were analyzed with light and electron microscopy for histological changes. Photoreceptor outer segment (POS) thickness in plastic sections was measured using the Adobe Photoshop CS4 extended ruler tool. Autophagic adaptor p62/SQSTM1 and markers for oxidatively damaged lipids, proteins, and DNA were examined with immunofluorescence staining of cryosections. Fluorescence signals were quantified using Image J software. RESULTS: Accumulation of p62/SQSTM1 reflecting autophagy deficiency was observed in the RPE of the Atg5ΔRPE and Atg7ΔRPE mice. 3-nitrotyrosine, advanced glycation end products (AGEs), and 8-hydroxy-2'-deoxyguanosine (8-OHdG), markers for oxidatively damaged proteins and DNA, were also found to accumulate in the RPE of these mice. We observed retinal degeneration in 35% of the Atg5ΔRPE mice and 45% of the Atg7ΔRPE mice at 8 to 24 months old. Degeneration severity and the number of mice with degeneration increased with age. The mean POS thickness of these mice was 25 µm at 8-12 months, 15 µm at 13-18 months, and 3 µm at 19-24 months, compared to 35 µm, 30 µm, and 24 µm in the wild-type mice, respectively. Early age-related macular degeneration (AMD)-like RPE defects were found in all the Atg5ΔRPE and Atg7ΔRPE mice 13 months old or older, including vacuoles, uneven RPE thickness, diminished basal infoldings, RPE hypertrophy/hypotrophy, pigmentary irregularities, and necrosis. The severity of the RPE defects increased with age and in the mice with retinal degeneration. RPE atrophy and choroidal neovascularization (CNV) were occasionally observed in the Atg5ΔRPE and Atg7ΔRPE mice with advanced age. CONCLUSIONS: Autophagy deficiency induced by RPE-specific deletion of Atg5 or Atg7 predisposes but does not necessarily drive the development of AMD-like phenotypes or retinal degeneration.

Antiprion Activity of DB772 and Related Monothiophene- and Furan-Based Analogs in a Persistently Infected Ovine Microglia Culture System.

The transmissible spongiform encephalopathies are fatal neurodegenerative disorders characterized by the misfolding of the native cellular prion protein (PrP(C)) into the accumulating, disease-associated isoform (PrP(Sc)). Despite extensive research into the inhibition of prion accumulation, no effective treatment exists. Previously, we demonstrated the inhibitory activity of DB772, a monocationic phenyl-furan-benzimidazole, against PrP(Sc) accumulation in sheep microglial cells. In an effort to determine the effect of structural substitutions on the antiprion activity of DB772, we employed an in vitro strategy to survey a library of structurally related, monothiophene- and furan-based compounds for improved inhibitory activity. Eighty-nine compounds were screened at 1 µM for effects on cell viability and prion accumulation in a persistently infected ovine microglia culture system. Eleven compounds with activity equivalent to or higher than that of DB772 were identified as preliminary hit compounds. For the preliminary hits, cytotoxicities and antiprion activities were compared to calculate the tissue culture selectivity index. A structure-activity relationship (SAR) analysis was performed to determine molecular components contributing to antiprion activity. To investigate potential mechanisms of inhibition, effects on PrP(C) and PrP(Sc) were examined. While inhibition of total PrP(C) was not observed, the results suggest that a potential target for inhibition at biologically relevant concentrations is through PrP(C) misfolding to PrP(Sc) Further, SAR analysis suggests that two structural elements were associated with micromolar antiprion activity. Taken together, the described data provide a foundation for deeper investigation into untested DB compounds and in the design of effective therapeutics.

Systemic toxoplasmosis in 2 domestic rabbits in Georgia, United States.

Toxoplasma gondii infections are common in a range of mammalian and avian species, but clinical disease has been reported only rarely in domestic rabbits. Two cases of toxoplasmosis in domestic rabbits from the same premises were submitted to a diagnostic pathology facility in Athens, GA, USA. Both rabbits died after exhibiting clinical signs of gastrointestinal stasis. The gross findings observed in both rabbits comprised miliary, random, white-to-tan, necrotic foci throughout the spleen, liver, and lungs. Histologically, tachyzoites were observed within necrotizing inflammatory foci in the spleens of both rabbits, and in various other organs (tracheobronchial lymph node, lung, heart, and cecal appendix) of one rabbit. In both cases, the tachyzoites were immunoreactive with anti-Toxoplasma gondii antibodies. In addition, T. gondii DNA was detected via PCR and sequencing from a fresh lung sample from one rabbit and formalin-fixed, paraffin-embedded spleen, liver, femoral bone marrow, and haired skin from the second rabbit. Given that T. gondii can cause disease in domestic rabbits and is also a concern for other potential intermediate hosts (e.g., humans, other domestic animals), this parasite warrants consideration in the diagnostic evaluation of lagomorph tissues with compatible lesions.

Pandemic Risk Assessment for Swine Influenza A Virus in Comparative In Vitro and In Vivo Models.

Swine influenza A viruses pose a public health concern as novel and circulating strains occasionally spill over into human hosts, with the potential to cause disease. Crucial to preempting these events is the use of a threat assessment framework for human populations. However, established guidelines do not specify which animal models or in vitro substrates should be used. We completed an assessment of a contemporary swine influenza isolate, A/swine/GA/A27480/2019 (H1N2), using animal models and human cell substrates. Infection studies in vivo revealed high replicative ability and a pathogenic phenotype in the swine host, with replication corresponding to a complementary study performed in swine primary respiratory epithelial cells. However, replication was limited in human primary cell substrates. This contrasted with our findings in the Calu-3 cell line, which demonstrated a replication profile on par with the 2009 pandemic H1N1 virus. These data suggest that the selection of models is important for meaningful risk assessment.

Discovery of genomic loci for liver health and steatosis reveals overlap with glutathione redox genetics.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver condition in the United States, encompassing a wide spectrum of liver pathologies including steatosis, steatohepatitis, fibrosis, and cirrhosis. Despite its high prevalence, there are no medications currently approved by the Food and Drug Administration for the treatment of NAFLD. Recent work has suggested that NAFLD has a strong genetic component and identifying causative genes will improve our understanding of the molecular mechanisms contributing to NAFLD and yield targets for future therapeutic investigations. Oxidative stress is known to play an important role in NAFLD pathogenesis, yet the underlying mechanisms accounting for disturbances in redox status are not entirely understood. To better understand the relationship between the glutathione redox system and signs of NAFLD in a genetically-diverse population, we measured liver weight, serum biomarkers aspartate aminotransferase (AST) and alanine aminotransferase (ALT), and graded liver pathology in a large cohort of Diversity Outbred mice. We compared hepatic endpoints to those of the glutathione redox system previously measured in the livers and kidneys of the same mice, and we screened for statistical and genetic associations using the R/qtl2 software. We discovered several novel genetic loci associated with markers of liver health, including loci that were associated with both liver steatosis and glutathione redox status. Candidate genes within each locus point to possible new mechanisms underlying the complex relationship between NAFLD and the glutathione redox system, which could have translational implications for future studies targeting NAFLD pathology.

PrPres in placental tissue following experimental transmission of atypical scrapie in ARR/ARR sheep is not infectious by Tg338 mouse bioassay.

Nor98-like atypical scrapie is a sporadic disease that affects the central nervous system of sheep and goats that, in contrast to classical scrapie, is not generally regarded as naturally transmissible. However, infectivity has been demonstrated via bioassay not only of brain tissue but also of certain peripheral nerves, lymphoid tissues, and muscle. This study examines placental tissue, a well characterized route of natural transmission for classical scrapie. Further, this study was conducted in sheep homozygous for the classical scrapie resistant ARR genotype and is the first to characterize the transmission of Nor98-like scrapie between homozygous-ARR sheep. Nor98-like scrapie isolated from a United States ARR/ARR sheep was transmitted to four ARR/ARR ewes via intracerebral inoculation of brain homogenate. These ewes were followed and observed to 8 years of age, remained non-clinical but exhibited progression of infection that was consistent with Nor98-like scrapie, including characteristic patterns of PrPSc accumulation in the brain and a lack of accumulation in peripheral lymphoid tissues as detected by conventional methods. Immunoblots of placental tissues from the infected ewes revealed accumulation of a distinct conformation of PrPres, particularly as the animals aged; however, the placenta showed no infectivity when analyzed via ovinized mouse bioassay. Taken together, these results support a low risk for natural transmission of Nor98-like scrapie in ARR/ARR sheep.

MYXOZOAN COMMUNITY COMPOSITION AND DIVERSITY IN CLINICAL CASES OF PROLIFERATIVE GILL DISEASE IN MISSISSIPPI CATFISH AQUACULTURE.

An abundance of morphologically variable Henneguya species complicates the understanding of disease relationships between ictalurid catfish and myxozoan (Phylum: Cnidaria) parasites on North American aquaculture operations. Henneguya ictaluri, the cause of proliferative gill disease (PGD) in channel and hybrid catfish, is arguably the most important parasite of commercial catfish aquaculture in the southeastern United States. While research indicates arrested development and limited sporogenesis of H. ictaluri in channel (Ictalurus punctatus) × blue (Ictalurus furcatus) hybrid catfish, incidents of PGD persist in hybrid production systems. This work investigated the influence of fish host on myxozoan community composition and diversity within naturally infected gill tissues from diagnostic case submissions to the Aquatic Research and Diagnostic Laboratory in Stoneville, Mississippi, from 2017 to 2019. Gills collected from farm-raised catfish with clinical PGD were subjected to metagenomic amplicon sequencing of the myxozoan 18S SSU rDNA gene diagnostic variable region 3 (DVR3). Myxozoan community composition significantly differed between channel and hybrid catfish PGD cases, with channel catfish having more diverse community structures. Channel catfish gills had a greater relative abundance of H. ictaluri in 2017 and 2019, while no differences were observed in 2018. Importantly, H. ictaluri was present in all channel and hybrid catfish PGD cases across all years; however, H. ictaluri was not the most abundant myxozoan in almost half the cases examined, suggesting other myxozoan species may also contribute to PGD pathology. The detection of numerous known and unclassified myxozoan sequences in addition to H. ictaluri provides evidence PGD may involve mixed species infections. Furthermore, the presence of numerous unclassified myxozoan sequences in gill samples from clinical PGD cases indicates the number of described species from U.S. farm-raised catfish vastly underestimates the true myxozoan diversity present within the varied pond microcosms associated with catfish aquaculture.

Comparable outcomes from long and short read random sequencing of total RNA for detection of pathogens in chicken respiratory samples.

Co-infections of avian species with different RNA viruses and pathogenic bacteria are often misdiagnosed or incompletely characterized using targeted diagnostic methods, which could affect the accurate management of clinical disease. A non-targeted sequencing approach with rapid and precise characterization of pathogens should help respiratory disease management by providing a comprehensive view of the causes of disease. Long-read portable sequencers have significant potential advantages over established short-read sequencers due to portability, speed, and lower cost. The applicability of short reads random sequencing for direct detection of pathogens in clinical poultry samples has been previously demonstrated. Here we demonstrate the feasibility of long read random sequencing approaches to identify disease agents in clinical samples. Experimental oropharyngeal swab samples (n = 12) from chickens infected with infectious bronchitis virus (IBV), avian influenza virus (AIV) and Mycoplasma synoviae (MS) and field-collected clinical oropharyngeal swab samples (n = 11) from Kenyan live bird markets previously testing positive for Newcastle disease virus (NDV) were randomly sequenced on the MinION platform and results validated by comparing to real time PCR and short read random sequencing in the Illumina MiSeq platform. In the swabs from experimental infections, each of three agents in every RT-qPCR-positive sample (Ct range 19-34) was detectable within 1 h on the MinION platform, except for AIV one agent in one sample (Ct = 36.21). Nine of 12 IBV-positive samples were assigned genotypes within 1 h, as were five of 11 AIV-positive samples. MinION relative abundances of the test agent (AIV, IBV and MS) were highly correlated with RT-qPCR Ct values (R range-0.82 to-0.98). In field-collected clinical swab samples, NDV (Ct range 12-37) was detected in all eleven samples within 1 h of MinION sequencing, with 10 of 11 samples accurately genotyped within 1 h. All NDV-positive field samples were found to be co-infected with one or more additional respiratory agents. These results demonstrate that MinION sequencing can provide rapid, and sensitive non-targeted detection and genetic characterization of co-existing respiratory pathogens in clinical samples with similar performance to the Illumina MiSeq.

Pathology in Practice.

In collaboration with the American College of Veterinary Pathologists.

Real-time, MinION-based, amplicon sequencing for lineage typing of infectious bronchitis virus from upper respiratory samples.

Infectious bronchitis (IB) causes significant economic losses in the global poultry industry. Control of IB is hindered by the genetic diversity of the causative agent, infectious bronchitis virus (IBV), which has led to the emergence of several serotypes that lack complete serologic cross-protection. Although serotyping requires immunologic characterization, genotyping is an efficient means to identify IBVs detected in samples. Sanger sequencing of the S1 subunit of the spike gene is currently used to genotype IBV; however, the universal S1 PCR was created to work from cultured IBV, and it is inefficient at detecting multiple viruses in a single sample. We describe herein a MinION-based, amplicon-based sequencing (AmpSeq) method that genetically categorized IBV from clinical samples, including samples with multiple IBVs. Total RNA was extracted from 15 tracheal scrapings and choanal cleft swab samples, randomly reverse transcribed, and PCR amplified using modified S1-targeted primers. Amplicons were barcoded to allow for pooling of samples, processed per manufacturer's instructions into a 1D MinION sequencing library, and then sequenced on the MinION. The AmpSeq method detected IBV in 13 of 14 IBV-positive samples. AmpSeq accurately detected and genotyped both IBV lineages in 3 of 5 samples containing 2 IBV lineages. Additionally, 1 sample contained 3 IBV lineages, and AmpSeq accurately detected 2 of the 3 lineages. Strain identification, including detection of different IBVs from the same lineage, was also possible with this AmpSeq method. Our results demonstrate the feasibility of using MinION-based AmpSeq for rapid and accurate identification and lineage typing of IBV from oral swab samples.

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.