Discovery and Genomic Characterization of a Novel Hepadnavirus from Asymptomatic Anadromous Alewife (Alosa pseudoharengus).

The alewife (Alosa pseudoharengus) is an anadromous herring that inhabits waters of northeastern North America. This prey species is a critical forage for piscivorous birds, mammals, and fishes in estuarine and oceanic ecosystems. During a discovery project tailored to identify potentially emerging pathogens of this species, we obtained the full genome of a novel hepadnavirus (ApHBV) from clinically normal alewives collected from the Maurice River, Great Egg Harbor River, and Delaware River in New Jersey, USA during 2015-2018. This previously undescribed hepadnavirus contained a circular DNA genome of 3146 nucleotides. Phylogenetic analysis of the polymerase protein placed this virus in the clade of metahepadnaviruses (family: Hepadnaviridae; genus: Metahepadnavirus). There was no evidence of pathology in the internal organs of infected fish and virions were not observed in liver tissues by electron microscopy. We developed a Taqman-based quantitative (qPCR) assay and screened 182 individuals collected between 2015 and 2018 and detected additional qPCR positives (n = 6). An additional complete genome was obtained in 2018 and it has 99.4% genome nucleotide identity to the first virus. Single-nucleotide polymorphisms were observed between the two genomes, including 7/9 and 12/8 synonymous vs nonsynonymous mutations across the polymerase and surface proteins, respectively. While there was no evidence that this virus was associated with disease in this species, alewives are migratory interjurisdictional fishes of management concern. Identification of microbial agents using de novo sequencing and other advanced technologies is a critical aspect of understanding disease ecology for informed population management.

Analysis of Emerging Variants of Turkey Reovirus using Machine Learning.

Avian reoviruses continue to cause disease in turkeys with varied pathogenicity and tissue tropism. Turkey enteric reovirus has been identified as a causative agent of enteritis or inapparent infections in turkeys. The new emerging variants of turkey reovirus, tentatively named turkey arthritis reovirus (TARV) and turkey hepatitis reovirus (THRV), are linked to tenosynovitis/arthritis and hepatitis, respectively. Turkey arthritis and hepatitis reoviruses are causing significant economic losses to the turkey industry. These infections can lead to poor weight gain, uneven growth, poor feed conversion, increased morbidity and mortality and reduced marketability of commercial turkeys. To combat these issues, detecting and classifying the types of reoviruses in turkey populations is essential. This research aims to employ clustering methods, specifically K-means and Hierarchical clustering, to differentiate three types of turkey reoviruses and identify novel emerging variants. Additionally, it focuses on classifying variants of turkey reoviruses by leveraging various machine learning algorithms such as Support Vector Machines, Naive Bayes, Random Forest, Decision Tree, and deep learning algorithms, including convolutional neural networks (CNNs). The experiments use real turkey reovirus sequence data, allowing for robust analysis and evaluation of the proposed methods. The results indicate that machine learning methods achieve an average accuracy of 92%, F1-Macro of 93% and F1-Weighted of 92% scores in classifying reovirus types. In contrast, the CNN model demonstrates an average accuracy of 85%, F1-Macro of 71% and F1-Weighted of 84% scores in the same classification task. The superior performance of the machine learning classifiers provides valuable insights into reovirus evolution and mutation, aiding in detecting emerging variants of pathogenic TARVs and THRVs.

Geographical Expansion of Avian Metapneumovirus Subtype B: First Detection and Molecular Characterization of Avian Metapneumovirus Subtype B in US Poultry.

Avian metapneumovirus (aMPV), classified within the Pneumoviridae family, wreaks havoc on poultry health. It typically causes upper respiratory tract and reproductive tract infections, mainly in turkeys, chickens, and ducks. Four subtypes of AMPV (A, B, C, D) and two unclassified subtypes have been identified, of which subtypes A and B are widely distributed across the world. In January 2024, an outbreak of severe respiratory disease occurred on turkey and chicken farms across different states in the US. Metagenomics sequencing of selected tissue and swab samples confirmed the presence of aMPV subtype B. Subsequently, all samples were screened using an aMPV subtype A and B multiplex real-time RT-PCR kit. Of the 221 farms, 124 (56%) were found to be positive for aMPV-B. All samples were negative for subtype A. Six whole genomes were assembled, five from turkeys and one from chickens; all six assembled genomes showed 99.29 to 99.98% nucleotide identity, indicating a clonal expansion event for aMPV-B within the country. In addition, all six sequences showed 97.74 to 98.58% nucleotide identity with previously reported subtype B sequences, e.g., VCO3/60616, Hungary/657/4, and BR/1890/E1/19. In comparison to these two reference strains, the study sequences showed unique 49-62 amino acid changes across the genome, with maximum changes in glycoprotein (G). One unique AA change from T (Threonine) to I (Isoleucine) at position 153 in G protein was reported only in the chicken aMPV sequence, which differentiated it from turkey sequences. The twelve unique AA changes along with change in polarity of the G protein may indicate that these unique changes played a role in the adaptation of this virus in the US poultry. This is the first documented report of aMPV subtype B in US poultry, highlighting the need for further investigations into its genotypic characterization, pathogenesis, and evolutionary dynamics.

Embolic necrosuppurative pneumonia in domestic cats induced by a novel Neisseria species.

Three cats, aged 2 to 11 years, presented to the University of Minnesota Veterinary Diagnostic Laboratory over a 3-year period following euthanasia or death due to respiratory distress. Thoracic radiographs revealed nodular, soft tissue opacities throughout the lung fields in all cases. On postmortem examination, approximately 60% to 80% of the lung parenchyma were expanded by multifocal to coalescing, well-demarcated, beige, semi-firm nodules. Histologically, large numbers of neutrophils, fewer macrophages, fibrin, and cellular and karyorrhectic debris effaced the pulmonary parenchyma. The inflammatory foci contained aggregates of gram-negative cocci. 16s rRNA Sanger sequencing and whole-genome sequencing identified the bacteria isolated from the lung of all cats under aerobic conditions as a novel Neisseria spp. Based on whole-genome sequence analysis, all 3 sequences shared 92.71% and 92.67% average nucleotide identity with closely related Neisseria animaloris NZ LR134440T and Neisseria animaloris GCA 002108605T, respectively. The in silico DNA-DNA hybridization identity compared to our isolates was 46.6% and 33.8% with strain DSM Neisseria zoodegmatis 21642 and strain DSM 21643, respectively. All 3 sequences have less than 95% average nucleotide identity and less than 70% DNA-DNA hybridization identity, suggesting that the 3 isolates are a novel species of the genus Neisseria. Infection with Neisseria spp. induces an embolic pneumonia in cats that radiographically and pathologically resembles a metastatic neoplastic process and should be considered among the etiologic differential diagnoses in cases of infectious pulmonary disease with a disseminated, nodular lung pattern.

Identification and characterization of porcine Rotavirus A in Chilean swine population.

Rotavirus A (RVA) is a common cause of diarrhea in newborn pigs, leading to significant economic losses. RVA is considered a major public health concern due to genetic evolution, high prevalence, and pathogenicity in humans and animals. The objective of this study was to identify and characterize RVA in swine farms in Chile. A total of 154 samples (86 oral fluids and 68 fecal samples) were collected, from 22 swine farms. 58 (38%) samples belonging to 14 farms were found positive for RVA by real-time RT-PCR. The samples with low Ct values (21) and the two isolates were selected for whole genome sequencing. Nearly complete genomes were assembled from both isolates and partial genomes were assembled from five clinical samples. BLAST analysis confirmed that these sequences are related to human and swine-origin RVA. The genomic constellation was G5/G3-P[7]-I5-R1-C1-M1-A8-N1-T1-E1-H1. Phylogenetic analysis showed that VP4, VP1, VP2, NSP2, NSP3, NSP4, and NSP5 sequences were grouped in monophyletic clusters, suggesting a single introduction. The phylogenies for VP7, VP6, VP3, and NSP1 indicated two different origins of the Chilean sequences. The phylogenetic trees showed that most of the Chilean RVA sequences are closely related to human and swine-origin RVA detected across the world. The results highlight the potential zoonotic nature of RVA circulating in Chilean swine farms. Therefore, it is important to continue RVA whole genome sequencing globally to fully understand its complex epidemiology and early detection and characterization of zoonotic strains.

Disease ecology and host range of Cyprinid herpesvirus 3 (CyHV-3) in CyHV-3 endemic lakes of North America.

Cyprinid herpesvirus-3 (CyHV-3) is an important pathogen of common carp (Cyprinus carpio, carp) causing significant economic and ecological impacts worldwide. The recent emergence of CyHV-3 in the Upper Midwest region of the United States has raised questions related to the disease ecology and host specificity of CyHV-3 in wild carp populations. To determine the prevalence of CyHV-3 in wild populations of fishes in Minnesota, we surveyed five lakes in 2019 in which the virus was known to have caused mass mortality events in carp from 2017 to 2018. A total of 28 species (n = 756 total fish) of native fishes and 730 carp were screened for the presence of CyHV-3 DNA using specific qPCR. None of the native fish tissues tested positive for CyHV-3 although the prevalence of CyHV-3 in carp was 10%-50% in the five lakes. A single lake (Lake Elysian) with a 50% DNA detection rate and evidence of ongoing transmission and CyHV-3-associated mortality was surveyed again in 2020 from April to September. During this period, none of the tissues from 24 species (n = 607 total fish) tested positive for CyHV-3 though CyHV-3 DNA and mRNA (indicating viral replication) was detected in carp tissues during the sampling period. CyHV-3 DNA was detected most often in brain samples without evidence of replication, potentially indicating that brain tissue is a site for CyHV-3 latency. Paired qPCR and ELISA testing for Lake Elysian in 2019-2020 identified young carp (especially males) to be the primary group impacted by CyHV-3-associated mortality and acute infections, but with no positive detections in juvenile carp. Seroprevalence of carp from Lake Elysian was 57% in 2019, 92% in April of 2020 and 97% in September 2020. These results further corroborate the host specificity of CyHV-3 to carp in mixed wild populations of fishes in Minnesota and provide additional insights into the ecological niche of CyHV-3 in shallow lake populations of carp in North America.

Characterization and Whole Genome Sequencing of Chromobacterium violaceum OUAT_2017: A Zoonotic Pathogen Found Fatal to a Wild Asiatic Elephant.

This study reports a rare fatal case of Chromobacterium violeceum OUAT_2017 strain infection in an Asiatic elephant calf in India. Necropsy revealed pus-filled nodules in liver, spleen, and lungs. Nutrient broth cultures of nodule content showed sediment of violet pigment whereas smooth, non-diffusible, violet-pigmented, homogeneous colonies appeared on nutrient agar. The organism was found to be non-haemolytic and resistant to 8 of the 24 antibiotics tested in vitro. Partial 16S rRNA gene sequence measuring 1410 bp revealed 97% homology with C. violeceum. The bacterial genome composed of 64.87% of G + C content with total size of 4,681,202 bp. The genome annotation has 42 genes responsible for multidrug antibiotic resistance with the presence of Aminoglycoside-modifying enzymes (AAC (6')) that targets streptomycin and spectinomycin. Our findings corroborated the lethal effect of C. violeceum in a new host (elephant) that enriched scientific information on epidemiological picture and whole genome sequencing as well. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-022-01047-4.

Infection and transmission dynamics of Turkey arthritis reovirus in different age Turkeys.

Turkey arthritis reovirus (TARV) has been established as a cause of lameness in meat type turkeys in the past decade. However, no information is available on the age susceptibility of TARV or its transmission dynamics. We conducted this study to determine the age at which turkey poults are susceptible to TARV infection and whether infected birds can horizontally transmit the virus to their non-infected pen mates (sentinels). Five groups of turkeys were orally inoculated with TARV (∼106 TCID50/ml) at 2, 7, 14, 21 and 28 days of age (DOA). Two days after each challenge, four uninfected sentinel turkeys of equal age were added to the virus-inoculated groups. At one- and two-weeks post infection, turkeys from each group, including two sentinels, were euthanized followed by necropsy. Inoculated birds in all age groups had TARV replication in the intestine and gastrocnemius tendon with no statistically significant variation at p < 0.5. Furthermore, the inoculated birds at different age groups showed consistently high gastrocnemius tendon histologic lesion scores while birds in the 28-days-old age group had numerically lower lesion scores at 14 days post inoculation (dpi). The sentinels, in turn, also showed virus replication in their intestines and tendons and histologic lesions in gastrocnemius tendons. The findings indicate that turkeys at the age of 28 days or less are susceptible to infection with TARV following oral challenge. It was also found that TARV-infected birds could transmit the infection to naïve sentinel turkeys of the same age.

Genetic Characterization of a Novel Bovine Rotavirus A G37P[52] Closely Related to Human Strains.

Bovine rotavirus A (boRVA) strains are common causative agents of diarrhea in calves, resulting in economic losses to the beef and dairy industry. Importantly, this virus has a zoonotic relevance due to its ability to reassort with human rotaviruses. In this study, fecal samples were collected from three calves with diarrhea during an outbreak on a dairy farm. The genetic material of boRVA was detected by real-time reverse transcription PCR (rtPCR) in two samples. Then the virus in one of these positive samples was identified as a novel boRVA genotype closely related with human rotavirus strains mainly from the USA based on whole-genome characterization. However, we consider the novel boRVA as the etiological agent of the outbreak due to the lesions associated with a rotavirus infection. Further studies are necessary to clarify the evolutionary advantages that novel rotavirus genotypes may have.

Detection of Multiple Lineages of PRRSV in Breeding and Growing Swine Farms.

The detection and co-circulation of multiple variants of porcine reproductive and respiratory syndrome virus (PRRSV) have been observed and reported in swine. However, the potential long-term impact of multiple prevailing PRRSV variants on pig-performance is not yet fully understood. The primary objective of this study was to describe the genetic variation of PRRSV in processing fluid (PF), oral fluid (OF), and tonsil scraping (TS) specimens from five swine farms with different production types and PRRS status over a period of time (~1 year). Furthermore, the association between PRRSV prevalence and production parameters was investigated. Results showed that PRRSV was detected by RT-qPCR in 21-25% of all types of specimens. In breeding farms, PRRSV detection in PF and/or TS samples was correlated with stillborn and mummified fetuses, and pre-weaning mortality throughout the study period. Although ORF5 sequences were obtained in <16% of all sample types, simultaneous detection of PRRSV variants including field and vaccine strains within a single sampling event was identified in both breeding and growing pig farms. Phylogenetic analyses based on the ORF5 sequence classified the detected field PRRSV into L1A and L1H, two sub-lineages of lineage 1 (L1). Our study demonstrated the presence of multiple PRRSV lineages, sub-lineages, and variants in swine herds and its potential association with swine reproductive performance under field conditions.

Adenoviral infection in 5 red-tailed hawks and a broad-winged hawk.

Adenoviral infections among raptors are best described in falcons and are characterized most commonly by necrotizing hepatitis and splenitis; only one case has been reported in a hawk. Five red-tailed hawks (Buteo jamaicensis) and a broad-winged hawk (Buteo platypterus) had an adenoviral infection based on history, histopathology, negative-stain electron microscopy, and PCR. All birds had acute onset of illness resulting in death; 3 had evidence of a concurrent bacterial infection. Microscopically, all 6 birds had solitary, pale eosinophilic-to-amphophilic, intranuclear inclusion bodies within presumed hematopoietic cells in bone marrow and macrophages in spleen. Five of the 6 birds had similar inclusions within hepatocytes and Kupffer cells. All but one bird had severe bone marrow necrosis. There was moderate splenic necrosis (3 of 6) and mild-to-marked hepatic necrosis (4 of 6). Negative-stain electron microscopy demonstrated adenoviral particles in bone marrow (5 of 6), liver (1 of 5), and/or spleen (1 of 5). PCR was positive for adenovirus in bone marrow (3 of 5), liver (1 of 3), spleen (4 of 6), and/or intestinal contents (2 of 3). Viral DNA polymerase gene sequences clustered within the Siadenovirus genus. There was 99% nucleotide identity to one another and 90% nucleotide identity with the closest related adenovirus (Harris hawk, EU715130). Our case series expands on the limited knowledge of adenoviral infections in hawks. The splenic and hepatic necrosis, and particularly the hitherto unreported bone marrow necrosis, suggest that adenoviral infection is clinically relevant and potentially fatal in hawks.

Comparative pathogenesis of turkey reoviruses.

Turkey reoviruses have been implicated in multiple disease syndromes resulting in significant economic losses to the turkey industry. It has been known for decades that turkey enteric reovirus (TERV) is involved in poult enteritis complex, but turkey arthritis reovirus (TARV), the causative agent of tenosynovitis in turkeys, emerged in 2011. In 2019, we isolated reovirus from several cases of hepatitis in turkeys and tentatively named it turkey hepatitis reovirus (THRV). The comparative pathogenesis of these viruses, and correlation with their genetic make-up (if any), is not known. In this study, we inoculated nine groups of 1-week-old turkey poults with two THRV, five TARV and two TERV via oral route. A tenth group served as a negative control. A subset of birds from each group was euthanised at 3, 5, 7, 14, 21, and 28 days post-inoculation (dpi). Tissues were collected for histology and real-time RT-PCR. All nine viruses were found to be enterotropic; the virus gene copy number in the intestine reached a peak at 5 dpi followed by a sharp decline at 7 dpi. All viruses caused a significant decline in body weight gain of birds as compared to the negative control group. Both TARV and THRV strains replicated in tendons and produced histologic lesions consistent with tenosynovitis. Hepatic lesions were produced by THRV only and the virus was re-isolated from liver and spleen of inoculated birds fulfilling Koch's postulates. The results of this study should be helpful in facilitating diagnosis and designing future mitigation plans.

Prevalence of Newcastle Disease Virus in Wild and Migratory Birds in Haryana, India.

Newcastle disease virus (NDV) can infect approximately 250 avian species and causes highly contagious Newcastle disease (ND) in domestic poultry, leading to huge economic losses. There are three different pathotypes of NDV, i.e., lentogenic, mesogenic, and velogenic. Wild resident (wild) and migratory birds are natural reservoirs of NDV and are believed to play a key role in transmitting the virus to domestic poultry. The present study was conducted to determine the prevalence of NDV in wild and migratory birds in the state of Haryana, India, during two migratory seasons (2018-19 and 2019-20). In total 1379 samples (1368 choanal swabs and 11 tissue samples) were collected from live (n = 1368) or dead birds (n = 4) belonging to 53 different avian species. These samples belonged to apparently healthy (n = 1338), sick (n = 30), and dead (n = 4) birds. All samples were tested for NDV by real-time reverse transcription-PCR using M gene specific primers and probe. Of the 1379 samples, 23 samples from wild birds [Columba livia domestica (n = 12, 52.17%), Pavo cristatus (n = 9, 39.13%), and Psittaciformes (n = 2, 8.69%)] were found positive for NDV. Only one of the 23 samples (from P. cristatus) was positive for F gene, indicating it to be a mesogenic/velogenic strain. These results indicate that both lentogenic and velogenic strains of NDV are circulating in wild birds in Haryana and that further studies are needed to characterize NDV strains from wild/migratory birds and domestic poultry to determine the extent of virus transmission among these populations. This study considers the disease transmission risk from domestic pigeons and parrots to commercial poultry and vice versa, and the results emphasize the need for strict biosecurity strategies to protect commercial poultry in the region.

Prevalencia del virus de la enfermedad de Newcastle en aves silvestres y migratorias en Haryana, India. El virus de la enfermedad de Newcastle (NDV) puede infectar aproximadamente a 250 especies de aves y causa la enfermedad de Newcastle (ND) altamente contagiosa en la avicultura comercial, lo que genera enormes pérdidas económicas. Hay tres patotipos diferentes del virus de Newcastle, que incluyen, lentogénico, mesogénico y velogénico. Las aves silvestres residentes (silvestres) y migratorias son reservorios naturales del virus de Newcastle y se cree que desempeñan un papel clave en la transmisión del virus a las aves domésticas comerciales. El presente estudio se realizó para determinar la prevalencia del virus de Newcastle en aves silvestres y migratorias en el estado de Haryana, India, durante dos temporadas migratorias (2018-19 y 2019-20). En total, se recolectaron 1379 muestras (1368 hisopos coanales y 11 muestras de tejido) de aves vivas (n = 1368) o muertas (n = 4) pertenecientes a 53 especies de aves diferentes. Estas muestras pertenecían a aves aparentemente sanas (n = 1338), enfermas (n = 30) y muertas (n = 4). Todas las muestras se analizaron para detectar al virus de Newcastle mediante transcripción reversa y PCR en tiempo real utilizando iniciadores y una sonda específicos del gene M. De las 1379 muestras, 23 muestras de aves silvestres [Columba livia domestica (n = 12, 52.17 %), Pavo cristatus (n = 9, 39.13 %) y Psittaciformes (n = 2, 8.69 %)] resultaron positivas para el virus de Newcastle. Solo una de las 23 muestras (de P. cristatus) fue positiva para el gene F, lo que indica que se trata de una cepa mesogénica/velogénica. Estos resultados indican que tanto las cepas lentogénicas como las velogénicas del virus de Newcastle están circulando en las aves silvestres de Haryana y que se necesitan más estudios para caracterizar las cepas del virus de Newcastle de las aves silvestres/migratorias y de las aves domésticas para determinar el alcance de la transmisión del virus entre estas poblaciones. Este estudio considera el riesgo de transmisión de la enfermedad de las palomasdomésticas y loros a las aves comerciales y viceversa, y los resultados enfatizan la necesidad de estrategias estrictas de bioseguridad para proteger las aves comerciales en la región.

Efficacy and Immunogenicity of Recombinant Pichinde Virus-Vectored Turkey Arthritis Reovirus Subunit Vaccine.

We created a recombinant live pichinde virus-vectored bivalent codon optimized subunit vaccine that expresses immunogenic Sigma C and Sigma B proteins of turkey arthritis reovirus. The vaccine virus could be transmitted horizontally immunizing the non-vaccinated pen mates. The vaccine was tested for efficacy against homologous (TARV SKM121) and heterologous (TARV O'Neil) virus challenge. Immunized poults produced serum neutralizing antibodies capable of neutralizing both viruses. The vaccinated and control birds showed similar body weights indicating no adverse effect on feed efficiency. Comparison of virus gene copy numbers in intestine and histologic lesion scores in tendons of vaccinated and non-vaccinated birds showed a decrease in the replication of challenge viruses in the intestine and tendons of vaccinated birds. These results indicate the potential usefulness of this vaccine.

Detection of Newcastle disease virus and assessment of associated relative risk in backyard and commercial poultry in Kerala, India.

BACKGROUND: Newcastle disease (ND) is an economically important viral disease affecting the poultry industry. In Kerala, a state in South India, incidences of ND in commercial and backyard poultry have been reported. But a systematic statewide study on the prevalence of the disease has not been carried out. OBJECTIVES: A cross-sectional survey was performed to detect the presence of Newcastle disease virus (NDV) in suspect cases and among apparently healthy commercial flocks and backyard poultry, in the state and to identify risk factors for NDV infection. METHODS: Real-time reverse transcription-PCR (RT-PCR) was used to detect the M gene of NDV in choanal swabs and tissue samples collected from live and dead birds, respectively and the results were statistically analysed. RESULTS: The predominant clinical signs of the examined birds included mild respiratory signs, huddling together and greenish diarrhoea. Nervous signs in the form of torticollis were noticed in birds in some of the affected flocks. On necropsy, many birds had haemorrhages in the proventriculus and caecal tonsils which were suggestive of ND. Of the 2079 samples tested, 167 (8.0%) were positive for the NDV M-gene by RT-PCR. Among 893 samples collected from diseased flocks, 129 (14.5%), were positive for M gene with pairwise relative risk (RR) of 15.6 as compared to apparently healthy flocks where 6 out of 650 (0.9%) samples were positive. All positive samples were from poultry; none of the ducks, pigeons, turkey and wild birds were positive. Commercial broilers were at higher risk of infection than commercial layers (RR: 4.5) and backyard poultry (RR: 4.9). Similarly, birds reared under intensive housing conditions were at a higher risk of being infected as compared to those reared under semi-intensive (RR: 6.7) or backyard housing (RR: 2.1). Multivariable analysis indicated that significantly higher risk of infection exists during migratory season and during ND outbreaks occurring nearby. Further, lower risk was observed with flock vaccination and backyard or semi-intensive housing when compared to intensive housing. When the M gene positive samples were tested by RT-PCR to determine whether the detected NDV were mesogenic/velogenic, 7 (4.2%) were positive. CONCLUSIONS: In Kerala, NDV is endemic in poultry with birds reared commercially under intensive rearing systems being affected the most. The outcome of this study also provides a link between epidemiologic knowledge and the development of successful disease control measures. Statistical analysis suggests that wild bird migration season and presence of migratory birds influences the prevalence of the virus in the State. Further studies are needed to genotype and sub-genotype the detected viruses and to generate baseline data on the prevalence of NDV strains, design better detection strategies, and determine patterns of NDV transmission across domestic poultry and wild bird populations in Kerala.

Multi-laboratory evaluation of the Illumina iSeq platform for whole genome sequencing of Salmonella, Escherichia coli and Listeria.

There is a growing need for public health and veterinary laboratories to perform whole genome sequencing (WGS) for monitoring antimicrobial resistance (AMR) and protecting the safety of people and animals. With the availability of smaller and more affordable sequencing platforms coupled with well-defined bioinformatic protocols, the technological capability to incorporate this technique for real-time surveillance and genomic epidemiology has greatly expanded. There is a need, however, to ensure that data are of high quality. The goal of this study was to assess the utility of a small benchtop sequencing platform using a multi-laboratory verification approach. Thirteen laboratories were provided the same equipment, reagents, protocols and bacterial reference strains. The Illumina DNA Prep and Nextera XT library preparation kits were compared, and 2×150 bp iSeq i100 chemistry was used for sequencing. Analyses comparing the sequences produced from this study with closed genomes from the provided strains were performed using open-source programs. A detailed, step-by-step protocol is publicly available via protocols.io (https://www.protocols.io/view/iseq-bacterial-wgs-protocol-bij8kcrw). The throughput for this method is approximately 4-6 bacterial isolates per sequencing run (20-26 Mb total load). The Illumina DNA Prep library preparation kit produced high-quality assemblies and nearly complete AMR gene annotations. The Prep method produced more consistent coverage compared to XT, and when coverage benchmarks were met, nearly all AMR, virulence and subtyping gene targets were correctly identified. Because it reduces the technical and financial barriers to generating WGS data, the iSeq platform is a viable option for small laboratories interested in genomic surveillance of microbial pathogens.

Prevalence of Newcastle disease and associated risk factors in domestic chickens in the Indian state of Odisha.

Newcastle disease (ND), caused by Newcastle disease virus (NDV), is a contagious disease that affects a variety of domestic and wild avian species. Though ND is vaccine-preventable, it is a persistent threat to poultry industry across the globe. The disease represents a leading cause of morbidity and mortality in chickens. To better understand the epidemiology of NDV among commercial and backyard chickens of Odisha, where chicken farming is being prioritized to assist with poverty alleviation, a cross-sectional study was conducted in two distinct seasons during 2018. Choanal swabs (n = 1361) from live birds (commercial layers, broilers, and backyard chicken) and tracheal tissues from dead birds (n = 10) were collected and tested by real-time reverse transcription polymerase chain reaction (RT-PCR) for the presence of matrix (M) and fusion (F) genes of NDV. Risk factors at the flock and individual bird levels (health status, ND vaccination status, geographical zone, management system, and housing) were assessed using multivariable logistic regression analyses. Of the 1371 samples tested, 160 were positive for M gene amplification indicating an overall apparent prevalence of 11.7% (95% CI 10.1-13.5%). Circulation of virulent NDV strains was also evident with apparent prevalence of 8.1% (13/160; 95% CI: 4.8-13.4%). In addition, commercial birds had significantly higher odds (75%) of being infected with NDV as compared to backyard poultry (p = 0.01). This study helps fill a knowledge gap in the prevalence and distribution of NDV in apparently healthy birds in eastern India, and provides a framework for future longitudinal research of NDV risk and mitigation in targeted geographies-a step forward for effective control of ND in Odisha.

Novel papillomavirus in a mallard duck with mesenchymal chondroid dermal tumors.

Papillomaviruses, which are epitheliotropic and may induce epithelial tumors, have been identified in several avian species, including ducks. An adult female mallard duck (Anas platyrhynchos) was admitted to a wildlife rehabilitation center with 2 beige, well-demarcated, firm masses: one in the subcutis under a wing, and the other on a digit of the right foot. After euthanasia, the masses were fixed in formalin for histologic examination. Both tumors had a lobular organization with cartilage cores surrounded by densely cellular interlacing bundles of spindle cells. Neoplastic chondroblasts in both masses, particularly the digital mass, contained basophilic intranuclear inclusion bodies, which consisted of assembly complexes of icosahedral virions of 44-nm diameter. Next-generation sequencing allowed whole genome assembly of a novel papillomavirus (Anas platyrhynchos papillomavirus 2) related most closely to Fulmarus glacialis papillomavirus 1 (59.49% nucleotide identity). Our case supports the observation that certain papillomaviruses can productively infect mesenchymal cells and induce neoplasia.

A longitudinal study on PRRSV detection in swine herds with different demographics and PRRSV management strategies.

Porcine reproductive and respiratory syndrome virus (PRRSV) has been one of the major health-related concerns in the swine production industry. Through its rapid transmission and mutation, the simultaneous circulation of multiple PRRSV strains can be a challenge in PRRSV diagnostic, control and surveillance. The objective of this longitudinal study was to describe the temporal detection of PRRSV in swine farms with different production types and PRRS management strategies. Tonsil scraping (n = 344) samples were collected from three breeding and two growing herds for approximately one year. In addition, processing fluids (n = 216) were obtained from piglet processing batches within the three breeding farms while pen-based oral fluids (n = 125) were collected in the two growing pig farms. Viral RNA extraction and reverse-transcription quantitative PCR (RT-qPCR) were conducted for all samples. The sample positivity threshold was set at quantification cycle (Cq) of ≤ 37. Statistical analyses were performed using generalized linear modelling and post hoc pairwise comparisons with Bonferroni adjustments using R statistical software. The results suggested a higher probability of detection in processing fluids compared to tonsil scraping specimens [odds ratio (OR) = 3.86; p = .096] in breeding farms whereas oral fluids were outperformed by tonsil scrapings (OR = 0.26; p < .01) in growing pig farms. The results described herein may lead to an improvement in PRRSV diagnostic and surveillance by selecting proper specimens.

Case Report: First Report and Phylogenetic Analysis of Porcine Astroviruses in Chile.

Porcine Astrovirus (PoAstV) causes mild diarrhea in young pigs and is considered an emerging virus in the swine industry worldwide. PoAstV has high genetic diversity and has been classified into five genetic lineages, PoAstV1-5. In Chile, only human astroviruses have been reported. This study aimed to determine the presence and genetic diversity of PoAstV circulating in intensive pig farms in Chile. Seventeen Chilean intensive swine farms from Valparaíso, Metropolitana, O'Higgins, Ñuble and Araucanía regions were sampled. A selection of oral fluid and fecal material samples from 1-80 days-old pigs were collected and analyzed using next-generation sequencing. The circulation of PoAstV was confirmed in all studied farms. We obtained complete or partial sequences of PoAstV-2 (n = 3), PoAstV-4 (n = 2), and PoAstV-5 (n = 7). In 15 out of 17 farms, we detected more than one lineage co-circulating. Phylogenetic analyses grouped the seven PoAstV-5 strains in a monophyletic cluster, closely related to the United States PoAstV-5 strains. The three PoAstV-2 were located into two separate sub-clusters. PoAstV-4 sequences are also grouped in two different clusters, all related to Japanese strains. Thus, our results indicate that PoAstV circulates in Chile with high frequency and diversity. However, the lack of reference sequences impairs local evolution patterns establishment and regional comparisons. This is the first contribution of PoAstV genomes in Latin America; more studies are needed to understand the diversity and impact of PoAstV on swine health.