Avibacterium paragallinarum is the causative agent of infectious coryza, a highly contagious respiratory disease in chickens. Given its fastidious nature, this bacterium is difficult to recover and identify, particularly from locations colonized by normal bacterial flora. Standard PCR methods have been utilized for detection but are labor-intensive and not feasible for high-throughput testing. We evaluated a real-time PCR (rtPCR) method targeting the HPG-2 region of A. paragallinarum, and validated a high-throughput extraction for this assay. Using single-tube extraction, the rtPCR detected 4 A. paragallinarum (ATCC 29545^T and 3 clinical) isolates with a limit of detection (LOD) of 10 cfu/mL and a PCR efficiency of 89-111%. Cross-reaction was not detected with 33 non-A. paragallinarum, all close relatives from the family Pasteurellaceae. Real-time PCR testing on extracts of 66 clinical samples (choana, sinus, or trachea) yielded 98.2% (35 of 36 on positives, 30 of 30 on negatives) agreement with conventional PCR. Duplicate samples tested in a 96-well format extraction in parallel with the single-tube method produced equivalent LOD on all A. paragallinarum isolates, and 96.8% agreement on 93 additional clinical samples extracted with both procedures. This A. paragallinarum rtPCR can be utilized for outbreak investigations and routine monitoring of susceptible flocks.

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

With the cost of next-generation sequencing (NGS) decreasing, this technology is rapidly being integrated into the workflows of veterinary clinical and diagnostic laboratories nationwide. The mission of the U.S. Department of Agriculture-National Animal Health Laboratory Network (NAHLN) is in part to evaluate new technologies and develop standardized processes for deploying these technologies to network laboratories for improving detection and response to emerging and foreign animal diseases. Thus, in 2018, the NAHLN identified the integration of NGS into the network as a top priority. In order to assess the current state of preparedness across NAHLN laboratories and to identify which have the capability for performing NGS, a questionnaire was developed by the NAHLN Methods Technical Working Group and submitted to all NAHLN laboratories in December 2018. Thirty of 59 laboratories completed the questionnaire, of which 18 (60%) reported having some sequencing capability. Multiple sequencing platforms and reagents were identified, and limited standardized quality control parameters were reported. Our results confirm that NGS capacity is available within the NAHLN, but several gaps remain. Gaps include not having sufficient personnel trained in bioinformatics and data interpretation, lack of standardized methods and equipment, and maintenance of sufficient computing capacity to meet the growing demand for this technology.

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.

In order to improve the analytic quality of respiratory specimens collected from cattle for nucleic acid-based diagnosis, a study was undertaken to verify realtime PCR efficiency of specimens collected and stabilized on FTA Cards™, filter paper which is treated chemically. Nucleic acids collected using FTA Cards without the need for a cold-chain or special liquid media handling provided realtime PCR results consistent (96.8% agreement, kappa 0.923 [95% CI=0.89-0.96]) with the same specimens collected using traditional viral transport media and shipped on ice using the U.S. Department of Transportation mandated liquid handling requirements. Nucleic acid stabilization on FTA Cards was evaluated over a temperature range (-27 °C to +46 °C) for up to 14 days to mimic environmental conditions for diagnostic sample handling between collection and processing in a routine veterinary laboratory. No significant difference (P≥0.05) was observed in realtime PCR cycle threshold values over the temperature range and time storage conditions for Bovine Viral Diarrhea virus, Bovine Respiratory Syncytial virus, Bovine Coronavirus, and Bovine Herpesvirus I. The four viruses evaluated in the study are associated with Bovine Respiratory Disease Complex where improvements in ease and reliability of specimen collection and shipping would enhance the diagnostic quality of specimens collected in the field, and ultimately improve diagnostic efficiency.

A group of Holstein first-calved heifers developed small pustules and ulcers on the vulva and in the vagina during the first 1-4 wk postpartum. The lesions varied from small red pinpoint foci to pustules and ulcers, 3-5 mm diameter. Some ulcers coalesced to form large ulcerated areas up to 15 mm diameter. In some animals, these ulcers progressed to become deep ulceration of the vaginal and vulvar mucosa with >50% of the mucosa involved. Vaginal biopsies from 4 heifers and vaginal individual swabs from 8 heifers for a combined sampling of 9 heifers were taken for clinical assessment. Six of the 9 heifers had parapoxvirus based on histopathology and/or PCR. Histologic examination of the biopsies of the pustules identified ballooning degeneration of the epithelium with degenerate epithelium containing eosinophilic intracytoplasmic inclusions consistent with a parapoxvirus in 3 of 4 biopsies. Testing for bovine herpesvirus 1, 2, and 4, bovine viral diarrhea virus, bovine papular stomatitis virus, and orf virus remained negative.

A 2-y-old female Grant's zebra ( Equus quagga [ burchellii] boehmi) was presented with a clinical history of depression, anorexia, and weakness of 1-wk duration. Postmortem examination identified ulcers on the tongue and palate; a large abscess adjacent to the larynx; left lung consolidation; mild swelling, darkening, and congestion of the liver with accentuation of the lobular pattern; and edema and congestion of the distal small and large intestines. Histologic examination identified necrotizing bronchopneumonia, necrotizing hepatitis, nephritis, and enterocolitis. Eosinophilic intranuclear inclusions were detected in syncytial cells and degenerate bronchial epithelium in the lungs and in some hepatocytes associated with necrotic foci. Bacterial cultures of the lung, liver, and laryngeal abscess failed to detect any significant pathogen. Lung and liver tested positive for equine herpesvirus with neuropathogenic marker by real-time PCR. Subsequently, equine herpesvirus was isolated in tissue culture, and the entire viral DNA polymerase gene (ORF30) was sequenced. The zebra lung isolate had a very close nucleotide and amino acid sequence identity to equid alphaherpesvirus 9 (EHV-9; 99.6% and 99.8%, respectively) in contrast to the neuropathogenic T953 strain of EHV-1 (94.7% and 96.6%, respectively). Although zebras are considered the natural host for EHV-9, we document an unusual acute systemic, fatal EHV-9 infection in a 2-y-old Grant's zebra.

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.

Ulcers of the oral cavity, esophagus, and gastric compartments of South American camelids are uncommon. Multifocal-to-coalescing ulcers were identified in the oral cavity, esophagus, and/or gastric compartments of 5 alpacas submitted for postmortem examination. Fusobacterium necrophorum was isolated from the lesions in all alpacas, in combination with other aerobic and anaerobic bacteria. In 4 of these cases, F. necrophorum-associated lesions were considered secondary to neoplasia or other chronic debilitating conditions; in 1 case, the alimentary ulcers were considered the most significant autopsy finding. It is not known if this agent acted as a primary or opportunistic agent in mucosal membranes previously damaged by a traumatic event, chemical insult, immunodeficiency, or any other debilitating condition of the host.

We reviewed case records from the California Animal Health and Food Safety (CAHFS) laboratory and the California Department of Fish and Wildlife (CDFW) spanning 25 years (1990-2014) for all deer accessions submitted to CAHFS for pathology and/or histopathology, with and without a diagnosis of adenoviral hemorrhagic disease (AHD), in order to determine the prevalence of AHD in California. We also examined spatial and temporal distribution, age, and mule deer subspecies in deer that died from AHD. Of 483 deer submitted to CAHFS for diagnostic testing in 1990-2014, 17.2% were diagnosed with confirmed AHD, and 26.5% were confirmed plus suspected cases of AHD. Columbian black-tailed deer ( Odocoileus hemionus columbianus), particularly fawns and juveniles, were most frequently affected. Deer adenovirus ( Odocoileus adenovirus 1; OdAdV-1) was detected by immunohistochemistry in archived CDFW formalin-fixed, paraffin-embedded tissues from deer that died in mortality events in 1981, 1983, and 1986-1987. OdAdV-1 is a common cause of hemorrhagic disease mortality events in California deer, and mortality as a result of AHD is documented as early as 1981.