National Veterinary Services Laboratories Research Articles

Surveillance for highly pathogenic avian influenza A (H5N1) in a raptor rehabilitation center-2022.

An ongoing, severe outbreak of highly pathogenic avian influenza virus (HPAI) A H5N1 clade 2.3.4.4b has been circulating in wild and domestic bird populations throughout the world, reaching North America in 2021. This HPAI outbreak has exhibited unique characteristics when compared to previous outbreaks. The global distribution of disease, prolonged duration, extensive number of species and individual wild birds affected, and the large impact on the global poultry industry have all exceeded historical impacts of previous outbreaks in North America. In this study, we describe the results of HPAI surveillance conducted at The Raptor Center, a wildlife rehabilitation hospital at University of Minnesota (Saint Paul, MN, U.S.A.), from March 28th-December 31, 2022. All wild raptors admitted to the facility were tested for avian influenza viruses using polymerase chain reaction (PCR) testing. All non-negative samples were submitted to the United States Department of Agriculture (USDA) Animal and Plant Health Inspection Service (APHIS) National Veterinary Services Laboratories for confirmatory HPAI testing and genetic sequencing. During the study period, 996 individual birds representing 20 different species were tested for avian influenza, and 213 birds were confirmed HPAI positive. Highly pathogenic avian influenza surveillance conducted at The Raptor Center contributed 75% of the HPAI positive raptor detections within the state of Minnesota, located within the Mississippi flyway, significantly augmenting state wildlife surveillance efforts. The viral genotypes observed in birds sampled at The Raptor Center were representative of what was seen in wild bird surveillance within the Mississippi flyway during the same time frame. Wildlife rehabilitation centers provide an opportune situation to augment disease surveillance at the human, wildlife and domestic animal interface during ongoing infectious disease outbreaks.

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.

Outbreak of Mycobacterium orygis in a Shipment of Cynomolgus Macaques Imported from Southeast Asia - United States, February-May 2023.

Nonhuman primates (NHP) can become infected with the same species of Mycobacteria that cause human tuberculosis. All NHP imported into the United States are quarantined and screened for tuberculosis; no confirmed cases of tuberculosis were diagnosed among NHP during CDC-mandated quarantine during 2013-2020. In February 2023, an outbreak of tuberculosis caused by Mycobacterium orygis was detected in a group of 540 cynomolgus macaques (Macaca fascicularis) imported to the United States from Southeast Asia for research purposes. Although the initial exposure to M. orygis is believed to have occurred before the macaques arrived in the United States, infected macaques were first detected during CDC-mandated quarantine. CDC collaborated with the importer and U.S. Department of Agriculture's National Veterinary Services Laboratories in the investigation and public health response. A total of 26 macaques received positive test results for M. orygis by culture, but rigorous occupational safety protocols implemented during transport and at the quarantine facility prevented cases among caretakers in the United States. Although the zoonotic disease risk to the general population remains low, this outbreak underscores the importance of CDC's regulatory oversight of NHP importation and adherence to established biosafety protocols to protect the health of the United States research animal population and the persons who interact with them.

Molecular characterization of the Newcastle disease virus that caused an outbreak in backyard birds in Costa Rica in 2015

Costa Rica gained its Newcastle Disease Virus NDV-free status with vaccination according to OIE proceedings in 1996, and its declaration as a country free of the velogenic, viscerotropic form of this disease (G/SPS/GEN/119) presented to the World Trade Organization (WTO) in 1999. On April 24th, 2015, SENASA (National Animal Health Service) attended a velogenic Newcastle disease outbreak that affected backyard chickens in a small town (Bellavista, Guanacaste) in the northern part of the country, near the Nicaraguan border. Sixty-five backyard birds died from a total of 84 exposed animals. Blood samples, cloacal swabs, tracheal swabs, cecal tonsils, lung and trachea tissues were collected for diagnosis at the National Veterinary Services Laboratory (LANASEVE). These samples were screened for Avian Influenza (AIV) and NDV. All samples were negative for Avian Influenza in ELISA test and RT-PCR. Serum samples were positive for NDV antibody by hemagglutination inhibition test, and tissue and swab samples were positive for NDV by conventional RT-qPCR targeting a 310 bp fragment of the virus fusion protein gene. The amino acid sequence of the protease cleavage site within the amplicon matched the sequence of a virulent strain (112RRQKRF117). The nucleotide sequence had a 98.7% identity and an e value of 4e-153 with a genotype V velogenic sequence from Belize (KF767467) and Honduras (JN872194) collected in 2008 and 2007, respectively, according to BLASTN. A total of 3604 backyard birds were euthanized in town and its surroundings (1 km), including 3495 chickens, 66 turkeys, 6 geese, and 37 ducks. The case was considered resolved, and OIE was notified in November 2015 following OIE guidelines. In April 2017, Costa Rica recovered its disease-free status through executive decree No. 40301-MAG.

189. Potential Tiger-to-Human Transmission of SARS-CoV-2 at a Tennessee Zoo: A One Health Approach to Outbreak Investigation

BackgroundHuman-to-feline and airborne transmission among cats of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has been described, though documented feline-to-human transmission has not been reported. In October 2020, all 3 Malayan tigers at a Tennessee AZA accredited zoo were diagnosed with symptomatic SARS-CoV-2 infection. We investigated to determine source and prevent further transmission.MethodsTiger nasal swab specimens were tested at the National Veterinary Services Laboratories (NVSL). An environmental assessment at the zoo was completed. We interviewed 18 staff who interacted with the tigers during the 2 weeks before animal symptom onset. Confirmed human cases were defined as persons testing positive for SARS-CoV-2 by RT-PCR during September 28–October 29, with tiger interaction during their 14-day incubation period. Interviewed staff had repeat SARS-CoV-2 RT-PCR and serum IgG testing on October 29. Tigers and staff testing positive had specimens sent to CDC for genomic sequencing. Tiger sequences were compared phylogenetically with 30 geographically associated human cases collected within 2 weeks of the outbreak and > 200 background sequences from TN.ResultsNVSL confirmed SARS-CoV-2 infection in all 3 tigers. Environmental assessment identified fencing between humans and animals allowing airflow and an open outdoor exhibit observation point above the habitat. Confirmed cases were identified in a tiger keeper and veterinary assistant; both developed symptoms after exposure to symptomatic tigers and one sample was genotyped. Staff did not report known contact with ill visitors. All staff were negative for SARS-CoV-2 IgG. The tigers and most temporally and geographically associated cases had genetic sequences in clade 20G and B.1.2. Tiger sequences were 3-6 single nucleotide polymorphisms different from the positive tiger keeper (Figure).Figure. Whole-genome phylogenetic analysis. Whole-genome phylogenetic analysis from a portion of clade 20G showing divergence estimates from SARS-CoV-2 Wuhan-Hu-1 reference genome with sequences from humans living in Tennessee and Malayan tigers sampled during the outbreak investigation in October 2020. Sequence analysis showed 3-6 single nucleotide polymorphisms (SNPs) differences between one human tiger keeper and all three tiger sequences. Differences are indicated by one-step edges (lines) between colored dots (individual SARS-CoV-2 sequenced infections). Numbers indicate unique sequences. Note not all analyzed sequences are shown in this figure.ConclusionUsing a One Health approach, we concluded the index tiger was likely infected via transmission from an ill visitor at an exhibit observation point or unidentified asymptomatic staff. Infection spread to the other 2 tigers and tiger-to-human transmission to 2 staff is possible thereafter. The zoo was advised on infection control practices for humans and animals, and no additional cases were identified.Disclosures William Schaffner, MD, VBI Vaccines (Consultant)

PREVALENCE AND ANTIMICROBIAL RESISTANCE PATTERNS OF SALMONELLA SPP. IN TWO FREE-RANGING POPULATIONS OF EASTERN BOX TURTLES (TERRAPENE CAROLINA CAROLINA).

Salmonellosis is an important zoonotic infection, and exposure to pet reptiles has been implicated in several human outbreaks. Although several studies report a low prevalence of salmonellae in free-ranging chelonians, they may serve as a reservoir. In spring and summer of 2013 and 2019, free-ranging eastern box turtles (Terrapene carolina carolina) from populations in Illinois (rural) and Tennessee (urban) were collected through canine and visual search. Cloacal swab samples were collected from each turtle, selectively enriched with tetrathionate broth, then plated on selective and differential media to isolate Salmonella spp. Genus was confirmed via MALDI-TOF MS and antibiotic sensitivities were performed. Isolates were serotyped by the National Veterinary Services Laboratory. Of the 341 turtles sampled, Salmonella spp. were detected in nine individuals (2.64%; 95% CI: 1.2-5.0%). The isolates included five different serovars: Anatum (n = 2), Newport (n = 2), Thompson (n = 1), Bareilly (n = 2), and Hartford (n = 2). Salmonella spp. were detected from six animals in 2013 (3.19%, 95% CI: 1.2-6.8%) and three in 2019 (1.96%, 95% CI: 0.4-5.6%). There was no significant difference in prevalence between state, (P = 0.115), Illinois locations (P = 0.224), season (P = 0.525), year (P = 0.297), sex (P = 0.435), or age class (P = 0.549). The health of Salmonella-positive and -negative turtles was not significantly different, as assessed through hematology and plasma biochemistry (P > 0.05), indicating asymptomatic carrier status. The low prevalence detected in this study likely concludes that free-ranging eastern box turtles play a minimal role in the spread of salmonellae. However, the identified serotypes are potentially human- and animal-pathogenic. Documenting the prevalence of Salmonella serotypes in animal indicators furthers our understanding of their spread between humans, animal agriculture, and the environment.

Hemagglutination Assay for Influenza Virus.

The hemagglutination assay (HA) is a tool used to screen cell culture supernatant or amniotic-allantoic fluid harvested from embryonated chicken eggs for hemagglutinating agents, such as type A influenza. The HA is not an identification assay, as other agents also have hemagglutinating properties. Live and inactivated viruses are detected by the HA test. Amplification by virus isolation in embryonated chicken eggs or cell culture is typically required before HA activity can be detected from a clinical sample. The test is, to some extent, quantitative as one hemagglutinating unit (HAU) is equal to approximately 5-6 log10 of virus. It is inexpensive and relatively simple to conduct. Several factors (e.g., the quality of chicken erythrocytes, laboratory temperature, laboratory equipment, technical expertise of the user) may contribute to slight differences in the interpretation of the test each time it is run. This chapter will describe the methods validated and used by the US National Veterinary Services Laboratories for screening and identification of hemagglutinating viruses.

Use of a multiagency approach to eradicate New World screwworm flies from Big Pine Key, Florida, following an outbreak of screwworm infestation (September 2016-March 2017).

On September 30, 2016, the US National Veterinary Services Laboratory confirmed an autochthonous case of New World screwworm infestation in a Key deer (Odocoileus virginianus clavium) from Big Pine Key, Fla. This case marked the first identification of a sustained and reproducing population of New World screwworm flies in the United States since 1966. Multiple federal, state, and local government agencies collaborated to initiate a response to the outbreak. Efforts were successful in eradicating the flies from Florida.

An investigation of Salmonella Fluntern illnesses linked to leopard geckos-United States, 2018.

Reptile contact can result in zoonotic non-typhoidal salmonellosis. In April 2018, Oregon Public Health Division contacted CDC about a cluster of four Salmonella serovar Fluntern (SF) illnesses in four states (OR, CA, IA, NY); patients reported contact with geckos, a popular reptile pet. PulseNet, the national molecular subtyping network of food-borne disease surveillance, subsequently identified additional SF clinical isolates. Twelve cases in 11 states were identified; median age was 5years (range: <1-58years). Three patients were hospitalized; no deaths were reported. Of those with exposure information (n=10), all reported reptile exposure; 9 (90%) specified contact with leopard geckos. No common source of geckos was identified from reported purchase locations. Los Angeles County (LAC) health officials isolated SF from one patient's leopard gecko. Five reptile/gecko isolates were identified from the USDA National Veterinary Services Laboratories (NVSL) from 2015 to 2018. Five countries responded to an Epidemic Intelligence Information System post by PulseNet; reptile isolate sequence data were received from Czech Republic. A clinical case from England was identified through the National Center for Biotechnology Information pathogen detection pipeline; the patient did not report contact with leopard geckos. Whole genome sequencing analysis revealed substantial genetic diversity between clinical and animal isolates; however, gecko and clinical isolates from LAC were highly related (1 allele difference). This investigation linking SF illnesses to leopard geckos highlights an important public health risk from pets. A better understanding of how geckos are distributed by the pet industry in the United States could improve traceability to points of origin and mitigate Salmonella transmission at gecko breeders. Earlier NVSL reports of SF isolates from geckos suggest the risk of human SF infection from geckos is not new. This investigation demonstrates a need to educate gecko breeders, retailers and gecko owners about the continued Salmonella infection risk from pet geckos.

Influenza A(H3N2) Variant Virus Outbreak at Three Fairs — Maryland, 2017

On September 17, 2017, the Maryland Department of Agriculture (MDA) was notified by fair and 4-H officials of ill swine at agricultural fair A, held September 14-17. That day, investigation of the 107 swine at fair A revealed five swine with fever and signs of upper respiratory tract illness. All five respiratory specimens collected from these swine tested positive for influenza A virus at the MDA Animal Health Laboratory, and influenza A(H3N2) virus was confirmed in all specimens by the U.S. Department of Agriculture National Veterinary Services Laboratory (NVSL). On September 18, MDA was notified by fair and 4-H officials that swine exhibitors were also ill. MDA alerted the Maryland Department of Health (MDH). A joint investigation with MDH and the local health department was started and later broadened to Maryland agricultural fairs B (September 13-17) and C (September 15-23). In total, 76 persons underwent testing for variant influenza, and influenza A(H3N2) variant (A(H3N2)v) virus infection was identified in 40 patients with exposure to swine at these fairs (Figure), including 30 (75%) who had more than one characteristic putting them at high risk for serious influenza complications; 24 (60%) of these were children aged <5 years. Twenty-six (65%) patients reported direct contact with swine (i.e., touching swine or swine enclosure), but 14 (35%) reported only indirect contact (e.g., walking through a swine barn). Two children required hospitalization; all patients recovered. This outbreak highlights the risk, particularly among children, for contracting variant influenza virus at agricultural fairs after direct or indirect swine contact. Publicizing CDC's recommendation that persons at high risk for serious influenza complications avoid pigs and swine barns might help prevent future variant influenza outbreaks among vulnerable groups (1).

Screwworm (Diptera: Calliphoridae) in the United States: Response to and Elimination of the 2016-2017 Outbreak in Florida.

Eradicating screwworm, Cochliomyia hominivorax (Coquerel), from continental North American via the sterile insect technique has provided huge economic benefit to livestock producers by eliminating screwworm myiasis. After confirmatory identification of fly samples from infested deer by the USDA National Veterinary Services Laboratory on September 30, 2016, an alert was issued that screwworm myiasis was discovered in the Florida Keys. Personnel from USDA Animal and Plant Health Inspection Service, Agricultural Research Service, the State of Florida, U.S. Fish and Wildlife Service and local officials responded to the outbreak focus on Big Pine Key. After witnessing infested Key deer (Odocoileus virginianus clavium Barboyr & Allen), screwworm adult sampling was initiated at 0930 h on October 5, 2016 using nets to collect flies arriving at putrid liver, with the first female collected within 1 h. Larval samples were collected from infested animals for DNA analyses and to develop a "Florida outbreak" colony to test mating compatibility with the mass-produced strain used for sterile fly releases. Ground release chambers for sterile screwworm releases were placed in favorable habitats based on satellite image analyses. Sterile pupae were first placed in the chambers on October 11, 2016. Further liver trapping showed that 13 Keys were infested. One case, presumably through animal movement, occurred near Homestead on the Florida mainland. Ultimately there were 35 sterile fly release stations, including 4 located around Homestead, but no further cases were identified. About 188 million sterile flies were released until successful eradication was declared on March 23, 2017. Containing the outbreak prevented economic losses to livestock producers and other wildlife on the mainland and kept eradication costs to a minimum.

65 Inactivation Kinetics of Epizootic Hemorrhagic Disease Virus (EHDV) By Ingredients Contained in Expect Healthy Deer Technology®.

Expect Healthy Deer Technology® is a novel feed additive that contains natural feed ingredients that are known to have anti-viral, anti-bacterial, and anti-fungal properties. Epizootic Hemorrhagic Disease (EHD), caused by EHD virus (EHDV), is a hemorrhagic disease spread by small biting midge gnats affecting certain species of animals, especially deer, although it can also affect elk and cattle. Death losses in deer from EHDV are very high, and in localized outbreaks, it can wipe out a large percentage of a deer herd. The aim of this study was to evaluate the virucidal effectiveness of a key ingredient contained in Expect Health Deer Technology® against EHDV. Briefly, serial dilutions of the key ingredient were mixed with equal amounts of EHDV. Samples were removed after various periods of contact and tested for the amount of surviving virus by inoculation in baby hamster kidney cells (BHK-21), which can support infection by EHDV. Serotype 1 of EHDV (EHDV-1) was obtained from the National Veterinary Services Laboratories, Ames, IA. To determine virucidal activity, titer levels of EHDV were calculated in triplicate for each dilution of the ingredient. The initial virus titer for the EHDV-1 was 3.50 log10 TCID50/0.1 ml. At the low dilution rate (1:2 dilution), the ingredient killed (P < 0.05) 3 logs (99.9%) of virus within 2 h and killed (P < 0.05) 2.5 logs at 12 and 24 h. At a medium dilution rate (1:20) the ingredient killed (P < 0.05) 3 logs (99.9%) of the EHD virus after 2 h, but killed (P < 0.05) 2.5 logs after 12 and 24 hours. At the high dilution rate (1:200) the ingredient killed (P < 0.05) 3 logs (99.9%) of virus after 2 h, but killed (P < 0.05) 2.5 logs after 12 and 24 h. At ultra-high dilution rates (1:2,000 and 1:20,000) the ingredient was less effective. In summary, the ingredient within Expect Healthy Deer Technology® demonstrated strong virucidal activity against EHDV under in-vitro (laboratory) conditions.

Genomic diversity of Taylorella equigenitalis introduced into the United States from 1978 to 2012.

Contagious equine metritis is a disease of worldwide concern in equids. The United States is considered to be free of the disease although sporadic outbreaks have occurred over the last few decades that were thought to be associated with the importation of horses. The objective of this study was to create finished, reference quality genomes that characterize the diversity of Taylorella equigenitalis isolates introduced into the USA, and identify their differences. Five isolates of T. equigenitalis associated with introductions into the USA from unique sources were sequenced using both short and long read chemistries allowing for complete assembly and annotation. These sequences were compared to previously published genomes as well as the short read sequences of the 200 isolates in the National Veterinary Services Laboratories’ diagnostic repository to identify unique regions and genes, potential virulence factors, and characterize diversity. The 5 genomes varied in size by up to 100,000 base pairs, but averaged 1.68 megabases. The majority of that diversity in size can be explained by repeat regions and 4 main regions of difference, which ranged in size from 15,000 to 45,000 base pairs. The first region of difference contained mostly hypothetical proteins, the second contained the CRISPR, the third contained primarily hemagglutinin proteins, and the fourth contained primarily segments of a type IV secretion system. As expected and previously reported, little evidence of recombination was found within these genomes. Several additional areas of interest were also observed including a mechanism for streptomycin resistance and other virulence factors. A SNP distance comparison of the T. equigenitalis isolates and Mycobacterium tuberculosis complex (MTBC) showed that relatively, T. equigenitalis was a more diverse species than the entirety of MTBC.

Study of an Outbreak of Highly Pathogenic Avian Influenza H5N8 in Commercial Pekin Ducks (Anas platyrhynchos domesticus) in California

A February 2015 outbreak of highly pathogenic avian influenza (HPAI) H5N8 in a flock of commercial Pekin ducks ( Anas platyrhynchos domesticus) in California was investigated in detail. Approximately 17,349 five-wk-old ducks experienced an increased mortality from a normal of eight birds per day to 24, 18, 24, 33, and 61 birds per day, respectively, in the last 5 days prior to flock depopulation. Clinically, there was decreased water and feed consumption, and approximately 1.0% of the affected flock exhibited neurologic signs. Necropsy of five clinically ill ducks revealed pale, patchy areas on the epicardium in two birds, pale foci of necrosis in the liver of one bird, and airsacculitis in three birds. Histopathology revealed multifocal nonsuppurative encephalomyelitis, myocarditis, myositis, pancreatitis, hepatitis, and glossitis. Immunohistochemistry revealed avian influenza virus (AIV) nucleoprotein in the nucleus and cytoplasm of various cells in the aforementioned organs, as well as in the skin and feathers. Eight of the 10 sera samples tested were positive for avian influenza antibodies by agar gel immunodiffusion serology. Oropharyngeal and cloacal swabs taken from 15 birds, as well as from the lungs, livers, pancreas, and spleen, were positive for AIV by real-time reverse transcriptase (rRT) PCR. AIV was isolated and typed as Eurasian lineage HPAI H5N8, clade 2.3.4.4, by the National Veterinary Services Laboratory, Ames, IA. Extensive surveillance of birds for AIV around the 10-km zone did not reveal any additional cases. Ducks on the affected premises were humanely euthanatized by foam and composted in-house, the houses were heated to 57 C for 4 days, and swabs were taken periodically from the compost to ensure negativity for AIV by rRT-PCR. The compost and litter were then removed, and the house was pressure cleaned, disinfected, and repopulated approximately 120 days after euthanatization of the ducks.

Ante-mortem detection of chronic wasting disease in recto-anal mucosa-associated lymphoid tissues from elk (Cervus elaphus nelsoni) using real-time quaking-induced conversion (RT-QuIC) assay: A blinded collaborative study

ABSTRACTPrion diseases are transmissible spongiform encephalopathies (TSEs) characterized by fatal, progressive neurologic diseases with prolonged incubation periods and an accumulation of infectious misfolded prion proteins. Antemortem diagnosis is often difficult due to a long asymptomatic incubation period, differences in the pathogenesis of different prions, and the presence of very low levels of infectious prion in easily accessible samples. Chronic wasting disease (CWD) is a TSE affecting both wild and captive populations of cervids, including mule deer, white-tailed deer, elk, moose, muntjac, and most recently, wild reindeer. This study represents a well-controlled evaluation of a newly developed real-time quaking-induced conversion (RT-QuIC) assay as a potential CWD diagnostic screening test using rectal biopsy sections from a depopulated elk herd. We evaluated 69 blinded samples of recto-anal mucosa-associated lymphoid tissue (RAMALT) obtained from USDA Veterinary Services. The results were later un-blinded and statistically compared to immunohistochemical (IHC) results from the USDA National Veterinary Services Laboratories (NVSL) for RAMALT, obex, and medial retropharyngeal lymph node (MRPLN). Comparison of RAMALT RT-QuIC assay results with the IHC results of RAMALT revealed 92% relative sensitivity (95% confidence limits: 61.52–99.8%) and 95% relative specificity (95% confidence limits: 85.13–99%). Collectively, our results show a potential utility of the RT-QuIC assay to advance the development of a rapid, sensitive, and specific prion diagnostic assay for CWD prions.

Changes in the prevalence, genotypes and antimicrobial resistance phenotypesofnon-typhoidal Salmonella recovered from mail-order hatchling poultry sold at US feed stores, 2013-2015.

Every year, multiple outbreaks of salmonellosis in humans are linked to contact with mail-order chicks and ducks. The objective of this study was to describe the temporal changes in the prevalence of serovars, genotypes and antimicrobial resistance (AMR) phenotypes of non-typhoidal Salmonella (NTS) recovered from shipped boxes of mail-order hatchling poultry in the United States during 2013 to 2015. In each year, a sample of feed stores belonging to a single national chain participated in the study. The store employees submitted swabs or hatchling pads from hatchling boxes and shipment tracking information of the arriving boxes to the investigators. NTS was cultured from the samples and isolates were sent to the National Veterinary Services Laboratories (Ames, IA) for serotyping, pulsed-field gel electrophoresis (PFGE) and AMR phenotyping. The PFGE patterns of Salmonella serovars isolated from hatchling boxes were compared with those from human outbreaks of salmonellosis linked to live poultry contact. The box-level prevalence of NTS was significantly higher in 2015 compared to 2014. Also, the population of Salmonella serovars recovered in 2015 was more diverse and substantially different from those recovered in the previous two years. Of PFGE patterns recovered from hatchling boxes, seven distinct patterns in 2015, three in 2014 and four in 2013 were indistinguishable from the PFGE patterns of human outbreaks-associated strains in the respective years. Importantly, a significant positive correlation was found between the box-level prevalence of PFGE patterns and the number of human illnesses associated with the same patterns. Also, the proportion of multidrug-resistant isolates was higher in 2014 and 2015 compared to that in 2013. The results demonstrate that shipments of mail-order hatchling poultry are frequently contaminated with Salmonella genotypes indistinguishable from human outbreaks-associated strains each year, and control efforts at hatchery level are likely to have an important public health impact.

The First 10 Years (2006–15) of Epizootic Hemorrhagic Disease Virus Serotype 6 in the USA

Epizootic hemorrhagic disease virus (EHDV) is a Culicoides biting midge-transmitted orbivirus (family Reoviridae) of wild and domestic ruminants and is an important pathogen of white-tailed deer (Odocoileus virginianus). Historically, only two serotypes, EHDV-1 and EHDV-2, have been known to be endemic in the US. However, in 2006, an exotic serotype (EHDV-6) was first detected in the US by a long-term passive surveillance system for EHDV and bluetongue viruses. Here we report EHDV-6 detections made through these passive surveillance efforts by the Southeastern Cooperative Wildlife Disease Study (University of Georgia, Athens, Georgia, USA) and the National Veterinary Services Laboratories (US Department of Agriculture, Ames, Iowa, USA) over a 10-yr period (2006-15). The results demonstrated that EHDV-6 was detected from ruminants every year since 2006 and was widespread in the central and eastern US, providing evidence that EHDV-6 is likely now established in the US.

Identification of Type A Influenza Viruses from Wild Birds on the Delmarva Peninsula, 2007-10.

Wild waterfowl and shorebirds in the Delaware-Maryland-Virginia (Delmarva) Peninsula region within the Atlantic Flyway were sampled as part of the Early Detection of Highly Pathogenic H5N1 Avian Influenza (AI) in Wild Migratory Birds program. The U.S. Department of Agriculture (USDA) and state wildlife agencies submitted 7858 samples for AI virus (AIV) testing by real-time reverse transcription PCR (rRT-PCR) to the University of Delaware Poultry Health System from April 2007 to March 2011. Virus isolation attempts were performed on samples with matrix gene cycle threshold (Ct) values ≤33.9. Using rRT-PCR, AIV was detected in 14% (1091/7857) of the samples. In species with sample sizes >100, American black duck ( Anas rubripes ; 28%), ruddy turnstone ( Arenaria interpres ; 27%), American green-winged teal ( Anas crecca ; 21%), semipalmated sandpiper ( Calidris pusilla ; 27%), greater snow goose ( Chen caerulescens atlanticus; 12%), mallard ( Anas platyrhynchos ; 10%), and northern pintail ( Anas acuta ; 14%) showed the highest rates of AIV detection. Forty-two AIVs were recovered from eight species: American black duck, mallard, ruddy turnstone, American green-winged teal, greater snow goose, Canada goose ( Branta canadensis ), ring-necked duck ( Aythya collaris ), and mallard × American black duck ( Anas platyrhynchos × Anas rubripes ). Recovered H5 (n = 2) and H7 (n = 2) viruses were found to be low pathogenicity by the USDA National Veterinary Services Laboratory. Additional AIVs represented a diversity of subtype combinations: H1-H4, H6, and H10 and H11 and N subtypes N1-N9 and N6-N9. The rate of AIV recovery from swabbings was inversely related to Ct value, ranging from 50% for Ct values of 16.0-18.9 to 5.1% for Ct values of 31-33.9.

Outbreak of highly pathogenic avian influenza in Minnesota in 2015.

The incursion of highly pathogenic avian influenza (HPAI) into the United States during 2014 resulted in an unprecedented foreign animal disease (FAD) event; 232 outbreaks were reported from 21 states. The disease affected 49.6 million birds and resulted in economic losses of $950 million. Minnesota is the largest turkey-producing state, accounting for 18% of U.S. turkey production. Areas with concentrated numbers of turkeys in Minnesota were the epicenter of the outbreak. The first case was presumptively diagnosed in the last week of February 2015 at the Minnesota Veterinary Diagnostic Laboratory (MVDL) and confirmed as HPAI H5N2 at the National Veterinary Services Laboratories on March 4, 2015. A total of 110 farms were affected in Minnesota, and the MVDL tested >17,000 samples from March to July 2015. Normal service was maintained to other clients of the laboratory during this major FAD event, but challenges were encountered with communications, staff burnout and fatigue, training requirements of volunteer technical staff, test kit validation, and management of specific pathogen-free egg requirements.

A Novel Mycoplasma sp. Associated with Phallus Disease in Goose Breeders: Pathological and Bacteriological Findings.

In April 2014, poor fertility in a major commercial goose breeder operation in California triggered the submission of six live affected Toulouse ganders ( Anser anser ) to the California Animal Health and Food Safety Laboratory, Turlock branch (University of California-Davis). Toulouse were principally affected among all breeds, and their egg fertility dropped from 65.7% to less than 33.9% in the first 40 days of the 2014 breeding season. The flock consisted of 410 adult birds, 90 males and 320 females, between 2 and 5 yr of age. Inspection of the flock revealed that 44.4% of the Toulouse ganders had severe phallic deformities that prevented them from mating. At postmortem examination, severe yellowish fibrocaseous exudate disrupted the architecture of the phallus and occasionally produced fistulating tracts through the wall of the organ. Microscopically, multifocal lymphoid nodules were noted in the mucosa and submucosa of the phallus and were associated with extensive granulomatous reaction, intralesional bacteria, and spermatozoa. Mycoplasma spp. were isolated from the phallus of affected and nonaffected birds, and PCR protocols targeting the 16S-23S ribosomal RNA intergenic spacer regions and the RNA polymerase beta subunit gene were performed to identify the isolates. Three distinct species were identified on sequencing and analysis using the National Center for Biotechnology Information basic local alignment search tool: Mycoplasma cloacale , Mycoplasma anseris , and an unknown novel Mycoplasma sp. Additionally, Pasteurella multocida , in combination with other bacteria, was also isolated from the phallic lesions and identified as serotype 3 with a DNA profile of 1511 (National Veterinary Service Laboratory). This is the first report of these Mycoplasma spp. and other bacteria associated with reproductive disease in ganders in the United States.