Serologic Survey of Selected Arthropod-Borne Pathogens in Free-Ranging Snowshoe Hares (Lepus americanus) Captured in Northern Michigan, USA.

Snowshoe hares (Lepus americanus) in the Upper Peninsula (UP) of Michigan, USA, occupy the southern periphery of the species' range and are vulnerable to climate change. In the eastern UP, hares are isolated by the Great Lakes, potentially exacerbating exposure to climate-change-induced habitat alterations. Climate change is also measurably affecting distribution and prevalence of vector-borne pathogens in North America, and increases in disease occurrence and prevalence can be one signal of climate-stressed wildlife populations. We conducted a serosurvey for vector-borne pathogens in snowshoe hares that were captured in the Hiawatha National Forest in the eastern UP of Michigan, USA, 2016-2017. The most commonly detected antibody response was to the mosquito-borne California serogroup snowshoe hare virus (SSHV). Overall, 24 (51%) hares screened positive for SSHV antibodies and of these, 23 (96%) were confirmed positive by plaque reduction neutralization test. We found a positive association between seroprevalence of SSHV and live weight of snowshoe hares. Additionally, we detected a significant effect of ecological land type group on seroprevalence of SSHV, with strong positive support for a group representing areas that tend to support high numbers of hares (i.e., acidic mineral containing soils with cedar, mixed swamp conifers, tamarack and balsam fir as common overstory vegetation). We also detected and confirmed antibodies for Jamestown Canyon virus and Silverwater virus in a single hare each. We did not detect antibodies to other zoonotic vector-borne pathogens, including Lacrosse encephalitis virus, West Nile virus, Borrelia burgdorferi, Powassan virus, and Francisella tularensis. These results provide a baseline for future serological studies of vector-transmitted diseases that may increase climate vulnerability of snowshoe hares in the UP of Michigan, as well as pose a climate-related zoonotic risk.

LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LAMP) ASSAY FOR DETECTION OF ASIAN FISH TAPEWORM, SCHYZOCOTYLE ACHEILOGNATHI (YAMAGUTI, 1934) [SYN. BOTHRIOCEPHALUS ACHEILOGNATHI].

The Asian fish tapeworm (Schyzocotyle acheilognathi syn. Bothriocephalus acheilognathi) (AFT) is an invasive parasite that can infect many species of fish, although most hosts are primarily members of Cyprinidae. Pathogenicity has most often been reported in aquaculture settings in fry and fingerling stages of carp (Cyprinus spp.). More recently, it has been shown to cause growth retardation in the endangered bonytail chub (Gila elegans) and found to be widespread in populations of endangered humpback chub (Gila cypha) in the Colorado River, Grand Canyon, Arizona. AFT spreads most often through the transport of infected fish, particularly baitfish. Despite its harmful potential, there is no efficient or accurate ante mortem test to detect AFT in water or fish samples before transport. Herein, we report on the development of a sensitive and specific loop-mediated isothermal amplification (LAMP) assay to detect the parasite in under 30 min from laboratory prepared samples. Six LAMP primers were designed to amplify a variable region of the 18S ribosomal RNA gene in AFT with the detection and quantification of DNA on a real-time fluorometer. The limit of detection was 1 × 101 copies/µl of DNA extracted from as few as 2 AFT eggs. Future application of our assay would be a low-cost test to rapidly and accurately detect AFT DNA from environmental samples on-site so that preventive actions can be taken to halt the spread of the AFT through the movement of infected fish.

Genetic Sequencing of Attwater's Prairie Chicken Avian Poxvirus and Evaluation of Its Potential Role in Reticuloendotheliosis Virus Outbreaks.

Efforts to breed Attwater's prairie chickens (APC; Tympanuchus cupido attwateri) in captivity to supplement wild populations of this endangered bird have been negatively affected by infections with Avipoxvirus and reticuloendotheliosis virus (REV). Because REV can be integrated into the genome of fowlpox virus (FPV) and may be transmitted in that manner, identifying the source of avipox disease in APC is important to mitigate the impact of this virus. Tissue samples from APC were collected from breeding programs in Texas from 2016 to 2020. These samples consisted of 11 skin lesions and three internal organs from a total of 14 different birds that died of unknown causes or were euthanized. Avipoxvirus was detected by PCR and isolation in embryonating chicken eggs in all skin lesion samples but was not detected in internal organs. Using sequence analysis of FPV polymerase and 4b genes, we determined that 10 out of 11 Avipoxvirus detections resided within the fowlpox clade and a single sample resided within the canarypox clade. REV sequences were detected in all FPV positive samples and in all internal organ tissues but were not detected in the sample matching the canarypox clade. Analysis of REV sequences and PCR detection showed the REV infecting APC was consistent with REV-A and had little variability on analysis of the U3 region of the long terminal repeat. The results of this study indicate control of REV in APC breeding colonies may benefit by a vaccination program targeting FPV and REV. However, a commercially available vaccine for REV is not available at this time.

Secuenciación genética de un virus de la viruela aviar de un gallo grande de las praderas Attwater y evaluación de su papel potencial en los brotes del virus de la reticuloendoteliosis. Los esfuerzos para criar gallos de las praderas grandes de Attwater (APC; Tympanuchus cupido attwateri) en cautiverio para complementar las poblaciones silvestres de esta ave en peligro de extinción se han visto afectados negativamente por infecciones con Avipoxvirus y con el virus de la reticuloendoteliosis (REV). Debido a que el virus de la reticuloendoteliosis puede integrarse en el genoma del virus de la viruela del pollo (FPV) y puede transmitirse de esa manera, identificar la fuente del virus pox en gallos de las praderas grandes es importante para mitigar el impacto de este virus. Se recolectaron muestras de tejido de gallos de las praderas grandes de programas de reproducción en Texas entre los años 2016 a 2020. Estas muestras consistieron en 11 lesiones cutáneas y tres órganos internos de un total de 14 aves diferentes que murieron por causas desconocidas o fueron sacrificadas. El Avipoxvirus se detectó mediante PCR y por aislamiento en huevos embrionados de pollo en todas las muestras de lesiones cutáneas, pero no se detectó en los órganos internos. Utilizando el análisis de secuencia de la polimerasa del virus de la viruela del pollo y de los genes 4b, se determinó que diez de las once detecciones de Avipoxvirus residían dentro del clado de la viruela aviar del pollo y una sola muestra residía dentro del clado de la viruela del canario. Se detectaron secuencias del virus de la reticuloendoteliosis en todas las muestras positivas para virus de la viruela de pollo y en todos los tejidos de órganos internos, pero no se detectaron en la muestra que coincidía con el clado de la viruela del canario. El análisis de las secuencias del virus de la reticuloendoteliosis y la detección por PCR mostró que los virus de reticuloendoteliosis que infectan a gallos de las praderas grandes eran compatible con virus de la reticuloendoteliosis A y tenía poca variabilidad en el análisis de la región U3 de la región repetida terminal larga. Los resultados de este estudio indican que el control del virus de la reticuloendoteliosis en colonias reproductoras de gallos de las praderas grandes puede beneficiarse de un programa de vacunación dirigido los virus de la viruela del pollo y de la reticuloendoteliosis. Sin embargo, una vacuna disponible comercialmente contra el virus de la reticuloendoteliosis no está disponible en este momento.

West Nile Virus Infection in American Singer Canaries: An Experimental Model in a Highly Susceptible Avian Species.

This study investigated the susceptibility of American singer canaries ( Serinus canaria) to West Nile virus (WNV) infection. Adult canaries were inoculated with 105, 102, and 101 plaque forming units (PFU) of WNV. All birds became infected and mortality occurred by 5 days postinoculation. The load of viral RNA as determined by RT-qPCR was dose dependent, and was higher at all doses than the level of viral RNA detected in American crows ( Corvus brachyrhynchos) challenged with 105 PFU of WNV. In a subset of birds, viremia was detected by virus isolation; canaries inoculated with 101 PFU of WNV developed viremia exceeding 1010 PFU/mL serum, a log higher than American crows inoculated with 105 PFU of virus. In canaries euthanized at 3 days postinoculation, WNV was isolated at >107 PFU of virus/100 mg of lung, liver, heart, spleen, and kidney tissues. Pallor of the liver and splenomegaly were the most common macroscopic observations and histologic lesions were most severe in liver, spleen, and kidney, particularly in canaries challenged with 102 and 101 PFU. Immunoreactivity to WNV was pronounced in the liver and spleen. IgG antibodies to WNV were detected in serum by enzyme immunoassay in 11 of 21 (52%) challenged canaries and, in 4 of 5 (20%) of these sera, neutralization antibodies were detected at a titer ≥ 1:20. American singer canaries provide a useful model as this bird species is highly susceptible to WNV infection.

Potential for Waterborne and Invertebrate Transmission of West Nile Virus in the Great Salt Lake, Utah.

In November and December of 2013, a large mortality event involving 15,000 to 20,000 eared grebes (Podiceps nigricollis) occurred at the Great Salt Lake (GSL), UT. The onset of the outbreak in grebes was followed by a mortality event in >86 bald eagles (Haliaeetus leucocephalus). During the die-off, West Nile virus (WNV) was detected by reverse transcription-PCR (RT-PCR) or viral culture in the carcasses of grebes and eagles submitted to the National Wildlife Health Center. However, no activity of mosquitoes, the primary vectors of WNV, was detected by the State of Utah's WNV monitoring program. The transmission of WNV has rarely been reported during the winter in North America in the absence of known mosquito activity; however, the size of this die-off, the habitat in which it occurred, and the species involved are unique. We experimentally investigated whether WNV could survive in water with a high salt content, as found at the GSL, and whether brine shrimp, the primary food of migrating eared grebes on the GSL, could have played a role in the transmission of WNV to feeding birds. We found that WNV can survive up to 72 h at 4°C in water containing 30 to 150 ppt NaCl, and brine shrimp incubated with WNV in 30 ppt NaCl may adsorb WNV to their cuticle and, through feeding, infect epithelial cells of their gut. Both mechanisms may have potentiated the WNV die-off in migrating eared grebes on the GSL.IMPORTANCE Following a major West Nile virus die-off of eared grebes and bald eagles at the Great Salt Lake (GSL), UT, in November to December 2013, this study assessed the survival of West Nile virus (WNV) in water as saline as that of the GSL and whether brine shrimp, the major food for migrating grebes, could have played a role as a vector for the virus. While mosquitoes are the major vector of WNV, under certain circumstances, transmission may occur through contaminated water and invertebrates as food.

Susceptibility and Antibody Response of the Laboratory Model Zebra Finch (Taeniopygia guttata) to West Nile Virus.

Since the introduction of West Nile virus (WNV) into North America in 1999 a number of passerine bird species have been found to play a role in the amplification of the virus. Arbovirus surveillance, observational studies and experimental studies have implicated passerine birds (songbirds, e.g., crows, American robins, house sparrows, and house finches) as significant reservoirs of WNV in North America, yet we lack a tractable passerine animal model for controlled studies of the virus. The zebra finch (Taeniopygia guttata) serves as a model system across a diversity of fields, and here we develop the zebra finch a songbird model for WNV. Like many natural hosts of WNV, we found that zebra finches developed sufficient viremia to serve as a competent host, yet in general resisted mortality from infection. In the Australian zebra finch (AZF) T. g. castanotis, we detected WNV in the majority of sampled tissues by 4 days post injection (dpi). However, WNV was not detected in tissues of sacrificed birds at 14 dpi, shortly after the development of detectable anti-WNV antibodies in the majority of birds indicating successful viral clearance. We compared susceptibility between the two zebra finch subspecies AZF and Timor zebra finch (TZF) T. g. guttata. Compared to AZF, WNV RNA was detected in a larger proportion of challenged TZF and molecular detection of virus in the serum of TZF was significantly higher than in AZF. Given the observed moderate host competence and disease susceptibility, we suggest that zebra finches are appropriate as models for the study of WNV and although underutilized in this respect, may be ideal models for the study of the many diseases carried and transmitted by songbirds.