Detection of Chronic Wasting Disease Prions in Prairie Soils from Endemic Regions.

Chronic wasting disease (CWD) is a contagious prion disease that affects cervids in North America, Northern Europe, and South Korea. CWD is spread through direct and indirect horizontal transmission, with both clinical and preclinical animals shedding CWD prions in saliva, urine, and feces. CWD particles can persist in the environment for years, and soils may pose a risk for transmission to susceptible animals. Our study presents a sensitive method for detecting prions in the environmental samples of prairie soils. Soils were collected from CWD-endemic regions with high (Saskatchewan, Canada) and low (North Dakota, USA) CWD prevalence. Heat extraction with SDS-buffer, a serial protein misfolding cyclic amplification assay coupled with a real-time quaking-induced conversion assay was used to detect the presence of CWD prions in soils. In the prairie area of South Saskatchewan where the CWD prevalence rate in male mule deer is greater than 70%, 75% of the soil samples tested were positive, while in the low-prevalence prairie region of North Dakota (11% prevalence in male mule deer), none of the soils contained prion seeding activity. Soil-bound CWD prion detection has the potential to improve our understanding of the environmental spread of CWD, benefiting both surveillance and mitigation approaches.

A PRELIMINARY INVESTIGATION OF PATHOGEN EXPOSURE OF ADULT FEMALE WHITE-TAILED DEER (ODOCOILEUS VIRGINIANUS) AND SURVIVAL OF ASSOCIATED FAWNS IN THE WESTERN DAKOTAS, USA.

Exposure of a dam to pathogens may potentially affect her fawns positively or negatively. Mammalian females transfer immunologic protection to their offspring via colostrum obtained while nursing. Conversely, chronic diseases in dams may potentially result in small and weak neonates, reduced milk production or quality, or infection. Little is known about how pathogen exposure in adult female white-tailed deer (Odocoileus virginianus) affects offspring survival. Our objective was to assess pathogen exposure for female white-tailed deer and subsequent survival rates of fawns in Dunn and Grant counties, North Dakota, and Perkins County, South Dakota, USA. We collected blood serum from 150 adult female deer during 2014. We compared survival of 49 fawns to maternal exposure to 10 pathogens from 37 of 150 adult females. There was no difference in fawn mass between dams based on antibody status and no difference in fawn survival for nine pathogens. The 12-wk survival for fawns born to mothers with antibodies against bovine herpesvirus 1 (BoHV-1, causing infectious bovine rhinotracheitis) was lower than for fawns born from mothers without antibodies against BoHV-1; however, the indirect or direct impacts of BoHV-1 exposure in mothers on fawn survival are unclear. Although our findings suggest that the cost of exposure to previous diseases may have minimal impact on short-term fawn survival for most pathogens, additional research with increased sample sizes is needed to confirm our findings.

Exposure to Select Pathogens in an Expanding Moose (Alces alces) Population in North Dakota, USA.

Forty female moose (Alces alces) captured in North Dakota, US, in March 2014 were tested for antibodies to a variety of pathogens. Antibodies to West Nile virus (WNV) were detected in 39 (98%) moose following a year with a high number of human cases, suggesting the population accurately reflects WNV activity. Fifteen percent of moose (6/40) had antibodies to Borrelia burgdorferi, implying expansion of the tick vector into the area. Antibodies to Anaplasma spp. were detected in 55% of moose (22/40), a higher rate than previously detected in cattle from the region. Low titers (100-400) to one or more serovars of Leptospira spp. were detected in 23% of moose (9/40), a common finding in wild ruminants. Exposure to other pathogens was uncommon (<8%; <3/40) or not documented. Survival and recruitment were high during the study period, suggesting a limited population-level impact at current levels of exposure and environmental co-stressors.

EXPERIMENTAL INFECTIONS AND SEROLOGY INDICATE THAT AMERICAN WHITE IBIS (EUDOCIUMUS ALBUS) ARE COMPETENT RESERVOIRS FOR TYPE A INFLUENZA VIRUS.

The American White Ibis (Eudocimus albus) is a nomadic wading bird common to wetland habitats in the southeastern US. In south Florida, US, habitat depletion has driven many ibis to become highly urbanized. Although they forage in neighborhood parks, artificial wetlands, backyards, and golf courses, the majority continue to nest in natural wetlands, often in dense, mixed species colonies. Adults and juveniles commonly disperse thousands of kilometers to other breeding colonies along the Gulf and southeast Atlantic coasts, presenting the potential for close contact with humans, domestic animals, and other wild bird species. Historically, wading birds were not considered to be significant hosts for influenza A virus (IAV), yet as ibis regularly move among various human, domestic animal, and wildlife interfaces, their potential to be exposed to or infected with IAV deserves attention. We experimentally challenged wild-caught, captive-reared White Ibis (n=20) with IAV, tested wild White Ibis for IAV, and serologically tested wild White Ibis for antibodies to IAV. White Ibis were highly susceptible to experimental challenge with H6N1 and H11N9 IAVs, with cloacal shedding lasting an average of 6 d. All 13 infected birds seroconverted by 14 d postinfection as determined by microneutralization. In contrast, no birds challenged with H3N8 were infected. We tested 118 swabs and 578 serum samples from White Ibis captured in southeastern Florida for IAV infection and antibodies to IAV, respectively. Although no IAVs were isolated, 70.4% serum samples were antibody positive by blocking enzyme-linked immunosorbent assay (bELISA). Neutralizing antibodies to H1-H12 were detected in 96.0% of a subset of bELISA positive birds (n=196) and 81.0% tested antibody positive to two or more hemagglutinin subtypes, indicating that exposure to multiple IAVs is common. These results provide evidence that White Ibis are susceptible and naturally infected with IAV and may represent a component of the IAV natural reservoir system.

SUSCEPTIBILITY OF LAUGHING GULLS (LEUCOPHAEUS ATRICILLA) AND MALLARDS (ANAS PLATYRHYNCHOS) TO RUDDY TURNSTONE (ARENARIA INTERPRES MORINELLA) ORIGIN TYPE A INFLUENZA VIRUSES.

Delaware Bay, US is the only documented location where influenza A virus (IAV) is consistently detected in a shorebird species, the Ruddy Turnstone (RUTU; Arenaria interpres morinella). Although IAV in shorebirds has been well studied at this site for decades, the importance of other species in the avian community as potential sources for the IAVs that infect RUTUs each spring remains unclear. We determined the susceptibility of Mallards (Anas platyrhynchos) and Laughing Gulls (Leucophaeus atricilla), to IAVs isolated from RUTUs in order to gain insight into the potential host range of these viruses. Captive-reared gulls were challenged with RUTU-origin H6N1, H10N7, H11N9, H12N4, and H13N6 IAV, as well as Mallard-origin H6N1 and H11N9. We challenged captive-reared Mallards with the same viruses, except for H13N6. At a biologically plausible challenge dose (104 50% embryo infective doses/0.1 mL), one of five gulls challenged with both H6N1 IAVs shed virus. The remaining gulls were resistant to infection with all viruses. In contrast, all Mallards were infected and shed virus. The H12N4 Mallard challenge group was an exception with no birds infected. These results indicated that Mallards are permissive to infection with viruses originating from a shorebird host and that interspecies transmission could occur. In contrast, host adaptation of IAVs to RUTUs may compromise their ability to be transmitted back to gulls.

Are Microneutralization and Hemagglutination Inhibition Assays Comparable? Serological Results from Influenza Experimentally Infected Mallard Ducks.

The hemagglutination inhibition (HI) assay is commonly used to assess the humoral immune response against influenza A viruses (IAV). However, the microneutralization (MN) assay has been reported to have higher sensitivity when testing sera from humans and other species. Our objective was to determine the agreement between MN and HI assays and compare the proportion of positive samples detected by both methods in sera of mallards primary infected with the A/mallard/MN/Sg-000169/ 2007 (H3N8) virus and subsequently inoculated with homosubtypic or heterosubtypic IAV. Overall, we found poor to fair agreement (prevalence-adjusted bias-adjusted kappa [PABAK], 0.03-0.35) between MN and HI assays in serum samples collected 2 weeks after H3N8 inoculation; the observed agreement increased to moderate or substantial in samples collected 4 to 5 weeks postinoculation (WPI) (PABAK, 0.52-0.75). The MN assay detected a higher proportion of positive samples compared with HI assays in serum samples collected 2 WPI (P = 0.01). This difference was not observed in samples collected 4 WPI. Also, a boosting effect in MN and HI titers was observed when birds were subsequently inoculated with IAV within the same H3 clade. This effect was not observed when birds were challenged with viruses that belong to a different HA clade. In summary, the agreement between assays varies depending on the postinfection sample collection time point and the similarity between the antigens used for the assays. Additionally, subsequent exposure of ducks to homosubtypic or heterosubtypic strains might affect the observed agreement.

¿Los ensayos de microneutralización e inhibición de la hemaglutinación son comparables? Resultados serológicos de patos de collar infectados experimentalmente con influenza. La prueba de inhibición de la hemaglutinación se usa rutinariamente para evaluar la respuesta inmune humoral contra los virus de influenza aviar, sin embargo, se ha reportado que la prueba de microneutralización tiene una mayor sensibilidad cuando se evalúan muestras de suero de humanos u otras especies. Este estudio tuvo como objetivo determinar la concordancia entre las pruebas de microneutralización e inhibición de la hemaglutinación en suero de patos de collar que fueron desafiados con el virus A/ mallard/MN/Sg-000169/2007(H3N8) y re-inoculados con virus de influenza aviar homosubtípicos o heterosubtípicos. Además, se comparó la proporción de muestras positivas detectadas por ambos métodos. En general, se observó un nivel de concordancia pobre a razonable (PABAK = 0.03 - 0.35) entre las pruebas de microneutralización e inhibición de la hemaglutinación en muestras de suero recolectadas dos semanas post-inoculación del virus H3N8. La concordancia se incrementó a moderada o sustancial en muestras recolectadas cuatro o cinco semanas después de la inoculación (PABAK = 0.52 - 0.75). Una mayor proporción de muestras recolectadas a las dos semanas después de la inoculación fueron positivas por microneutralización en comparación con inhibición de la hemaglutinación (P = 0.01), estas diferencias no fueron observadas con las muestras recolectadas a las cuatro semanas después de la inoculación. Adicionalmente, se observó un incremento en los títulos de anticuerpos cuando las aves fueron re-inoculadas con virus de influenza aviar pertenecientes al mismo clado H3 de la hemaglutinina. Este efecto no fue observado en los patos re-inoculados con virus de influenza aviar pertenecientes a un clado distinto. En resumen, la concordancia entre los ensayos varía según el momento de recolección de la muestra y la similitud entre los antígenos utilizados para los ensayos. Además, la re-inoculación de patos con una cepa homosubtípica or heterosubtípica podría afectar el nivel de concordancia observada.

NEUTRALIZING ANTIBODIES TO TYPE A INFLUENZA VIRUSES IN SHOREBIRDS AT DELAWARE BAY, NEW JERSEY, USA.

Influenza A virus (IAV) infections in shorebirds at Delaware Bay, New Jersey, US, have historically included avian hemagglutinin (HA) subtypes H1-13 and H16. In a given year, infections are characterized by a limited number of HA and neuraminidase subtypes and a dominant HA subtype that often represents >50% of all isolates. Predominant HA subtypes shift between consecutive years. In addition, infection prevalence is consistently higher in Ruddy Turnstones (RUTU; Arenaria interpres morinella) compared to Red Knots (REKN; Calidris canutus rufa), despite comparable rates of exposure. To investigate a potential immunological basis for this phenomenon, a virus microneutralization assay was used to detect subtype-specific, neutralizing antibodies to H1-H12 in sera collected from RUTUs from 2012-16 and REKNs in 2012, 2013, and 2016. Neutralizing antibodies to one or more subtypes were detected in 36% (222/611) of RUTUs. Prevalence of antibodies to subtypes H6 and H11 remained high throughout the study, and these virus subtypes were isolated every year, suggesting a continual source of exposure. Antibody prevalence was intermediate for most IAV subtypes that were isolated in 2-3 of 5 yr (H1, H3, H5, H9, H10, and H12) but was low for H7 viruses, despite the isolation of this virus subtype in 3 of 5 yr. This suggests a reduced antigenicity of H7 IAVs compared to other subtypes. Antibody prevalence was low for H4 virus that was isolated once, and H2 and H8 viruses that were never isolated. Neutralizing antibodies were detected in 66% (169/257) of REKNs and subtype-specific antibody prevalences were higher in REKNs than RUTUs with few exceptions. The results suggest that population immunity influences which species is infected at Delaware Bay, indicate that IAV dynamics are subtype-dependent, and demonstrate the utility of the microneutralization assay as a supportive tool for field research.

Heterosubtypic immunity increases infectious dose required to infect Mallard ducks with Influenza A virus.

Previous field and experimental studies have demonstrated that heterosubtypic immunity (HSI) is a potential driver of Influenza A virus (IAV) prevalence and subtype diversity in mallards. Prior infection with IAV can reduce viral shedding during subsequent reinfection with IAV that have genetically related hemagglutinins (HA). In this experiment, we evaluated the effect of HSI conferred by an H3N8 IAV infection against increasing challenge doses of closely (H4N6) and distantly (H6N2) related IAV subtypes in mallards. Two groups of thirty 1-month-old mallards each, were inoculated with 105.9 50% embryo infectious doses (EID50) of an H3N8 virus or a mock-inoculum. One month later, groups of five birds each were challenged with increasing doses of H4N6 or H6N2 virus; age-matched, single infection control ducks were included for all challenges. Results demonstrate that naïve birds were infected after inoculation with 103 and 104 EID50 doses of the H4N6 or H6N2 virus, but not with 102 EID50 doses of either IAV. In contrast, with birds previously infected with H3N8 IAV, only one duck challenged with 104 EID50 of H4N6 IAV was shedding viral RNA at 2 days post-inoculation, and with H6N2 IAV, only birds challenged with the 104 EID50 dose were positive to virus isolation. Viral shedding in ducks infected with H6N2 IAV was reduced on days 2 and 3 post-inoculation compared to control birds. To explain the differences in the dose necessary to produce infection among H3-primed ducks challenged with H4N6 or H6N2 IAV, we mapped the amino acid sequence changes between H3 and H4 or H6 HA on predicted three-dimensional structures. Most of the sequence differences occurred between H3 and H6 at antigenic sites A, B, and D of the HA1 region. These findings demonstrate that the infectious dose necessary to infect mallards with IAV can increase as a result of HSI and that this effect is most pronounced when the HA of the viruses are genetically related.

Introduction and Establishment of Raccoon Rabies on Islands: Jekyll Island, Georgia, USA as a Case Study.

The introduction of rabies virus (RABV) to barrier islands, which are often popular tourist destinations with resource-rich habitats and connectivity and proximity to the mainland, is especially concerning because it can easily become endemic due to factors like dense rabies-vector populations (e.g., raccoons [ Procyon lotor]), high inter- and intraspecies contact rates, and anthropogenic activities such as supplemental feeding of feral cats ( Felis catus). In January 2013, a neurologic raccoon found on the Jekyll Island (JI), Georgia, US causeway tested positive for rabies. Mortality investigations of 29 raccoons have been conducted between December 2012-May 2017. The two most common diagnoses were RABV ( n=11) and canine distemper virus (CDV; n=8). Parvoviral enteritis was diagnosed in four raccoons but no coinfections were diagnosed. There was no apparent seasonality for rabies cases, but all CDV cases occurred in spring-fall. Most (64%) rabies submissions came from residential or recreational use areas located near feral cat feeding stations. Jekyll Island is a popular destination where tourists engage in numerous outdoor activities which facilitate human-wildlife interactions. Concerns regarding public and animal health highlight the importance of rabies surveillance, prevention, and control on islands. This is the first report of rabies on JI and emphasizes the importance of disease investigations because the assumption that neurologic raccoons have CDV, an endemic pathogen, can miss the establishment of novel pathogens such as RABV.

Management Factors Associated with Operation-Level Prevalence of Antibodies to Cache Valley Virus and Other Bunyamwera Serogroup Viruses in Sheep in the United States.

A cross-sectional study was performed to identify operation-level risk factors associated with prevalence of antibody to Bunyamwera (BUN) serogroup viruses in sheep in the United States. Sera were obtained from 5150 sheep in 270 operations located in 22 states (three in the west, nine central states, and 10 in the east) and tested at a dilution of 1:20 by a plaque reduction neutralization test (PRNT) using Cache Valley virus (CVV). Antibodies that neutralized CVV were identified in 1455 (28%) sheep. Animal-level seroprevalence was higher in the east (49%) than the central (17%) and western (10%) states. A convenient subset (n = 509) of sera with antibodies that neutralized CVV was titrated and further analyzed by PRNT using all six BUN serogroup viruses that occur in the United States: CVV, Lokern virus (LOKV), Main Drain virus (MDV), Northway virus (NORV), Potosi virus (POTV), and Tensaw virus (TENV). Antibodies to CVV and LOKV were identified in sheep in all three geographic regions; MDV and POTV activity was detected in the central and eastern states, NORV activity was restricted to the west, and antibodies to TENV were not detected in any sheep. Several management factors were significantly associated with the presence of antibodies to BUN serogroup viruses. For instance, sheep housed during the lambing season inside structures that contained four walls and a roof and a door closed most of the time were more likely to be seropositive than other sheep. In contrast, herded/open-range sheep were less likely to be seropositive than their counterparts. These data can be used by producers to implement strategies to reduce the likelihood of BUN serogroup virus infection and improve the health and management practices of sheep.

Monitoring sheep and Culicoides midges in Montana for evidence of Bunyamwera serogroup virus infection.

INTRODUCTION: A serological and entomological investigation was performed to monitor for potential Bunyamwera (BUN) serogroup virus activity in Montana. RESULTS: To facilitate the serological investigation, sera were collected from 104 sheep in 2013 and 2014 and assayed by plaque reduction neutralization test using all six BUN serogroup viruses known to occur in the United States: Cache Valley virus (CVV), Lokern virus (LOKV), Main Drain virus (MDV), Northway virus, Potosi virus and Tensaw virus. BUN serogroup virus-specific antibodies were detected in 41 (39%) sheep. Of these, three were seropositive for MDV, one was seropositive for CVV, one was seropositive for LOKV and 36 had antibodies to an undetermined BUN serogroup virus. Additionally, 30,606 Culicoides sonorensis were collected in 2013 using Centers for Disease Control and Prevention (CDC) light traps and assayed for cytopathic virus by virus isolation in African Green Monkey kidney (Vero) cells. All midges were negative. Almost one-third of the midges were further tested by reverse transcription-polymerase chain reaction using BUN serogroup virus-reactive primers and all were negative. CONCLUSIONS: We provide evidence of BUN serogroup virus infection in sheep but not C. sonorensis in Montana in 2013-2014. This study also provides the first evidence of CVV, MDV and LOKV activity in Montana.