Paul C. Wolf

Characterization of Newcastle Disease Viruses Isolated from Cormorant and Gull Species in the United States in 2010

SUMMARY. Newcastle disease virus (NDV), a member of the genus Avulavirus of the family Paramyxoviridae, is the causative agent of Newcastle disease (ND), a highly contagious disease that affects many species of birds and which frequently causes significant economic losses to the poultry industry worldwide. Virulent NDV (vNDV) is exotic in poultry in the United States; however, the virus has been frequently associated with outbreaks of ND in cormorants, which poses a significant threat to poultry species. Here, we present the characterization of 13 NDV isolates obtained from outbreaks of ND affecting cormorants and gulls in the states of Minnesota, Massachusetts, Maine, New Hampshire, and Maryland in 2010. All 2010 isolates are closely related to the viruses that caused the ND outbreaks in Minnesota in 2008, following the new evolutionary trend observed in cormorant NDV isolates since 2005. Similar to the results obtained with the 2008 isolates, the standard United States Department of Agriculture F-gene real-time reverse-transcription PCR (RRT-PCR) assay failed to detect the 2010 cormorant viruses, whereas all viruses were detected by a cormorant-specific F-gene RRT-PCR assay. Notably, NDV-positive gulls were captured on the eastern shore of Maryland, which represents a significant geographic expansion of the virus since its emergence in North America. This is the first report of vNDV originating from cormorants isolated from wild birds in Maryland and, notably, the first time that genotype V vNDV has been isolated from multiple wild bird species in the United States. These findings highlight the need for constant epidemiologic surveillance for NDV in wild bird populations and for consistent biosecurity measures to prevent the introduction of the agent into domestic poultry flocks.

Characterization of the risk of deer-cattle interactions in Minnesota by use of an on-farm environmental assessment tool

OBJECTIVE To characterize the risk of interactions that may lead to the transmission of Mycobacterium bovis between cattle and white-tailed deer (Odocoileus virginianus) on farms in northern Minnesota. SAMPLE 53 cattle farms in northwestern Minnesota adjacent to an area where bovine tuberculosis-infected cattle and deer were detected. PROCEDURES A semiquantitative deer-cattle interaction assessment tool was used for the 53 cattle herds. Farm risk scores were analyzed on the basis of deer damage to stored feed. RESULTS 27 (51%) farms reported deer damage to stored cattle feeds within the year previous to the farm visit. A strong association was found between increases in the percentage of land that could serve as deer cover and deer damage to stored feeds on a farm. The total risk score was significantly associated with the probability of a farm having deer damage. By use of a logistic regression model, the total risk score and proportion of nonagricultural land around a farm could be used to predict the likelihood of deer damage to stored feeds. CONCLUSIONS AND CLINICAL RELEVANCE Management practices on many farms in northwestern Minnesota allowed potential deer-cattle interactions. The on-farm risk assessment tool served as a valuable tool for prioritizing the biosecurity risks for farms. Continued development of biosecurity is needed to prevent potential transmission of bovine tuberculosis between deer and cattle, especially on farms that have a higher risk of deer damage.

Antibodies to Avian Influenza Viruses in Canada Geese (Branta canadensis): A Potential Surveillance Tool?

Traditionally, the epidemiology of avian influenza viruses (AIVs) in wild birds has been defined by detection of virus or viral RNA through virus isolation or reverse-transcription polymerase chain reaction. Our goals were to estimate AIV antibody prevalence in Canada geese (Branta canadensis) and measure effects of age and location on these estimates. We collected 3,205 samples from nine states during June and July 2008 and 2009: Georgia, Massachusetts, Minnesota, Mississippi, New Jersey, North Carolina, Pennsylvania, Washington, and West Virginia. Serum samples were tested for AIV antibodies with the use of a commercial blocking enzyme-linked immunosorbent assay. Overall, 483 (15%) Canada geese had detectable antibodies to AIV. Significantly higher prevalences were detected in geese collected from northeastern and upper midwestern states compared with southeastern states. This trend is consistent with results from virus isolation studies reporting AIV prevalence in North American dabbling ducks. Within Pennsylvania, significantly higher antibody prevalences were detected in goose flocks sampled in urban locations compared to flocks sampled in rural areas. Antibody prevalence was significantly higher in after-hatch-year geese compared to hatch-year geese. No significant differences in prevalence were detected from 10 locations sampled during both years. Results indicate that Canada geese are frequently exposed to AIVs and, with resident populations, may potentially be useful as sentinels to confirm regional AIV transmission within wild bird populations.

A genetic signature of the evolution of loss of flight in the Galapagos cormorant

Loss of flight in the Galapagos cormorant Although rare among existing birds, the loss of flight appears to have occurred multiple times in evolutionary history. However, the genetic changes that ground avian species are not well understood. Burga et al. sequenced genomes from three cormorant species and compared them with that of the flightless Galapagos cormorant (see the Perspective by Cooper). They identified variants in genes involved in primary ciliogenesis. Functional analyses of these variants suggest that the impaired function of the genes may be responsible for skeletal changes associated with the Galapagos cormorant’s loss of flight. Science, this issue p. eaal3345; see also p. 904 Genomic analysis points to CUX1 and cilia-related genes likely underlying a loss of flight in the Galapagos cormorant. INTRODUCTION Changes in the size and proportion of limbs and other structures have played a key role in the evolution of species. One common class of limb modification is recurrent wing reduction and loss of flight in birds. Indeed, Darwin used the occurrence of flightless birds as an argument in favor of his theory of natural selection. Loss of flight has evolved repeatedly and is found among 26 families of birds in 17 different orders. Despite the frequency of these modifications, we have a limited understanding of their underpinnings at the genetic and molecular levels. RATIONALE To better understand the evolution of changes in limb size, we studied a classic case of recent loss of flight in the Galapagos cormorant (Phalacrocorax harrisi). Cormorants are large water birds that live in coastal areas or near lakes, and P. harrisi is the only flightless cormorant among approximately 40 extant species. The entire population is distributed along the coastlines of Isabela and Fernandina islands in the Galapagos archipelago. P. harrisi has a pair of short wings, which are smaller than those of any other cormorant. The extreme reduction of the wings and pectoral skeleton observed in P. harrisi is an attractive model for studying the evolution of loss of flight because it occurred very recently; phylogenetic evidence suggests that P. harrisi diverged from its flighted relatives within the past 2 million years. We developed a comparative and predictive genomics approach that uses the genome sequences of P. harrisi and its flighted relatives to find candidate genetic variants that likely contributed to the evolution of loss of flight. RESULTS We sequenced and de novo assembled the whole genomes of P. harrisi and three closely related flighted cormorant species. We identified thousands of coding variants exclusive to P. harrisi and classified them according to their probability of altering protein function based on conservation. Variants most likely to alter protein function were significantly enriched in genes mutated in human skeletal ciliopathies, including Ofd1, Evc, Wdr34, and Ift122. We carried out experiments in Caenorhabditis elegans to confirm that a missense variant present in the Galapagos cormorant IFT122 protein is sufficient to affect ciliary function. The primary cilium is essential for Hedgehog (Hh) signaling in vertebrates, and individuals affected by ciliopathies have small limbs and ribcages, mirroring the phenotype of P. harrisi. We also identified a 4–amino acid deletion in the regulatory domain of Cux1, a highly conserved transcription factor that has been experimentally shown to regulate limb growth in chicken. The four missing amino acids are perfectly conserved in all birds and mammals sequenced to date. We tested the consequences of this deletion in a chondrogenic cell line and showed that it impairs the ability of CUX1 to transcriptionally up-regulate cilia-related genes (some of which contain function-altering variants in P. harrisi) and to promote chondrogenic differentiation. Finally, we show that positive selection may have played a role in the fixation of the variants associated with loss of flight in P. harrisi. CONCLUSION Our results indicate that the combined effect of variants in genes necessary for the correct transcriptional regulation and function of the primary cilium likely contributed to the evolution of highly reduced wings and other skeletal adaptations associated with loss of flight in P. harrisi. Our approach may be generally useful for identification of variants underlying evolutionary novelty from genomes of closely related species. Comparative and predictive genomics of loss of flight. Comparison of the genomes of four closely related cormorant species allowed us to predict function-altering variants exclusively affecting the Galapagos cormorant and to test their functional consequences. Our results implicate ciliary dysfunction as a likely contributor to the evolution of loss of flight. We have a limited understanding of the genetic and molecular basis of evolutionary changes in the size and proportion of limbs. We studied wing and pectoral skeleton reduction leading to flightlessness in the Galapagos cormorant (Phalacrocorax harrisi). We sequenced and de novo assembled the genomes of four cormorant species and applied a predictive and comparative genomics approach to find candidate variants that may have contributed to the evolution of flightlessness. These analyses and cross-species experiments in Caenorhabditis elegans and in chondrogenic cell lines implicated variants in genes necessary for transcriptional regulation and function of the primary cilium. Cilia are essential for Hedgehog signaling, and humans affected by skeletal ciliopathies suffer from premature bone growth arrest, mirroring skeletal features associated with loss of flight.

Surveillance for highly pathogenic H5 avian influenza virus in synanthropic wildlife associated with poultry farms during an acute outbreak

In November 2014, a Eurasian strain H5N8 highly pathogenic avian influenza virus was detected in poultry in Canada. Introduced viruses were soon detected in the United States and within six months had spread to 21 states with more than 48 million poultry affected. In an effort to study potential mechanisms of spread of the Eurasian H5 virus, the United States Department of Agriculture coordinated several epidemiologic investigations at poultry farms. As part of those efforts, we sampled synanthropic birds and mammals at five infected and five uninfected poultry farms in northwest Iowa for exposure to avian influenza viruses. Across all farms, we collected 2,627 samples from 648 individual birds and mammals. House mice were the most common mammal species captured while house sparrows, European starlings, rock pigeons, swallows, and American robins were the most commonly captured birds. A single European starling was positive for Eurasian H5 viral RNA and seropositive for antibodies reactive to the Eurasian H5 virus. Two American robins were also seropositive. No mammal species showed evidence of infection. These results indicate synanthropic species merit further scrutiny to better understand potential biosecurity risks. We propose a set of management practices aimed at reducing wildlife incursions.

Serologic evidence of various arboviruses detected in white-tailed deer (Odocoileus virginianus) in the United States

Abstract. White-tailed deer (Odocoileus virginianus) are an abundant mammal with a wide geographic distribution in the United States, which make them good sentinels for monitoring arboviral activity across the country. Exposure to various arboviruses has been detected in white-tailed deer, typically in conjunction with another diagnostic finding. To better assess the exposure of white-tailed deer to seven arboviruses, we tested 1,508 sera collected from 2010 to 2016 for antibodies to eastern equine encephalitis (2.5%), Powassan (4.2%), St. Louis encephalitis, (3.7%), West Nile (6.0%), Maguari (19.4%), La Crosse (30.3%), and bluetongue (7.8%) viruses. At least one arbovirus was detected in 51.3%, and exposure to more than one arbovirus was identified in 17.6% of the white-tailed deer sampled.

Surveillance for Highly Pathogenic Avian Influenza Virus in Wild Birds during Outbreaks in Domestic Poultry, Minnesota, 2015.

In 2015, a major outbreak of highly pathogenic avian influenza virus (HPAIV) infection devastated poultry facilities in Minnesota, USA. To understand the potential role of wild birds, we tested 3,139 waterfowl fecal samples and 104 sick and dead birds during March 9–June 4, 2015. HPAIV was isolated from a Cooper’s hawk but not from waterfowl fecal samples.

Antibody Detection and Molecular Characterization of Toxoplasma gondii from Bobcats (Lynx rufus), Domestic Cats (Felis catus), and Wildlife from Minnesota, USA

Little is known of the epidemiology of toxoplasmosis in Minnesota. Here, we evaluated Toxoplasma gondii infection in 50 wild bobcats (Lynx rufus) and 75 other animals on/near 10 cattle farms. Antibodies to T. gondii were assayed in serum samples or tissue fluids by the modified agglutination test (MAT, cut‐off 1:25). Twenty nine of 50 bobcats and 15 of 41 wildlife trapped on the vicinity of 10 farms and nine of 16 adult domestic cats (Felis catus) and six of 14 domestic dogs resident on farms were seropositive. Toxoplasma gondii oocysts were not found in feces of any felid. Tissues of all seropositive wild animals trapped on the farm were bioassayed in mice and viable T. gondii was isolated from two badgers (Taxidea taxus), two raccoons (Procyon lotor), one coyote (Canis latrans), and one opossum (Didelphis virginiana). All six T. gondii isolates were further propagated in cell culture. Multi‐locus PCR‐RFLP genotyping using 10 markers (SAG1, SAG2 (5′‐3′SAG2, and alt.SAG2), SAG3, BTUB, GRA6, c22‐8, c29‐2, L358, PK1, and Apico), and DNA from cell culture derived tachyzoites revealed three genotypes; #5 ToxoDataBase (1 coyote, 1 raccoon), #1 (1 badger, 1 raccoon, 1 opossum), and #2 (1 badger). This is the first report of T. gondii prevalence in domestic cats and in bobcats from Minnesota, and the first isolation of viable T. gondii from badger.

SPATIAL AND TEMPORAL PATTERNS OF AVIAN PARAMYXOVIRUS-1 OUTBREAKS IN DOUBLE-CRESTED CORMORANTS (PHALACROCORAX AURITUS) IN THE USA

Abstract Morbidity and mortality events caused by avian paramyxovirus-1 (APMV-1) in Double-crested Cormorant (DCCO; Phalacrocorax auritus) nesting colonies in the US and Canada have been sporadically documented in the literature. We describe APMV-1 associated outbreaks in DCCO in the US from the first reported occurrence in 1992 through 2012. The frequency of APMV-1 outbreaks has increased in the US over the last decade, but the majority of events have continued to occur in DCCO colonies in the Midwestern states. Although morbidity and mortality in conesting species has been frequently reported during DCCO APMV-1 outbreaks, our results suggest that isolation of APMV-1 is uncommon in species other than DCCO during APMV-1 outbreaks and that the cause of mortality in other species is associated with other pathogens. Populations of DCCO do not appear to have been significantly affected by this disease; however, because at least 65% of the APMV-1 outbreaks in DCCO in the US have involved APMV-1 strains classified as virulent to poultry (virulent Newcastle disease virus), its persistence and increased occurrence in DCCO warrants continued research and surveillance.

Surveillance for Highly Pathogenic Avian Influenza in Wild Turkeys (Meleagris gallopavo) of Minnesota, USA during 2015 Outbreaks in Domestic Poultry

Abstract An outbreak of a novel reassortant of highly pathogenic avian influenza A (H5N2) virus (HPAIV) decimated domestic turkeys (Meleagris gallopavo) from March through mid-June, 2015 in the state of Minnesota, US. In response, as part of broader surveillance efforts in wild birds, we designed a pilot effort to sample and test hunter-harvested Wild Turkeys (Meleagris gallopavo) for HPAIV in Minnesota counties with known infected poultry facilities. We also collected opportunistic samples from dead Wild Turkeys or live Wild Turkeys showing neurologic signs (morbidity and mortality samples) reported by the public or state agency personnel. Cloacal and tracheal samples were collected from each bird and screened for avian influenza virus (AIV) RNA by real-time reverse transcription PCR. From 15 April to 28 May 2015, we sampled 84 hunter-harvested male Wild Turkeys in 11 Minnesota counties. From 7 April 2015 through 11 April 2016, we sampled an additional 23 Wild Turkeys in 17 Minnesota counties. We did not detect type A influenza or HPAIV from any samples, and concluded, at the 95% confidence level, that apparent shedding prevalence in male Wild Turkeys in central Minnesota was between 0% and 2.9% over the sampling period. The susceptibility of wild turkeys to HPAIV is unclear, but regular harvest seasons make this wild gallinaceous bird readily available for future AIV testing.