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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.

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.

The recently emerged novel coronavirus, SARS-CoV-2, is phylogenetically related to bat coronaviruses (CoVs), specifically SARS-related CoVs from the Eurasian bat family Rhinolophidae. As this human pandemic virus has spread across the world, the potential impacts of SARS-CoV-2 on native North American bat populations are unknown, as is the ability of North American bats to serve as reservoirs or intermediate hosts able to transmit the virus to humans or to other animal species. To help determine the impacts of the pandemic virus on North American bat populations, we experimentally challenged big brown bats (Eptesicus fuscus) with SARS-CoV-2 under BSL-3 conditions. We inoculated the bats both oropharyngeally and nasally, and over the ensuing three weeks, we measured infectivity, pathology, virus concentrations in tissues, oral and rectal virus excretion, virus transmission, and clinical signs of disease. We found no evidence of SARS-CoV-2 infection in any examined bat, including no viral excretion, no transmission, no detectable virus in tissues, and no signs of disease or pathology. Based on our findings, it appears that big brown bats are resistant to infection with the SARS-CoV-2. The potential susceptibility of other North American bat species to SARS-CoV-2 remains to be investigated.

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.

West Nile virus (WNV) is maintained cryptically primarily in avian (passerine) populations, where it is transmitted by Culex spp. mosquitoes. Mosquito-control measures currently include physical activities to reduce mosquito-breeding sites and the application of mosquito larvicides or aerosolized insecticides to kill adults (adulticides) when arboviral diseases such as WNV or Zika virus are detected in mosquito populations. Organochlorine, organophosphorus, carbamate, and pyrethroid insecticides are often used. Previous work suggests an effect of pyrethroids on the immune system in a variety of vertebrates. We examined the effects of exposure to aerosolized Permanone® 30:30 insecticide (permethrin and piperonyl butoxide in soy oil vehicle) at approximately 103 to 106 times potential environmental concentrations on the response of captive zebra finches (Taeniopygia guttata) to experimental challenge with WNV. Compared to vehicle control birds, WNV outcome was unchanged (65% of birds produced a viremia) in the "low" exposure (9.52 ± 3.13 mg/m3 standard deviation [SD] permethrin) group but reduced in the "high" exposure (mean 376.5 ± 27.9 mg/m3 SD permethrin) group (30% were viremic; p < 0.05). After clearing WNV infection, birds treated with Permanone regained less body mass than vehicle-treated birds (p < 0.001). The present study suggests that exposure to aerosolized Permanone insecticide at levels exceeding typical application rates has the potential to not change or to mildly enhance a bird's resistance to WNV. Environ Toxicol Chem 2017;36:3376-3386. Published 2017 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.

West Nile virus (WNV) was first detected in New York in 1999 and, during its expansion across the continental US, southern Canada, and Mexico, members of the Corvidae (ravens, crows, magpies, and jays) were frequently infected and highly susceptible to the virus. As part of a behavioral study of Steller's Jays (Cyanocitta stelleri) conducted from 2011–14 in the coastal California counties of San Mateo and Santa Cruz, 380 Steller's Jays were captured and tested for antibodies to WNV. Using the wild bird immunoglobulin G enzyme linked immunoassay, we failed to detect antibodies to WNV, indicating either that there was no previous exposure to the virus or that exposed birds had died.

Serological tests play an important role in the detection of wildlife diseases. However, while there are many commercial assays and reagents available for domestic species, there is a need to develop efficient serological assays for wildlife. In recent years, marine mammals have represented a wildlife group with emerging infectious diseases, such as influenza, brucellosis, and leptospirosis. However, with the exception of disease-agent-specific assays or functional assays, few reports describe the use of antibody detection assays in marine mammals. In an indirect enzyme-linked immunoassay (EIA) or an immunofluorescence assay, antibody is detected using an antitarget species secondary conjugated antibody. The sensitivity of the assay depends on the avidity of the binding reaction between the bound antibody and the detection antibody. A commercial polyclonal antidog IgG conjugated antibody was tested in an EIA for its ability to sensitively detect the IgG of seven marine mammals including sea otter ( Enhydra lutris ), polar bear ( Ursus maritimus ), grey seal ( Halichoerus grypus ), harbor seal ( Phoca vitulina ), northern elephant seal ( Mirounga angustirostris ), California sea lion ( Zalophus californianus ), Pacific walrus ( Odobenus rosmarus ) and one freshwater mammal: Asian small-clawed otter ( Aonyx cinerea ). With the exception of Asian small-clawed sea otters, the detection of IgG in these marine mammals either exceeded or was nearly equal to detection of dog IgG. The use of the tested commercial antidog IgG antibody may be a valid approach to the detection of antibody response to disease in sea mammals.

Detection of West Nile virus (WNV) in ducks has been reported in North America in isolated cases of mortality in wild waterbirds and following outbreaks in farmed ducks. Although the virus has been noted as an apparent incidental finding in several species of ducks, little is known about the prevalence of exposure or the outcome of infection with WNV in wild ducks in North America. From 2004-06, we collected sera from 1,406 wild-caught American Wigeon ( Anas americana ), Mallard ( Anas platyrhynchos ), and Northern Pintail ( Anas acuta ) ducks at national wildlife refuges (NWRs) in North Dakota and Wood Ducks ( Aix sponsa ) at NWRs in South Carolina and Tennessee. We measured the prevalence of previous exposure to WNV in these ducks by measuring WNV antibodies and evaluated variation in exposure among species, age, and year. Additionally, we evaluated the performance of a commercial antibody to wild bird immunoglobulin in duck species that varied in their phylogenetic relatedness to the bird species the antibody was directed against. As determined by a screening immunoassay and a confirmatory plaque reduction neutralization assay, the prevalence of WNV antibody was 10%. In light of experimental studies that show ducks to be relatively resistant to mortality caused by WNV, the antibody prevalence we detected suggests that wild ducks may be less-frequently exposed to WNV than expected for birds inhabiting wetlands where they may acquire infection from mosquitoes.

Domesticated Australian and Timor zebra finches (Nota de investigación- Diferencias poblacionales en la susceptibilidad a Plasmodium relictum en diamantes cebra Taeniopygia guttata. Se inocularon diamantes cebra de Australia y de Timor (Taeniopygia guttata castanotis y Taeniopygia guttata guttata, respectivamente) con sangre de canario silvestre (Serinus canaria) que contenía un aislado hawaiano de Plasmodium relictum (linaje GRW04), que es un hemoparásito que causa la malaria aviar. En dos ensayos experimentales, los diamantes cebra de Timor desarrollaron una parasitemia detectada mediante un examen microscópico de frotis de sangre, pero los diamantes cebra australianos no desarrollaron dicha parasitemia. En el segundo ensayo, en el que se utilizaron métodos de detección molecular, un solo pinzón australiano y cinco de las seis aves de Timor desafiadas resultaron positivas para el parásito. Además, se detectó el ADN de P. relictum en múltiples muestras de sangre obtenidas de las aves de Timor durante 28 días posteriores al desafío. Los diamantes cebra de Timor pueden proporcionar un modelo de laboratorio útil y de fácil mantenimiento para el estudio de las interacciones de Plasmodium en passeriformes, pero áun son inferiores en comparación con los canarios, que son utilizados como modelo de infección por malaria aviar tradicionalmente usado en términos de apoyo a las infecciones con alta parasitemia.

West Nile virus (WNV) was first detected in North America in 1999. Alaska and Hawaii (HI) remain the only U.S. states in which transmission of WNV has not been detected. Dead bird surveillance has played an important role in the detection of the virus geographically, as well as temporally. In North America, corvids have played a major role in WNV surveillance; however, the only corvid in HI is the endangered Hawaiian crow that exists only in captivity, thus precluding the use of this species for WNV surveillance in HI. To evaluate the suitability of alternate avian species for WNV surveillance, we experimentally challenged seven abundant non-native bird species present in HI with WNV and compared mortality, viremia, oral shedding of virus, and seroconversion. For detection of WNV in oral swabs, we compared viral culture, reverse-transcriptase polymerase chain reaction, and the RAMP(®) test. For detection of antibodies to WNV, we compared an indirect and a competitive enzyme-linked immunoassay. We found four species (house sparrow, house finch, Japanese white-eye, and Java sparrow) that may be useful in dead bird surveillance for WNV; while common myna, zebra dove, and spotted dove survived infection and may be useful in serosurveillance.

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.

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.

Greater sage-grouse(Centrocercusurophasianus;sage-grouse) arehighlysusceptible toinfectionwith West Nile virus (WNV), with substantial mortality reported in wild populations and in experimentally infected birds. Although sage-grouse are hunted throughout much of their range, they have also recently been considered for protection under the Endangered Species Act. We used blood samples collected on filter-paper strips during the 2006-2010 Oregon, USA, annual sage-grouse hunt to survey for specific WNV-neutralizing antibodies that indicate a previous infection with WNV. During this period, hunters submitted 1,880 blood samples from sage- grouse they harvested. Samples obtained were proport ional for all 12 Oregon sage-grouse hunting units. Laboratory testing of 1,839 samples by the WNV epitope-blocking enzyme-linked immunosorbent assay (bELISA) followed by plaque reduction neutralization test on bELISA-positive samples yielded 19 (1%) and 1 (0.05%) positive samples, respectively. These data provided early baseline information for future comparisons regarding the prevalence of WNV-specific neutralizing antibodies in sage-grouse in Oregon. This methodology mayprovideotherstateswheresage-grouse(orotherspecies)populationsarehuntedandwhereWNVconstitutes a species conservation concern with a viable option to track the relative prevalence of the virus in populations. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

The sagebrush biome in the western United States is home to the imperiled greater sage-grouse ( Centrocercus urophasianus ) and encompasses rangelands used for cattle production. Cattle grazing activities have been implicated in the range-wide decline of the sage-grouse, but no studies have investigated the relationship between the physiological condition of sage-grouse and the presence of grazing cattle. We sampled 329 sage-grouse across four sites (two grazed and two ungrazed) encompassing 13 600 km 2 during the spring and late summer–early autumn of 2005 to evaluate whether demographic factors, breeding status, plasma protein levels, and residence in a cattle-grazed habitat were associated with the stress hormone corticosterone. Corticosterone was measured in feces as immunoreactive corticosterone metabolites (ICM). Males captured during the lekking season exhibited higher ICM levels than all others. Prenesting female sage-grouse captured in a grazed site had higher ICM levels than those in ungrazed sites and prenesting female plasma protein levels were negatively correlated with ICM concentrations. With the use of a small-scale spatial model, we identified a positive correlation between cattle pat count and sage-grouse ICM levels. Our model indicated that ICM levels increased by 2.60 ng · g -1 dry feces for every increase in the number of cow pats found in the vicinity. Management practices will benefit from future research regarding the consistency and mechanism(s) responsible for this association and, importantly, how ICM levels and demographic rates are related in this species of conservation concern.

West Nile virus (WNV) causes significant mortality of American White Pelican chicks at northern plains colonies. We tested oropharyngeal/cloacal swabs from moribund chicks for shed WNV. Such shedding could enable chick-to-chick transmission and help explain why WNV spreads rapidly in colonies. WNV was detected on swabs from 11% of chicks in 2006 and 52% of chicks in 2007; however, viral titers were low. Before onset of WNV mortality, we tested blood from < 3-week-old chicks for antibodies to WNV; 5% of chicks were seropositive, suggesting passive transfer of maternal antibodies. Among near-fledged chicks, 41% tested positive for anti-WNV antibodies, indicating that they survived infection. Among years and colonies, cumulative incidence of WNV in chicks varied from 28% to 81%, whereas the proportion of chicks surviving WNV (i.e., seropositive) was 64–75%. Our data revealed that WNV kills chicks that likely would fledge in the absence of WNV, that infection of chicks is pervasive, and that significant numbers of chicks survive infection.

West Nile virus (WNV) is maintained cryptically primarily in avian (passerine) populations, where it is transmitted by Culex spp. mosquitoes. Mosquito-control measures currently include physical activities to reduce mosquito-breeding sites and the application of mosquito larvicides or aerosolized insecticides to kill adults (adulticides) when arboviral diseases such as WNV or Zika virus are detected in mosquito populations. Organochlorine, organophosphorus, carbamate, and pyrethroid insecticides are often used. Previous work suggests an effect of pyrethroids on the immune system in a variety of vertebrates. We examined the effects of exposure to aerosolized Permanone® 30:30 insecticide (permethrin and piperonyl butoxide in soy oil vehicle) at approximately 10

Immunological reagents for wild, non-model species are limited or often non-existent for many species.In this study, we compare the reactivity of a new anti-passerine IgY secondary antibody with existing secondary antibodies developed for use with birds. Samples from 41 species from the following six avian orders were analysed: Anseriformes (1 family, 1 species), Columbiformes (1 family, 2 species), Galliformes (1 family, 1 species), Passeriformes (16 families, 34 species), Piciformes (1 family, 2 species) and Suliformes (1 family, 1 species). Direct ELISAs were performed to detect total IgY using goat anti-passerine IgY, goat anti-chicken IgY or goat anti-bird IgY secondary antibodies.The anti-passerine antibody exhibited significantly higher IgY reactivity compared to the anti-chicken and/or anti-bird antibodies in 80% of the passerine families tested. Birds in the order Piciformes (woodpeckers) and order Suliformes (cormorants) were poorly detected by all three secondary antibodies. A comparison of serum and plasma IgY levels was made within the same individuals for two passerine species (house finch and white-crowned sparrow), and serum exhibited significantly more IgY than the plasma for all three secondary antibodies. This result indicates that serum may be preferred to plasma when measuring total antibody levels in blood.This study indicates that the anti-passerine IgY secondary antibody can effectively be used in immunological assays to detect passerine IgY for species in most passerine families and is preferred over anti-chicken and anti-bird secondary antibodies for the majority of passerine species. This anti-passerine antibody will allow for more accurate detection and quantification of IgY in more wild bird species than was possible with previously available secondary antibodies.

West Nile virus (WNV) spread to the US western plains states in 2003, when a significant mortality event attributed to WNV occurred in Greater Sage-grouse ( Centrocercus urophasianus ). The role of avian species inhabiting sagebrush in the amplification of WNV in arid and semiarid regions of the North America is unknown. We conducted an experimental WNV challenge study in Vesper Sparrows ( Pooecetes gramineus ), a species common to sagebrush and grassland habitats found throughout much of North America. We found Vesper Sparrows to be moderately susceptible to WNV, developing viremia considered sufficient to transmit WNV to feeding mosquitoes, but the majority of birds were capable of surviving infection and developing a humoral immune response to the WNV nonstructural 1 and envelope proteins. Despite clearance of viremia, after 6 mo, WNV was detected molecularly in three birds and cultured from one bird. Surviving Vesper Sparrows were resistant to reinfection 6 mo after the initial challenge. Vesper sparrows could play a role in the amplification of WNV in sagebrush habitat and other areas of their range, but rapid clearance of WNV may limit their importance as competent amplification hosts of WNV.

Serum samples from a total of 71 healthy captive birds belonging to 18 species were collected in July of 2008 in Medellin (Colombia) and tested for flaviviruses. Eighteen of 29 samples from American Flamingoes (Phoenicopterus ruber) were positive for West Nile virus (WNV) by reverse transcription-polymerase chain reaction. Selected positive samples were serially passaged and WNV was confirmed by immunofluorescence. Two isolates (524/08, 9835/08) were characterized in vitro and in vivo. Sequence analysis revealed WNV with 16 nucleotide substitutions resulting in six amino acid changes when compared with the NY99 strain. Colombian (COL) viruses were more closely related to Louisiana isolates from 2001. When compared with attenuated strains isolated from Texas, COL isolates differed in their plaque size and temperature sensitivity phenotype. The COL viruses were pathogenic in embryonated chicken eggs and Balb/c mice.

West Nile virus, a mosquito-vectored flavivirus of the Japanese encephalitis serogroup, was first detected in North America following an epizootic in the New York City area in 1999. In the intervening 11 years since the arrival of the virus in North America, it has crossed the contiguous USA, entered the Canadian provinces bordering the USA, and has been reported in the Caribbean islands, Mexico, Central America and, more recently, South America. West Nile virus has been reported in over 300 species of birds in the USA and has caused the deaths of thousands of birds, local population declines of some avian species, the clinical illness and deaths of thousands of domestic horses, and the clinical disease in over 30 000 Americans and the deaths of over 1000. Prior to the emergence of West Nile virus in North America, St. Louis encephalitis virus and Dengue virus were the only other known mosquito-transmitted flaviviruses in North America capable of causing human disease. This review will discuss the North American experience with mosquito-borne flavivirus prior to the arrival of West Nile virus, the entry and spread of West Nile virus in North America, effects on wild bird populations, genetic changes in the virus, and the current state of West Nile virus transmission.

Little is known about West Nile virus (WNV) in non-domestic (i.e., wild or feral) horses ( Equus caballus ) beyond serologic surveys that have reported WNV antibody prevalence from < 1% to 63% of animals tested. 1– 4 The Bureau of Land Management currently manages an estimated 33,700 wild horses in 10 of the western United States. 5 As part of the management of these herds, non-domestic horses are routinely rounded up or “gathered” by natural resource management agencies to reduce populations. We report five non-consecutive years of study to determine seroprevalence against WNV in feral horses on Sheldon National Wildlife Refuge in Nevada, United States, by using serum samples obtained during horse gathers.

We investigated the prevalence of West Nile virus (WNV)-neutralizing antibodies and infectious virus, and the occurrence of overwinter transmission in two raptor species during January and March 2006 at the Salton Sea, Imperial County, California. We captured 208 American Kestrels (Falco sparverius) (January, n=100; March, n=108) and 116 Burrowing Owls (Athene cunicularia) (January, n=52; March, n=64). Laboratory analysis revealed that 83% of American Kestrels and 31% of Burrowing Owls were positive for WNV-neutralizing antibodies. Additionally, two seroconversions were detected in Burrowing Owls between January and March. Infectious WNV, consistent with acute infection, was not detected in any bird.

Public health agencies utilize aerial insecticides to interrupt an active West Nile virus (WNV) transmission cycle, which may expose WNV-infected birds to these agents. Although resmethrin has been considered benign to birds, no studies have evaluated whether the environmentally employed form of resmethrin with PBO synergist (synergized resmethrin (SR)) can suppress avian immunity to WNV infection and enhance a bird's host competence. Recognizing that wild birds confront toxicological stressors in the context of various physiological states, we exposed four groups ( n = 9–11) of 9-week-old chickens ( Gallus domesticus ) to drinking water with either SR (three alternate days at 50 μg/l resmethrin + 150 μg/l piperonyl butoxide), CORT (10 days at 20 mg/l to induce subacute stress), the combination of SR and CORT, or 0.10% ethanol vehicle coincident with WNV infection. Compared to controls, SR treatment did not magnify but extended viremia by 1 day, and depressed IgG; CORT treatment elevated (mean, 4.26 log 10 PFU/ml) and extended viremia by 2 days, enhanced IgM and IgG, and increased oral virus. The combination of SR and CORT increased the number of chickens that shed oral virus compared to those treated with CORT alone. None of the chickens developed a readily infectious viremia to mosquitoes (none ≥5 log 10 PFU/ml), but viremia in a CORT-exposed chicken was up to 4.95 log 10 PFU/ml. Given that SR is utilized during WNV outbreaks, continued work toward a complete risk assessment of the potential immunotoxic effects of SR is warranted. This would include parameterization of SR exposures with immunological consequences in wild birds using both replicating (in the laboratory) and non-replicating (in the field) antigens. As a start, this study indicates that SR can alter some immunological parameters, but with limited consequences to primary WNV infection outcome, and that elevated CORT mildly enhances SRs immunotoxicity in chickens.

SUMMARY. West Nile virus (WNV) causes morbidity and mortality in humans, horses, and in more than 315 bird species in North America. Currently approved WNV vaccines are designed for parenteral administration and, as yet, no effective oral WNV vaccines have been developed. WNV envelope (E) protein is a highly antigenic protein that elicits the majority of virus-neutralizing antibodies during a WNV immune response. Leghorn chickens were given three vaccinations (each 2 wk apart) of E protein orally (20 m go r 100mg/dose), of E protein intramuscularly (IM, 20 mg/dose), or of adjuvant only (control group) followed by a WNV challenge. Viremias were measured post-WNV infection, and three new enzyme-linked immunosorbent assays were developed for quantifying IgM, IgY, and IgA-mediated immune response of birds following WNV infection. WNV viremia levels were significantly lower in the IM group than in both oral groups and the control group. Total WNV E protein-specific IgY production was significantly greater, and WNV nonstructural 1-specific IgY was significantly less, in the IM group compared to all other treatment groups. The results of this study indicate that IM vaccination of chickens with E protein is protective against WNV infection and results in a significantly different antibody production profile as compared to both orally vaccinated and nonvaccinated birds.

Introduction of the West Nile virus (WNV) to Hawai’i will undoubtedly devastate many populations of critically endangered avian species indigenous to Hawai’i. The protective efficacy of a protein-based WNV subunit vaccine formulated with adjuvant was evaluated in domestic geese as a surrogate species for the endangered Nēnē, the state bird of Hawai’i. Prevention of viremia following viral infection of vaccinated birds was used as the clinical endpoint of protection. ELISA and plaque reduction neutralization tests demonstrate that significant levels of vaccine antigen-specific antibody were produced in groups of birds vaccinated with 5 or 10 μg of the WN-80E antigen formulated with ISA720 adjuvant. Moreover, after challenge with WNV, no viremia was detected in vaccinated birds, whereas viremia was detected up to 4 days after and virus was detected by oral swab for 6 days after infection among control groups. Safe and effective vaccination of managed or captive endangered bird populations will protect species with critically low numbers that could not survive the added mortality of introduced disease.

American white pelicans (Pelecanus erythrorhynchos) are colonial-nesting birds and their breeding sites are concentrated in a few small areas, making this species especially vulner- able to factors that can influence productivity, such as disease, disturbance, predation, weather events and loss of nesting habitat. Nearly half of the American white pelican pop- ulation breeds at four colonies in the northern plains: Chase Lake National Wildlife Refuge (NWR) in North Dakota, Bitter Lake (Waubay NWR) in South Dakota, Medicine Lake NWR in Montana, and Marsh Lake in Minnesota. Thus, sustained productivity at these colonies is crucial to the health of the entire species. During the latter half of the 2002 and 2003 breed- ing seasons, unusually high mortality of pelican chicks was observed at these colonies. West Nile virus (WNv) was identified as one source of these losses. In 2004-2007 we mon- itored three major colonies in the northern plains to assess mortality of chicks during the late breeding season. We documented severe weather events, disturbance, and WNv as fac- tors contributing to chick mortality. Before WNv arrived in the region in 2002, chick mortal- ity after mid-July was 64%, and then jumped to as high as 44% in the years since WNv arrived. WNv kills older chicks that are no longer vulnerable to other common mortality factors (e.g., severe weather, gull predation) and typically would have survived to fledge; thus WNv appears to be an additive mortality factor. Persistence of lower productivity at American white pelican colonies in the northern plains might reduce the adult breeding population of this species in the region.

Background: Argon plasma coagulation has been rapidly accepted for endoscopic obliteration of vascular lesions and superficial tumors. Depth of injury is thought to be limited through preferential discharge of energy to nondesiccated tissue. However, argon plasma coagulation-induced injury has not been well characterized. The aim of this study was to characterize argon plasma coagulation-induced colonic injury by using a porcine model. Methods: Laparotomy was performed in 6 female swine and the colon exteriorized with the subjects under general anesthesia. Lesions were made with an argon plasma coagulation probe held perpendicular and 2 mm from the mucosa. Variables studied were as follows: power (45 W, 60 W, and 75 W) and duration (1, 2, or 3 seconds; n=11 for each power/duration combination). Injury was graded as either superficial or deep, involving the muscularis propria. Results: Circular muscle layer injury correlated closely with power ( p = 0.02), duration ( p = 0.001), and total energy delivered ( r = 0.977). Longitudinal muscle damage was associated with duration of burn ( p = 0.001) and total energy delivered ( r = 0.855), but correlated poorly with power ( p = 0.40). No perforations occurred. Submucosal injection of saline solution had a protective effect with reductions in circular (90% to 10%, p = 0.002) and longitudinal muscle injury (50% to 0%, p = 0.1). Conclusions: Injury to the muscularis propria occurs at recommended settings for argon plasma coagulation. Injury correlates with power setting, duration of burn, and total energy delivery. Protective arcing to nondesiccated tissue does not appear to be significant in vivo. Submucosal injection of saline solution protects against deep injury. (Gastrointest Endosc 2002;55:631-6.)

Globally, Avipoxvirus species affect over 230 species of wild birds and can significantly impair survival. During banding of nine grassland songbird species (n=346 individuals) in southwestern Wisconsin, USA, we noted species with a 2-6% prevalence of pox-like lesions (possible evidence of current infection) and 4-10% missing digits (potential evidence of past infection). These prevalences approach those recorded among island endemic birds (4-9% and 9-20% for the Galapagos and Hawaii, respectively) for which Avipoxvirus species have been implicated as contributing to dramatic population declines. Henslow's Sparrow Ammodramus henslowii (n=165 individuals) had the highest prevalence of lesions (6.1%) and missing digits (9.7%). Among a subset of 26 Henslow's Sparrows from which blood samples were obtained, none had detectable antibody reactive to fowlpox virus antigen. However, four samples (18%) had antibody to canarypox virus antigen with test sample and negative control ratios (P/N values) ranging from 2.4 to 6.5 (median 4.3). Of four antibody-positive birds, two had lesions recorded (one was also missing a digit), one had digits missing, and one had no signs. Additionally, the birds with lesions or missing digits had higher P/N values than did the antibody-positive bird without missing digits or recorded lesions. This study represents an impetus for considering the impacts and dynamics of disease caused by Avipoxvirus among North American grassland bird species.

Infection of different strains of laboratory mice with the agent of Lyme disease, Borrelia burgdorferi, results in arthritis, the severity of which has been correlated with the dominance of Th1 cytokines. In this study, we demonstrate that changes in B. burgdorferi-specific immunologic responses associated with pregnancy can alter the outcome of Lyme arthritis in mice. Whereas nonpregnant female C3H mice consistently developed severe Lyme arthritis, pregnant mice had a marked reduction in arthritis severity that was associated with a slight reduction in IFN-γ and markedly increased levels of IL-4 production by B. burgdorferi-specific T cells. Similar reductions in arthritis severity and patterns of cytokine production were observed in nonpregnant, progesterone-implanted mice. Ab neutralization of IL-4 in progesterone-implanted mice resulted in severe arthritis. Our results are consistent with the known shift toward Th2 cytokine expression at the maternal-fetal interface, and are the first to show a pregnancy-related therapeutic effect in an infectious model.

We determined if the ear biopsy location affected detection of Borrelia burgdorferi when either culture or the polymerase chain reaction (PCR) was used among 50 white-footed mice (Peromyscus leucopus), live-captured in a Lyme disease enzootic area in Maryland (USA) between March and October of 1991 and 1992. The infection status of individual mice was determined by organ culture; ear biopsy samples were obtained from the peripheral and central part of the ear for detection of B. burgdorferi by culture and by PCR. Overall, B. burgdorferi was cultured from one or more tissue samples in 33 (66%) of 50 captured mice. Among infected mice, B. burgdorferi was detected by culture in 29 (88%) of 33 peripheral and 28 (85%) of 33 central ear biopsy samples. By PCR it was detected in 24 (73%) of 33 peripheral and all 33 central samples (P = 0.002). Detection of B. burgdorferi by culture was independent of the ear biopsy location; however, the organism was detected by PCR with greater frequency in central ear biopsy samples as compared to peripheral samples. Agreement between culture and PCR was moderate (Kappa = 0.64) on peripheral ear samples and excellent (Kappa = 0.79) on central samples. We propose that when ear biopsy samples are used to detect B. burgdorferi by PCR in wild-caught P. leucopus, removal of biopsy samples from the central part of the ear will achieve maximum sensitivity and will achieve the highest concordance between assays when both culture and PCR of ear biopsy samples are conducted in parallel.

We determined the antibody prevalence to Ehrlichia spp., in white-tailed deer (Odocoileus virginianus) and the geographic distribution of seropositive animals in 84 counties in Alabama, Arkansas, Florida, Georgia, Illinois, Kentucky, Louisiana, Maryland, Massachusetts, Mississippi, Missouri, North Carolina, South Carolina, Tennessee, Texas, Virginia, and West Virginia (USA). Using an indirect fluorescent antibody test we detected antibodies (or = 1:128) to this bacterium in 544 (43%) of 1269 deer. Presence of antibodies to Ehrlichia spp. was related to a southerly latitude, low elevation, and resulting milder climatic conditions. It appears that white-tailed deer were naturally infected with Ehrlichia spp.; the infection was widely distributed throughout the southeastern United States. Based on these data, we propose that white-tailed deer play a role in the natural history of Ehrlichia spp. infection in the United States.

Five southern red-backed voles (Myodes gapperi) of the first generation of a wild-caught breeding colony were presented with lesions at the maxillary incisors consistent with elodontoma. The affected animals had a history of chronic weight loss, were >16 months of age, and were siblings. Radiographs of the head showed multiglobular to irregularly outlined mineral opacity masses at the apices of the maxillary incisors. On necropsy, maxillary incisor teeth were not grossly visible, and a gingival ulceration was observed at the expected site of eruption. Microscopically, the apical region of the maxillary incisors was thickened or replaced by irregular dental tissue masses consistent with elodontoma. This is the first report to describe elodontoma in red-backed voles.

Culture and the polymerase chain reaction (PCR) were compared for detection of Borrelia burgdorferi infection in wild-caught Peromyscus leucopus and experimentally inoculated C.B-17 scid/scid (severe combined immunodeficient) mice. PCR targeted highly conserved regions of the ospA gene and could detect one to five cultured organisms and 10 to 50 copies of molecularly cloned ospA DNA. Organs (kidney, spleen, and urinary bladder) and/or ear biopsy samples were obtained from 108 captured P. leucopus mice, and tissues were obtained from 7 experimentally inoculated mice. A simple sample-processing procedure with proteinase K and detergent treatment was used in the PCR analysis. Overall, B. burgdorferi was detected in 29 of 108 (27%) P. leucopus mice by culture and in 31 of 108 (29%) mice by PCR. As assessed by the kappa statistic, agreement between PCR and culture was high for ear and bladder (kappa = 0.80 and 0.65, respectively) and low for kidney and spleen (kappa = 0.37 and 0.03, respectively). While concordant results were obtained from 98 animals, PCR detected B. burgdorferi from 6 additional mice for which cultures were negative and culture detected B. burgdorferi from 4 animals which were PCR negative. Further phenol-chloroform extraction of DNA in a limited number of samples improved the sensitivity of PCR compared with that of culture. These results indicate that PCR may be as sensitive as culture for detecting B. burgdorferi in ear samples and that PCR analysis is suitable for establishing the infection status of animals in mark-release-recapture studies.

Introduced mosquito-borne avian disease is a major limiting factor in the recovery and restoration of native Hawaiian forest birds. Annual epizootics of avian pox (Avipoxvirus) and avian malaria (Plasmodium relictum) likely led to the extinction of some species and continue to impact populations of susceptible Hawaiian honeycreepers (Drepanidinae). The introduction of a novel pathogen, such as West Nile virus (WNV), could result in further population declines and extinctions. During September and October 2004, we infected Hawai'i' Amakihi (Hemignathus virens) with a North American isolate of WNV by needle inoculation and mosquito bite to observe susceptibility, mortality, and illness in this endemic passerine, and to determine the vector competence of the co-occurring, introduced mosquito Culex quinquefasciatus. All experimentally infected Hawai'i ;Amakihi became viremic, with a mean titer10(5) plaque-forming units (PFU)/ml, and they experienced clinical signs ranging from anorexia and lethargy to ataxia. The fatality rate among needle-inoculated Hawai'i' Amakihi (n=16) was 31.3%, but mortality in free-ranging birds is likely to increase due to predation, starvation, thermal stress, and concomitant infections of avian malaria and pox. Surviving Hawai'i' Amakihi seem to clear WNV from the peripheral blood by 7-10 days postinfection (DPI), and neutralizing antibodies were detected from 9 to 46 DPI. In transmission trials, Hawaiian Cx. quinquefasciatus proved to be a competent vector and Hawai'i Amakihi an adequate amplification host of WNV, suggesting that epizootic WNV could readily become an additional limiting factor of some native Hawaiian bird populations.

Investigators of wildlife populations often utilize demographic indicators to understand the relationship between habitat characteristics and population viability. Assessments of corticosterone may enable earlier detection of populations at risk of decline because physiological adjustments to habitat disturbance occur before reproductive diminutions. Noninvasive methods to accomplish these assessments are important in species of concern, such as the greater sage grouse (GRSG). Therefore, we validated a radioimmunoassay that measures immunoreactive corticosterone metabolites (ICM) in fecal samples and used it to characterize the adrenocortical response of 15 GRSG exposed to capture, intravenous injection of 50 IU/kg adrenocorticotrophic hormone (ACTH) or saline, and 22 h of confinement. Those animals injected with ACTH exhibited a more sustained (P = 0.0139) and less variable (P = 0.0012) response than those injected with saline, indicating different levels of adrenocortical activity. We also found that potential field-collection protocols of fecal samples did not alter ICM concentrations: samples held at 4 degrees C for up to 16 h contained similar levels of ICM as those frozen (-20 degrees C) immediately. This study demonstrates a multiphasic adrenocortical response that varied with the level of stimulation and indicates that the assay used to measure this phenomenon is applicable for studies of wild GRSG.

Background: Submucosal saline solution injection may limit the depth of thermal injury to the gut wall by acting as a heat-sink and by increasing the distance between burn and serosa. The aim of this study was to determine the effect of submucosal saline solution injection on depth of colonic thermal injury produced by commonly used endoscopic thermal modalities. Methods: Longitudinal colotomy incisions were made on the antimesenteric colonic border of anesthetized swine. Lesions were made by using a bipolar device (20 W, 2 seconds), heat probe (30 J); monopolar contact with hot biopsy forceps (20 W, 2 seconds), and monopolar noncontact with argon plasma coagulation (45 W, 3 seconds). Ten or more lesions were created with each device. Lesions were made with or without prior submucosal injection of 2 mL of normal saline solution. After 24 hours the lesions were excised for histologic analysis. Injury was assessed in relation to the severity of damage to the deep (longitudinal) muscle layer. Results: The proportions of control lesions (without submucosal saline solution injection) in which deep injury was evident were as follows: argon plasma coagulation, 86%; hot biopsy forceps, 64%; heat probe, 50%; bipolar device, 18%. Submucosal saline solution injection significantly reduced the proportions of lesions with deep injury for argon plasma coagulation (p = 0.009) and heat probe (p = 0.03), but not hot biopsy forceps or bipolar device (argon plasma coagulation, 86% to 21%; heat probe, 50% to 0%; hot biopsy forceps, 64% to 50%; bipolar device, 18% to 9%). Conclusions: At equivalent energy outputs, the bipolar device results in less deep injury than the monopolar or heat probe. Submucosal saline solution injection reduced injury to the muscularis propria caused by both heat probe and argon plasma coagulation, but not hot biopsy forceps. Despite submucosal saline solution injection, caution should be exercised when using prolonged monopolar cautery. (Gastrointest Endosc 2002;56:95-9.)

Background: The current understanding of the inflammation associated with Lyme disease directly involves infection caused by Borrelia burgdorferi within specific target tissues, accompanied by a significant host immunologic component driving the inflammatory process. The measurement of spirochetal tissue burden may thus be useful for studying animal models of Lyme disease pathogenesis. Widely available methods based on the culture of spirochetes from tissues do not provide quantitative information. Methods: We developed and evaluated a quantitative-competitive polymerase chain reaction assay based on amplification of the B. burgdorferi flagellin gene. The assay makes use of a competitive internal standard and a commercially available enzyme-linked immunosorbent assay detection kit. Results and Conclusion: The assay clearly discriminated between infected and uninfected mouse tissues, and an accurate quantitation range of 500 to 20,000 spirochetes per milligram of tissue was obtained. C3H mice were shown to harbor greater amounts of spirochetal genomic DNA than BALB/c mice. Normalization of samples by tissue weight and genomic DNA content both provided acceptable results. These data indicate this assay can be used to provide reliable and meaningful measurements of spirochetal infectious burden, which will be extremely useful for the study of Lyme disease pathogenesis in the murine model.

The maintenance of Borrelia burgdorferi in a population of Peromyscus leucopus was investigated from 202 mark and recapture mice and 61 mice that were removed from a site in Baltimore County, Maryland. Borrelia burgdorferi infection was detected by culture and polymerase chain reaction (PCR) of ear tissue, and exposure to the spirochete was quantified by serology. Overall prevalence of B. burgdorferi, as determined by culture and PCR of ear tissue at first capture, was 25% in the longitudinal sample and 42% in the cross-sectional sample. Significantly more juvenile mice were captured in the longitudinal sample (18%) than in the cross-sectional sample (0%). Among 36 captured juvenile mice, only one was infected with B. burgdorferi; this contributed to a significant trend for infection with B. burgdorferi with age. Recovery from infection with B. burgdorferi was not detected among 77 mice followed for an average of 160 days. The incidence rate of infection with B. burgdorferi was 10 times greater in mice captured during two periods of high risk of exposure to nymphal Ixodes scapularis ticks compared with a period of low risk. Maintenance of B. burgdorferi in this population was dependent on indirect transmission of the organism from infected ticks to susceptible mice and development of chronic infection with the spirochete, which had no measurable effect on the survival of infected mice.

Thirty isolates of Borrelia burgdorferi sequentially cultured from 15 naturally infected white-footed mice (Peromyscus leucopus) were examined for variability in protein and plasmid profiles. Heterogeneity was detected in OspB and OspC and in proteins between 18.0 and 28.0 kDa by PAGE. Plasmid profiles were heterogeneous in the first isolate from 11 mice (73%) and between the first and last sequential isolate from 13 mice (87%). Comparison of the first and last isolates showed increased expression of OspC in 6 mice (40%) and was associated in each case with a shift in mobility of a 16.0-kb plasmid, suggesting that regulatory elements of ospC may reside on this plasmid. Hybridization studies suggested that individual mice may have been infected by a heterogeneous population of spirochetes and that changes in the protein and plasmid profiles between the first and last sequential isolates from some mice may have been the result of clonal selection.