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The Effect of Swab Sample Choice on the Detection of Avian Influenza inApparently Healthy Wild DucksAuthor(s): Hon S. Ip, Robert J. Dusek and Dennis M. HeiseySource: Avian Diseases, 56(1):114-119. 2012.Published By: American Association of Avian PathologistsDOI: http://dx.doi.org/10.1637/9832-061311-Reg.1URL: http://www.bioone.org/doi/full/10.1637/9832-061311-Reg.1

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The Effect of Swab Sample Choice on the Detection of Avian Influenza in ApparentlyHealthy Wild Ducks

Hon S. Ip,A* Robert J. Dusek,* and Dennis M. Heisey*

AU.S. Geological Survey, National Wildlife Health Center, 6006 Schroeder Road, Madison, WI 53711

Received 16 June 2011; Accepted and published ahead of print 6 October 2011

SUMMARY. Historically, avian influenza viruses have been isolated from cloacal swab specimens, but recent data suggest thatthe highly pathogenic avian influenza (HPAI) H5N1 virus can be better detected from respiratory tract specimens. To betterunderstand how swab sample type affects the detection ability of low pathogenic avian influenza (LPAI) viruses we collected andtested four swab types: oropharyngeal swabs (OS), cloacal swabs (CS), the two swab types combined in the laboratory (LCS), andthe two swab types combined in the field (FCS). A total of 1968 wild waterfowl were sampled by each of these four methods andtested for avian influenza virus using matrix gene reverse-transcription (RT)-PCR. The highest detection rate occurred with theFCS (4.3%) followed by the CS (4.0%). Although this difference did not achieve traditional statistical significance, Bayesiananalysis indicated that FCS was superior to CS with an 82% probability. The detection rates for both the LCS (2.4%) and the OS(0.4%) were significantly different from the FCS. In addition, every swab type that was matrix RT-PCR positive was also tested forrecovery of viable influenza virus. This protocol reduced the detection rate, but the ordering of swab types remained the same:1.73% FCS, 1.42% CS, 0.81% LCS, and 0% OS. Our data suggest that the FCS performed at least as well as any other swab typefor detecting LPAI viruses in the wild ducks tested. When considering recent studies showing that HPAI H5N1 can be betterdetected in the respiratory tract, the FCS is the most appropriate sample to collect for HPAI H5N1 surveillance while notcompromising LPAI studies.

RESUMEN. Efecto de la muestra recolectadas con hisopos para la deteccion de la influenza aviar en patos silvestresaparentemente sanos.

Historicamente, los virus de la influenza aviar han sido aislados de muestras cloacales recolectadas con hisopos, pero los datosrecientes sugieren que la influenza aviar altamente patogena (HPAI) H5N1 pueden se detectados de una mejor forma de muestrasrespiratorias. Para comprender mejor como el tipo hisopo afecta a la capacidad de deteccion del virus de la influenza aviar de bajapatogenicidad (LPAI), se recolectaron y analizaron cuatro tipos de hisopos: hisopos orofarıngeos (OS), hisopos cloacales (CS), losdos tipos de hisopos combinados en el laboratorio (LCS), y los dos tipos de hisopos combinados en el campo (FCS). Semuestrearon un total de 1968 aves acuaticas silvestres mediante cada uno de estos metodos y la prueba de transcripcion reversa yreaccion en cadena de la polimerasa RT-PCR para el virus de la influenza aviar, amplificando al gene de la matriz viral. La mayortasa de deteccion se produjo con las muestras combinadas en el campo (4.3%), seguida por los hisopos cloacales (4.0%). Aunqueesta diferencia no mostro una significancia estadıstica tradicional, el analisis bayesiano indico que las muestras mezclada en el campofueron superiores a las muestras cloacales, con una probabilidad del 82%. Las tasas de deteccion tanto para las muestras mezcladasen el laboratorio (2.4%) y las muestras orofarıngeas (0.4%) fueron significativamente diferentes de las muestras mezcladas en elcampo. Ademas, cada tipo de hisopo que fue positivo al prueba de RT-PCR fue analizado para la recuperacion de los virus de lainfluenza viables. Este protocolo de redujo la tasa de deteccion, pero el orden de los tipos de algodon sigue siendo la misma: 1.73%para las muestras mezcladas en el campo, un 1.42% paral los hisopos cloacales, 0.81% para las muestras mezcladas en el laboratorioy 0% para las muestras de hisopos orofarıngeos. Nuestros datos sugieren que las muestras mezcladas en el campo que se recolectande la misma manera como cualquier otro hisopo para la deteccion del virus de la influenza aviar de baja patogenicidad en patossilvestres. Al considerar los estudios recientes que muestran que la influenza aviar H5N1 puede ser detectada de mejor forma por lasvıas respiratorias, el las muestras mezcladas en el campo fueron las mas adecuadas para recolectar a los virus de la influenza aviarH5N1 y que no comprometieron los estudios con el virus de baja patogenicidad.

Key words: avian influenza, wild birds, surveillance, sampling methods

Abbreviations: AIV 5 avian influenza virus; CS 5 cloacal swab sample; FCS 5 field-combined swab sample; HPAI 5 highpathogenicity avian influenza; LCS 5 laboratory-combined swab sample; LPAI 5 low pathogenicity avian influenza; NWR 5 Na-tional Wildlife Refuge; OS 5 oropharyngeal swab sample; rRT-PCR 5 real-time reverse-transcriptase PCR; VTM 5 viral transportmedium

Wild birds are the primary reservoir for influenza viruses (27,44).For example, the Department of the Interior surveillance program inthe United States has found all HA and NA subtypes from wild birdscollected in the US between 2006 and 2008 (9,20; National WildlifeHealth Center, unpublished data), with the exception of H14 andH15, which are only known from Europe (45). In contrast, only H1,H2, and H3 and N1 and N2 subtypes are typically found in seasonaland pandemic human influenza viruses (30). A similar restriction of

subtypes is found in the influenza viruses that infect other taxa(39,44). In poultry, influenza viruses such as H5 and H7 subtypeswhen associated with large-scale morbidity and mortality and aretermed high pathogenicity avian influenza (HPAI) viruses.

From 1996 to 2005, the HPAI H5N1 virus that was first detectedin Hong Kong circulated only in Southeast Asia. In May, 2005, thefirst indication of a change in the distribution of HPAI H5N1 wasits detection in the mortality event involving over 6000 wild birds inQinghai, China. The virus was subsequently detected in CentralAsia, Europe, and Africa during the latter half of 2005 and early2006 (16). The relatively short duration of this large-scale

ACorresponding author. E-mail: hip@usgs.gov*All authors contributed equally to this work.

AVIAN DISEASES 56:114–119, 2012

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geographic expansion prompted many to ask whether wild birds,especially migratory birds, played a role in the spread of the virusinto new geographical regions (31,32,34). HPAI H5N1 has beendetected in a growing list of apparently healthy wild birds includingsparrows (22), ducks (6), geese (23), gulls (33), and swans (14,38);however, only a limited number of apparently healthy wild birdshave been found to be infected with HPAI H5N1 in activesurveillance programs (14,17).

Virus isolation in embryonating chicken eggs remains the ‘‘goldstandard’’ for influenza virus detection (46,47). But the process islengthy, taking weeks to complete, and is labor intensive. The abilityto analyze a large number of samples in a short period of time hasmade the detection of viral RNA by real-time reverse-transcriptasePCR (rRT-PCR) the preferred method of HPAI H5N1 screening(14,17,28). With this method, results can be available within a day,and the volume of testing can be rapidly scaled up to accommodateincreased surveillance activities to provide up-to-date situationalassessment as to the extent of the outbreak.

The need to conduct surveillance to detect HPAI H5N1 virus inwild birds has led to large-scale programs to survey wild birds for thisvirus (11,28,42). The large number of samples collected and theneed for rapid assessment of possible HPAI H5N1 introduction intonew regions means that all these programs are based on an initialscreening using RT-PCR with only positive samples followed up byvirus isolation. Most of these programs initially collected only cloacalswabs from wild birds (14,28). Unlike infections in mammals inwhich influenza infection is primarily respiratory in nature, influenzainfection in wild birds replicates in the digestive tract and is shed infeces (27,36,44). Cloacal swabs have thus become the standardspecimen for the detection and isolation of avian influenza viruses(AIVs) from wild birds (46,47). However, some AIVs, particularlyhighly pathogenic viruses, including HPAI H5N1, replicate in therespiratory tract of birds (1,41). HPAI H5N1 virus shedding fromthe oropharynx can begin earlier and be shed at higher titer and for alonger duration than from the digestive tract (1,5). These findingshave led to the recommendation that a tracheal or oropharyngealswab be collected to detect HPAI H5N1 (13,47). The collection ofan oropharyngeal swab in addition to the cloacal swab could doublethe time, cost, and labor necessary and has prompted some tocombine both swabs from the same bird into a single tube for testing(3,14). Pooling swab samples in this manner has historically beendone for AI testing of poultry (4,40), but to the best of ourknowledge, no data are available on whether the ability to detect AIusing rRT-PCR testing or virus isolation in samples collected fromwild birds is affected when both swabs are combined.

Numerous countries and organizations around the world areinvolved in the collection of samples from wild birds in order todetect the presence of HPAI H5N1 and low pathogenicity AI (LPAI)viruses. In most cases when these groups added the oropharyngealswab samples to their surveillance strategy they combined thesesamples with a cloacal swab sample from the same bird, either in thelaboratory or the field, without consideration of how this mightaffect the ability to detect LPAI viruses or the HPAI H5N1 virus. Inthis study, we analyzed four swab sample types (or combination ofsample types) and compared the LPAI detection results betweensample types when using the large sample size paradigm of initialrRT-PCR testing followed by virus isolation of positives (11,28,42).We compared oropharyngeal swab alone (OS), cloacal swab alone(CS), OS and CS combined in the laboratory (LCS), and OS andCS combined directly in the field (FCS) for both the detection ofLPAI viral RNA as well as for the preservation of viable AIVs fromrepresentative species of wild birds.

MATERIALS AND METHODS

Field methods. Birds were sampled at three study sites in California,North Dakota, and Colorado in 2007–2008. In California, sampleswere collected from hunter-killed birds at Delevan National WildlifeRefuge (NWR) (39.306uN, 122.110uW), January 24–28, 2007. Allsamples were collected from consenting hunters as they checked out ofthe refuge at the end of their hunt day. In North Dakota, samples werecollected from waterfowl during annual trapping operations at J. ClarkSalyer NWR (48.795uN, 100.892uW), September 6–14, 2007.Waterfowl at this location were trapped at four separate trappinglocations by baiting a site for up to 5 days prior to a capture attempt andthen using a rocket-net for capture (2). In Colorado, samples werecollected from waterfowl at Monte Vista NWR (37.476uN,106.048uW), March 14–21, 2008. Waterfowl at this location werecaptured using baited swim-in traps (2). Traps were baited in theevening and checked early the next morning, and all captured birds wereprocessed on site. All live captured and released waterfowl in NorthDakota and Colorado were banded with uniquely numbered U.S.Geological Survey bird bands prior to release.

All sampled birds were each swabbed four times, twice from the cloacaand twice from the oropharyngeal cavity, each time using a clean sterileswab. One CS and one OS were placed in separate 1.8-ml cryovialscontaining 1 ml of viral transport media (VTM) (8). In addition, asecond CS and second OS were combined and placed in either a 1.8-mlcryovial containing 1 ml of VTM (California) or a 4-ml cryovialcontaining 2 ml of VTM (North Dakota and Colorado) and constitutedthe FCS. For each bird, the order of each CS and each OS was alternatedas to which went first into the individual swab cryovial or combinedswab cryovial. Immediately after sampling, the samples were placed onwet ice until transferred to liquid nitrogen dry shippers or dry ice within8 hr. Once in the laboratory, samples were transferred to 270 C freezersuntil analyzed. Prior to testing all tubes were vortexed to ensure adequatemixing and the LCS was created by combining 25 ml each from the OSand CS specimens in a 96-well microtiter plate prior to extraction (seebelow).

Laboratory methods. The presence of AIV in swab specimens wasdetermined by rRT-PCR and followed the same testing procedures asthose used for AI surveillance by the U.S. Department of Agricultureand the U.S. Department of the Interior (42). All four swab specimentypes (CS, OS, LCS, and FCS) were extracted using this procedure. Inbrief, viral RNA was extracted from 50-ml aliquots of each sample usingthe Ambion MagMax RNA extraction kit (Austin, TX) according tomanufacturer’s instructions. The recovered RNA was quantitated in thematrix gene RT-PCR amplification in a Stratagene MX3005P real timethermal cycler (Santa Clara, CA) (20).

Viable AIVs were isolated from each specimen by filtering thesample through a 0.2-mm filter and inoculating 0.2 ml into three8-day-old specific-pathogen-free (SPF) embryonating chicken eggs.After incubating for 3 days at 37 C, allantoic fluids were collected, andthe presence of AIV was determined by hemagglutination (HA) usingrooster and turkey red blood cells. HA-positive samples were furthertested to determine if the hemagglutinating agent was AIV by thematrix gene RT-PCR test. HA-negative samples were re-inoculated inadditional eggs and passaged a second time before a sample wasdetermined negative.

Statistical methods. McNemar’s test was used to compare thepairwise detection rates of the various sample types; calculations wereperformed using SAS PROC FREQ (SAS Institute, Inc., Cary, NC).McNemar’s test considers only pairwise-discordant samples (onepositive and the other negative) and tests whether discordant samplesoccur more frequently for one sample type than the other. Let P be theprobability that for a discordant sample, FCS was positive. If P equals0.5, a discordant sample is equally likely to be a FCS-positive, or analternative sample type-positive, so the detection rates for FCS and thealternative are ‘‘equivalent.’’ We then used a Bayesian analysis todetermine the probability that P . 0.5 by computing and examining theposterior distribution of P. This was performed on the logit scale,assigning the uniform (improper) prior to the logit, and conditioning on

Effects of swab sample choice on avian influenza detection 115

the total number of discordant samples. Calculations were performedwith WinBUGS software (24).

RESULTS

Distribution of species sampled. A total of 1968 wild waterfowl(order Anseriformes) were sampled to obtain specimens for each ofthe four sample types. Ten species were represented in the sampleincluding: mallard (Anas platyrhynchos, n 5 794), northern pintail(A. acuta, n 5 433), green-winged teal (A. crecca, n 5 223),northern shoveler (A. clypeata, n 5 178), gadwall (A. strepara, n 5

151), American wigeon (A. americana, n 5 107), cinnamon teal (A.cyanoptera, n 5 55), wood duck (Aix sponsa, n 5 20), ring-neckedduck (Aythaya collaris, n 5 4), and redhead (A. americana, n 5 3)(Table 1).

Detection of viral RNA in swab samples by rRT-PCR. Of thewaterfowl sampled, a total of 84 (4.27%) FCS, 48 (2.44%) LCS, 79(4.01%) CS, and 8 (0.41%) OS samples were positive for AIV RNAaccording to the matrix gene rRT-PCR test (Table 2). Concordanceanalysis revealed that FCS detected 17 positives that CS did not, andCS detected 12 positives that FCS did not (Table 3). This observedsuperiority of FCS over CS did not achieve statistical significance(FCS vs. CS; P 5 0.35, McNemar’s test), but a Bayesian analysisindicated that the probability that FCS was superior to CS was 82%(see Statistical Methods). FCS detected 43 positives that LCS didnot, and LCS detected seven positives that FCS did not (Table 3)(FCS vs. LCS; P , 0.0001, McNemar’s test). FCS detected 82positives that OS did not, and OS detected six positives that FCS didnot (Table 3) (FCS vs. OS; P ,0.0001, McNemar’s test).

Detection of viable influenza virus in swab samples. For everyrRT-PCR positive sample, we attempted recovery of viable virus. Atotal of 34 (1.73%) FCS, 16 (0.81%) LCS, 28 (1.42%) CS, and 0OS samples had AIVs recovered from positive matrix gene rRT-PCRswabs (Table 4). Concordance analysis revealed that FCS detected17 positives that CS did not, and CS detected 11 positives that FCSdid not (Table 3). This observed superiority of FCS over CS did notachieve statistical significance (FCS vs. CS; P 5 0.26, McNemar’stest); however, a Bayesian analysis indicated that the probability that

FCS was superior to CS was 88% (see Statistical Methods). FCSdetected 25 positives that LCS did not, and LCS detected sevenpositives that FCS did not (Table 3) (FCS vs. LCS; P 5 0.0015,McNemar’s test). FCS detected 34 positives that OS did not, andOS detected 0 positives that FCS did not (Table 3) (FCS vs. OS; P, 0.0001, McNemar’s test). Conditional on having first tested RT-PCR positive, the rate of viable virus isolation in FCS, LCS, and CSwas fairly similar across sample types at 39%, 33%, and 35%,respectively (Table 4). No viable virus was recovered from OS, buteight samples were initially positive by RT-PCR.

DISCUSSION

Molecular detection of AIV in infected birds. Our study foundthe highest AIV prevalence detection rates in sample types thatcontained the CS as one of the components. The FCS and CSsample types had the highest overall RT-PCR detection rate at 4.3%(n 5 84) and 4.0% (n 5 79) respectively. In contrast, only eightbirds (0.4%) had detectable AI viral RNA in the OS specimen. Alower prevalence rate of LPAI virus in the OS versus CS by rRT-PCR was also found by Ellstrom et al. (10), who found that of 534mallards studied, 85 (15.7%) were positive by CS, while only 27(5.1%) were positive by OS. In contrast, Gronesova et al. (15) foundin a survey of passerines in western Slovakia (n 5 105), 18% of OSsamples were rRT-PCR-positive and 18% of the CS samples werepositive. Our data support the idea that LPAI virus replicationoccurs primarily in the gastrointestinal and to a lesser extent in therespiratory tract in the species of wild ducks tested. For decades,influenza infection in birds was assumed to be a respiratory infectionsimilar to that in pigs and people (44). Slemons and Easterday (36)first showed LPAI viruses in wild ducks are more readily detectedfrom CS rather than tracheal swabs, and other studies supported thisconclusion (35,48). The amount of virus shed in the feces can be ashigh as 108.7 50% embryo infectious doses per gram of feces shedduring the infectious period in waterfowl (44). Even if ducks areinfected artificially using an aerosol route, the digestive tract remainsthe main site of LPAI virus replication (18,37). Whether theshedding pattern changes if ducks or other birds are infected at

Table 1. Waterfowl sampled by location in the United States. The total number of birds that were avian influenza matrix RT-PCR positive by atleast one swab sample is indicated in parentheses.

Species

Location

California North Dakota Colorado

Green-winged teal (Anas crecca) 193 (25) 22 (2) 8 (0)American wigeon (Anas americana) 60 (0) 45 (0) 2 (0)Cinnamon teal (Anas cyanoptera) 53 (7) 2 (0)Gadwall (Anas strepera) 151 (3)Mallard (Anas platyrhynchos) 39 (0) 550 (31) 205 (5)Northern pintail (Anas acuta) 82 (0) 246 (11) 105 (1)Northern shoveler (Anas clypeata) 178 (18)Redhead (Aythya americana) 3 (0)Ring-necked duck (Aythya collaris) 4 (0)Wood duck (Aix sponsa) 20 (0)Total 756 (53) 883 (44) 329 (6)

Table 2. Effect of swab types on avian influenza virus rRT-PCR detection in U.S. waterfowl.

Swab types Field combined Lab combined Cloacal Oropharyngeal

Positive (%) 84 (4.3) 48 (2.4) 79 (4.0) 8 (0.4)Negative 1884 1920 1889 1960Total 1968 1968 1968 1968

116 H. S. Ip et al.

different times of the year or when population density changes isunclear at the present time.

We found that the procedure for combining OS and CS samplesis also important. When the OS and CS were collected separately inthe field and combined in the laboratory, only 2.4% (n 5 48) of theLCS specimens were rRT-PCR positive. In contrast, 84 (4.3%) ofthe FCS specimens were rRT-PCR positive. Moreover the averageRT-PCR value of the positive LCS specimens was 2.55 Ct higher(poorer signal) than the equivalent matched positive FCS samples.Whether the differences in detection rate are due to 1) dilutioneffects, 2) LCS undergoing an additional freeze–thaw cycle, 3) abetter opportunity for viruses in FCS to be resuspended into thetransport media mechanically because they are in tubes with largerinternal volume, or 4) other factors remains to be determined.

By traditional hypothesis testing, FCS and CS were notsignificantly different (P 5 0.35). However this should not betaken as a basis for concluding the two are equivalent; one is almostcertainly superior to the other, and concluding equivalence wouldalmost certainly be a Type II error (19). In order to further compareFCS and CS, we performed a Bayesian analysis, which showed theprobability that FCS has a higher detection rate than CS to be 82%.

Recovery of viable virus following rRT-PCR screening. SincerRT-PCR detects the presence of AIV matrix gene RNA, the test canbe positive not only when there is viable virus present but also whenthe virus is inactivated, when the viral RNA is partially degraded, orwhen there is immature virus. Our rates of viable virus recovery forFCS (39%), LCS (33%), and CS (35%) are within the rangereported by others. For example, Ferro et al. (12) isolated 136 AIVsfrom 541 rRT-PCR positive samples (25%), Munster et al. (26)isolated 482 AIVs from 1483 rRT-PCR positive samples (33%), andWallensten et al. (43) isolated 129 AIVs from 213 rRT-PCR positivesamples (60%).

As with rRT-PCR, FCS and CS were not statistically significantlydifferent (P 5 0.26). Again, it would be inappropriate to use this asthe basis for concluding the two are equivalent. Our Bayesiananalysis indicates the probability that FCS has a higher detection ratethan CS to be 88%.

Sample type concordance. It is expected that a CS and an OSsample from the same bird would give discordant results, whetherfor rRT-PCR screening or viable virus detection following screening(see Tables 2 and 4). The explanation for the substantialdiscordances between the FCS and the other samples is less clear.Unique detections in uncombined samples might be explained bydilution effects in combined samples, or perhaps interference orcompetition in the combined sample as well. Unique detections inthe combined samples seem more difficult to explain, but perhapsthe swab order—whether the first swab was used as a combined swabor an individual swab, or perhaps combining swabs results in somesort of protective effect. In a study of wild birds in Slovakia, none ofthe 187 waterfowl tested were positive for AIVs in oropharyngeal,cloacal, and fecal swabs at the same time (25). In a separate study,only 6.6% of the 105 passerines tested were positive when the CSand OS specimens were tested for AIV at the same time when,individually however, 18% of the CS and 18% of the OS werepositive (15). Domestic ducks infected with AIVs can also have atransitory respiratory phase (21). In a study of rRT-PCR detectionin CS versus OS of waterfowl in Sweden, 3% of the samples wereonly positive by OS and would have been missed if only CS wereexamined (10), and AIVs were detected more often in OS than CSspecimens in experimentally infected wood ducks, redheads, andlaughing gulls (Leucophaeus atricilla) (7).

Among the samples tested, our data indicate that the FCS isprobably better than any of the other sample types for both the rRT-PCR and viable virus detection of LPAI virus in wild ducks. A reviewof the available literature on HPAI H5N1 infection in wild birdsalso suggests that FCS would be appropriate samples for thedetection of the HPAI virus as well. While no single specimen isperfect and testing of multiple samples from an individual bird mayyield a low number of additional positives, we suggest that whentime, labor, and/or financial resources are limited, a single fieldcombined sample containing an OS and a CS is appropriate for thedetection of HPAI H5N1 and LPAI in wild birds. This is also theconclusion reached by a recently published study based on the wildbird surveillance program in Canada (29).

Table 3. Concordance between swab types for avian influenza detection by matrix RT-PCR and virus isolation in U.S. waterfowl sampled in thecurrent study.

Field combined

RT-PCR Virus isolationA

Negative Positive Negative Positive

Cloacal

Negative 1872 12 1923 11Positive 17 67 17 17

Lab combined

Negative 1877 7 1927 7Positive 43 41 25 9

Oropharyngeal

Negative 1878 6 1934 0Positive 82 2 34 0AVirus isolation attempted on RT-PCR positive samples only.

Table 4. Effect of swab types collected from U.S. waterfowl on AIV isolation following RT-PCR screening.

Swab types Field combined Lab combined Cloacal Oropharyngeal

Positive 34 (39%) 16 (33%) 28 (35%) 0 (0%)Negative 50 32 51 8Total 84 48 79 8

Effects of swab sample choice on avian influenza detection 117

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ACKNOWLEDGMENTS

We would like to thank the many people who assisted with thisproject. R. Zane, D. Goldberg, J. Teslaa, L. Clark, B. Iko, and L. Baetenprovided assistance with field sampling. E. Jansen, M. Houfe, K.Griffin, A. Hauser, J. Montez, J. Messer and other members of theNational Wildlife Health Center provided assistance with sampleanalysis. In addition, J. Gammonley, Colorado Division of Wildlife,provided logistical support. We also gratefully acknowledge theinstitutions that allowed access to their properties and providedinvaluable assistance in obtaining samples including: Delevan NationalWildlife Refuge (NWR) and the Sacramento NWR Complex, MonteVista NWR, and J. Clark Salyer NWR. This study was funded by theU.S. Geological Survey. Use of trade or product names does not implyendorsement by the United States government.

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