Assays to detect West Nile virus in dead birds.Using oral swab samples to detect West Nile virus West Nile virus, microorganism and the infection resulting from it, which typically produces no symptoms or a flulike condition. The virus is a flavivirus and is related to a number of viruses that cause encephalitis. in dead birds, we compared the Rapid Analyte Measurement Platform (RAMP) assay with VecTest and real-time reverse-transcriptase-polymerase chain reaction. The sensitivities of RAMP and VecTest for testing corvid species were 91.0% and 82.1%, respectively. ********** Since the discovery of West Nile virus (WNV WNV West Nile Virus WNV World Net Visions ) in New York New York, state, United States New York, Middle Atlantic state of the United States. It is bordered by Vermont, Massachusetts, Connecticut, and the Atlantic Ocean (E), New Jersey and Pennsylvania (S), Lakes Erie and Ontario and the Canadian province of in 1999, an integral part of monitoring has been testing dead bird tissue by using real-time and standard reverse-transcriptase-polymerase chain reaction (RT-PCR RT-PCR reverse transcriptase-polymerase chain reaction. See PCR1. ) (1-3). The detection limit for WNV by both methods is as low as 0.08 PFU PFU plaque-forming unit; in virology, areas of cell lysis (CPE) in monolayer cell culture, under overlay conditions, initiated by infection with a single virus particle. (1.9 [log.sub.10] PFU/mL), which indicates that RT-PCR is more sensitive than cell culture and more accurately indicates infection, since RNA RNA: see nucleic acid. RNA in full ribonucleic acid One of the two main types of nucleic acid (the other being DNA), which functions in cellular protein synthesis in all living cells and replaces DNA as the carrier of genetic is more stable than infectious virus in tissues (3). Recent studies have assessed potential time- and cost-saving alternatives such as VecTest (Medical Analysis Systems Camarillo, CA, USA) (4-9). Although studies have found that VecTest, with a detection limit in mosquitoes of 5.17 [log.sub.10] PFU/mL (10), is less sensitive than RT-PCR for detecting WNV, test sensitivity was generally high when testing swab samples from corvid species (4,6,8,9) and certain noncorvid species such as House Sparrows (Passer domesticus) (4) and North American North American named after North America. North American blastomycosis see North American blastomycosis. North American cattle tick see boophilusannulatus. Owls (family Strigidae Noun 1. family Strigidae - a family of nocturnal birds of the order Strigiformes Strigidae bird family - a family of warm-blooded egg-laying vertebrates characterized by feathers and forelimbs modified as wings ) (7). Disadvantages were occasional atypical results, including false-positives (4). In this study we evaluated another alternative for WNV detection, the Rapid Analyte Measurement Platform (RAMP, Response Biomedical bi·o·med·i·cal adj. 1. Of or relating to biomedicine. 2. Of, relating to, or involving biological, medical, and physical sciences. Corp, Burnaby, British Columbia “Burnaby” redirects here. For persons sharing this surname, see Burnaby (surname). Burnaby, British Columbia, Canada, is the city immediately east of Vancouver. , Canada). Limited studies conducted at the Centers for Disease Control and Prevention Centers for Disease Control and Prevention (CDC), agency of the U.S. Public Health Service since 1973, with headquarters in Atlanta; it was established in 1946 as the Communicable Disease Center. and the Canadian National Microbiology Laboratory The National Microbiology Laboratory (NML) is located in the Canadian Science Centre for Human and Animal Health in Winnipeg, Manitoba. This modern state-of-the-art facility houses the NML's Biological Safety Level 4 (BSL-4) containment laboratory, currently Canada's only BSL-4 indicated that the RAMP WNV test, with detection limits in mosquitoes as low as 3.17 [log.sub.10] PFU/mL, was more sensitive than VecTest (10). Both tests incorporate immunochromatographic test strips by using labeled antibodies to detect antigen in samples. VecTest uses antibodies bound to gold sol particle labels, while the RAMP test uses antibodies bound to fluorescently labeled latex particles. Development of a visible reddish-purple line in both the test and control zones on the VecTest strip indicates a positive result. The RAMP test strip, enclosed within a cartridge, is inserted into a reader that calculates the ratio between the fluorescence emitted at the test and control zones and displays the results as RAMP units. Values above a background threshold are recorded as positive. This study compared WNV results from the RAMP and VecTest on oral swab samples from dead birds, with RT-PCR on brain tissue as the standard. Brain swab samples were also tested as an alternate antigen source in the RAMP and VecTest. The Study Birds included in this study were received from mid-May to late November 2004 and from mid-February through May 2005 from counties in New York There are sixty-two counties in the State of New York. Five of these are boroughs of New York City and do not have functioning county governments. New York City encompasses five counties, and is the county seat of all five of them: New York County (Manhattan), Kings County (Brooklyn), State. Oral swab samples for the RAMP and VecTest were collected with 2 sterile, polyester fiber-tipped plastic applicators held together and moved around the oral cavity oral cavity n. The part of the mouth behind the teeth and gums that is bounded above by the hard and soft palates and below by the tongue and the mucous membrane connecting it with the inner part of the mandible. and proximal esophagus esophagus (ĭsŏf`əgəs), portion of the digestive tube that conducts food from the mouth to the stomach. When food is swallowed it passes from the pharynx into the esophagus, initiating rhythmic contractions (peristalsis) of the . One swab sample was twirled in 1.0 mL of VecTest buffer solution in a 5-mL plastic tube. The second swab sample was either twirled in 1.0 mL of RAMP buffer solution in a separate 5-mL plastic tube or placed in an empty 5-mL plastic tube, capped, and frozen at -20[degrees]C for later testing. RAMP tests were run the same day on flesh material or later on frozen samples. Before being tested, all frozen samples were thawed at room temperature; swabs not previously mixed in solution and swabs from thawed carcasses were then mixed in RAMP buffer solution. Samples were taken from the brains of a subset of corvid species by swabbing cerebral parenchyma Parenchyma A ground tissue of plants chiefly concerned with the manufacture and storage of food. The primary functions of plants, such as photosynthesis, assimilation, respiration, storage, secretion, and excretion—those associated with living and processing as for oral samples. The RAMP and VecTest were run according to according to prep. 1. As stated or indicated by; on the authority of: according to historians. 2. In keeping with: according to instructions. 3. manufacturers' directions in a class II biosafety cabinet at the New York State Department of Environmental Conservation's Wildlife Pathology Unit. RAMP test values [greater than or equal to] 50, calculated by the RAMP reader, were recorded as positive. Differences in test performance were assessed by chi-square analysis. Data are expressed as a percentage in text and tables only when n is [greater than or equal to] 10. Brain samples for RT-PCR were taken at necropsy necropsy /nec·rop·sy/ (nek´rop-se) examination of a body after death; autopsy. nec·rop·sy n. See autopsy. necropsy examination of a body after death. See also autopsy. and frozen at -20[degrees]C. Brain tissue was analyzed at the Arbovirus arbovirus Any of a large group of viruses that develop in arthropods (chiefly mosquitoes and ticks). The name derives from “arthropod-borne virus.” The spheroidal virus particle is encased in a fatty membrane and contains RNA; it causes no apparent harm to the Laboratory, Wadsworth Center, New York State Department of Health, as described previously (2,3). RT-PCR was repeated on 54 birds for which results from RAMP, VecTest, or both, contrasted with RT-PCR results. Retests of 6 birds yielded different results from the original tests. Three of these were initially positive and retested negative; the original values were low, which indicated infectivity infectivity ability of an agent to infect. was focal and undetected on a different sample, and the level was below RAMP and VecTest limits of detection. Three originally negative samples retested positive; 2 were highly positive, which indicated a technical error, and 1 kidney tissue sample was positive, although results of a retest re·test tr.v. re·test·ed, re·test·ing, re·tests To test again. n. A second or repeated test. with brain tissue were negative. In this study, oral samples from 679 birds were tested 193 (28.4%) were WNV-positive by RT-PCR. RAMP sensitivity was 80.8%, compared to 71.0% for VecTest (Table 1). For corvid species (n = 156), RAMP sensitivity (91.0%) was significantly greater than that of VecTest (82.1%) (p [less than or equal to] 0.025). With smaller sample sizes at the species level, sensitivity between RAMP and VecTest did not differ significantly (p>0.05) for 128 American Crows (Corvus brachyrhynchos) (91.4% and 84.4%, respectively) and 27 Blue Jays (Cyanocitta cristata) (88.9% and 70.4%, respectively) tested, nor for interspecies differences within each test. The detection thresholds of these tests, coupled with viral titers of specimens, may explain these different results. RAMP confirmed more Common Grackles (Quiscalus quiscula) (3/3) and House Sparrows (5/6) as positive than did VecTest (2/3 and 3/6, respectively). With the exception of a few species, both tests performed poorly overall on small sample sizes of other noncorvid species. To determine if RAMP results were affected by freezing the sample, samples from 13 corvids (10 positive, 3 negative) were retested by using swabs taken from frozen carcasses. Six initially were tested with fresh swabs and 7 with frozen swabs; all retests yielded results similar to initial results. The same results for fresh versus frozen samples were obtained with VecTest (4). VecTest specificity with oral swabs was excellent in correctly identifying all 486 RT-PCR-negative birds, returning no false-positive results (Table 2). RAMP had high specificity for American Crows (98.5%), Blue Jays (90.9%), and noncorvid species (98.9%). Brain swab samples from 39 corvids were tested; 27 (69.2%) were RT-PCR-positive. Both RAMP and VecTest performed well, with sensitivities of 92.6% and 88.9%, respectively, and no false-positive results. Conclusions Although RAMP was more sensitive than VecTest, both appear adequate for WNV surveillance in dead corvids. RAMP also performed well with oral swabs from Common Grackles and House Sparrows, although sample sizes were small. These findings are similar to previous results for VecTest, which also tested well with House Finches (Carpodacus mexicanus), Northern Cardinals (Cardinalis cardinalis), and American Kestrels (Falco sparverius) (4). As in the previous study, both tests did poorly in RT-PCR-positive raptors. In the previous New York study, VecTests successfully tested brain, kidney, blood, feather pulp, and cloacal cloacal emanating from or pertaining to cloaca. cloacal kiss the contact which occurs during insemination in birds when the vent of the female is everted exposing the cloacal mucosa against which the phallus of the male is pressed. samples from corvids and House Sparrows (4). In the current study, RAMP and VecTest worked well with brain as the antigen source. Brain swab samples may be the preferred antigen source when the oral cavity is compromised. Further testing of alternative swab samples is warranted and may identify a superior antigen source; however, testing internal organs may pose greater risks and may not be applicable in field work and nonlaboratory-based surveillance. In addition, further testing, including immunohistochemical tests, on noncorvids should be conducted to accurately assess these tests and identify the distribution of WNV in the oral cavity and internal tissues. In this study, VecTest produced no false-positive results. Although its specificity was high, RAMP produced 8 false-positive results (range 50.9-147.6). Four of these were near the [greater than or equal to] 50 positive indicator level and may have been due to other sources of fluorescence. The remaining 4 false-positives (3 American Crows and 1 Blue Jay), with scores from 74.4 to 147.6, came from birds with oral cavities compromised by blood or fly eggs, which may have biased results. VecTest results are easily distinguished when a true WNV-positive reaction occurs, but the reddish-purple line may appear faint or thin in other cases and may be subject to interpretation (4). RAMP quantitative results eliminate subjective interpretation, which helps assure replication but limits confidence in lower RAMP-positive scores. The RAMP system requires an initial purchase of an electronic reader ([approximately equal to] US $3,500), and materials cost $13-$15 per test; VecTest costs $8 per test. If large numbers of specimens are tested, the cost of the RAMP reader per test is minimal. The RAMP test requires a minimum of 1.5 h to run because of the required cartridge drying time; VecTest takes 15-30 min to run after the test strip is placed in the sample solution. In conclusion, both RAMP and VecTest are useful alternatives to RT-PCR for WNV surveillance in dead corvids and some passerine passerine Any perching bird. All passerines belong to the largest order of birds, Passeriformes, and have feet specialized for holding onto a horizontal branch (perching). The passerine foot has three forward-directed toes and one backward-directed toe. species when immediate turn-around of large numbers of specimens is valuable. Testing with RAMP is advantageous because of its increased sensitivity; however, follow-up testing with RT-PCR is recommended for low RAMP-positive results near the positive indicator level. Using both tests in a system in which initial testing is conducted with VecTest may also be useful; RAMP could be reserved for high-priority cases in which VecTest results are negative. RT-PCR should still be used to confirm initial viral activity in a new period and area and for research requiring more definitive results. Acknowledgments We thank Kevin Hynes, Joe Okoniewski, and Darci Dougherty for their technical assistance; Dave Galinski for bird necropsy, swab and tissue collection, performance of RAMP, and VecTest; Susan Jones and Mary Franke for real-time RT-PCR assays; county health departments, New York City New York City: see New York, city. New York City City (pop., 2000: 8,008,278), southeastern New York, at the mouth of the Hudson River. The largest city in the U.S. Department of Health and Mental Hygiene mental hygiene, the science of promoting mental health and preventing mental illness through the application of psychiatry and psychology. A more commonly used term today is mental health. , Yoichiro Hagiwara, Richard Chipman, Richard A. Watt and other animal control officers, wildlife rehabilitators, and the concerned public for coordinating and participating in dead bird reporting and submission. The New York State Department of Environmental Conservation and the New York State Department of Health jointly supported this research. Work on this study was partially supported by federal funds Federal Funds Funds deposited to regional Federal Reserve Banks by commercial banks, including funds in excess of reserve requirements. Notes: These non-interest bearing deposits are lent out at the Fed funds rate to other banks unable to meet overnight reserve from the Centers for Disease Control and Prevention under cooperative agreement numbers U50/ CCU CCU abbr. 1. coronary care unit 2. critical care unit CCU critical care unit. CCU Critical care unit, see there 223671 and U90/CCU216988. References (1.) Lanciotti RS. Molecular amplification assays for the detection of flaviviruses. Adv Virus Res. 2003;61:67-99. (2.) Kauffman EB, Jones SA, Dupuis AP, Ngo KA, Bernard KA, Kramer LD. Virus detection protocols for West Nile virus in vertebrate vertebrate, any animal having a backbone or spinal column. Verbrates can be traced back to the Silurian period. In the adults of nearly all forms the backbone consists of a series of vertebrae. All vertebrates belong to the subphylum Vertebrata of the phylum Chordata. and mosquito specimens. J Clin Microbiol. 2003;41:3661-7. (3.) Shi P-Y, Kauffman EB, Ren P, Felton A, Tai JH, Dupuis II AP, et al. High-throughput detection of West Nile virus RNA. J Clin Microbiol. 2001:39:1264-71. (4.) Stone WB, Okoniewski JC, Therrien JE, Kramer LD, Kaufman EB, Eidson M. VecTest as diagnostic and surveillance tool for West Nile virus in dead birds. Emerg Infect Dis. 2004;10:2175-81. (5.) Siirin M, Sargent C, Langer RC, Parsons Parsons, city (1990 pop. 11,924), Labette co., SE Kans.; inc. 1871. It is a shipping point for dairy products, grain, and livestock. Manufactures include ammunition, wire and paper products, plastics, and appliances. R, Vanlandingham DL, Higgs S, et al. Comparative sensitivity of the VecTest antigen-capture assay, reverse transcriptase-PCR, and cell culture for detection of West Nile virus in dead birds. Vector-borne and Zoonotic Diseases Zoonotic diseases Diseases caused by infectious agents that can be transmitted between (or are shared by) animals and humans. This can include transmission through the bite of an insect, such as a mosquito. Mentioned in: West Nile Virus . 2004;4:204-9. (6.) Henson G, Hicock E Rapid detection of West Nile virus in bird using the VecTest WNV antigen assay. Clin Lab CLIN LAB Clinical Laboratory / Klinisches Labor (Journal) Sci. 2004; 17:218-20. (7.) Gancz AY, Campbell DG, Barker IK, Lindsay R, Hunter B. Detecting West Nile virus in owls and raptors by an antigen-capture assay. Emerg Infect Dis. 2004; 10:2204-6. (8.) Lindsay R, Barker I, Nayar G, Drebot M, Calvin S, Scammell C, et al. Rapid antigen-capture assay to detect West Nile virus in dead corvids. Emerg Infect Dis. 2003;9:1406-10. (9.) Yaremych SA, Warner RE, Van de Wyngaerde MT, Ringia AM, Lampman R, Novak RJ. West Nile virus detection in American Crows. Emerg Infect Dis. 2003;9:1319-21. (10.) Burkhalter KL, Lindsay R, Anderson R, Dibernardo A, White H, Drebot M, et al. undated un·dat·ed adj. 1. Not marked with or showing a date: an undated letter; an undated portrait. 2. . Evaluation of commercial assays for detecting West Nile virus antigen. Burnaby, British Columbia, Canada: Response Biomedical Corporation. Ward B. Stone, * Joseph E. Therrien, * Robert Benson Robert Benson may mean:
n. A reference mark (  ) used in printing and writing. Also called diesis.Noun 1. ]) * New York State Department of Environmental Conservation, Albany, New York For other uses, see Albany. Albany is the capital of the State of New York and the county seat of Albany County. Albany lies 136 miles (219 km) north of New York City, and slightly to the south of the juncture of the Mohawk and Hudson Rivers. , USA; ([dagger]) New York State Department of Health, Albany, New York, USA; and ([double dagger]) Suffolk County Suffolk County may refer to:
Dr Stone has been the wildlife pathologist for the New York State Department of Environmental Conservation for >36 years. He also is an adjunct professor at the State University of New York (body) State University of New York - (SUNY) The public university system of New York State, USA, with campuses throughout the state. College at Cobleskill and the College of St. Rose. Dr Stone's main research interests are in infectious and parasitic diseases, toxicology toxicology, study of poisons, or toxins, from the standpoint of detection, isolation, identification, and determination of their effects on the human body. Toxicology may be considered the branch of pharmacology devoted to the study of the poisonous effects of drugs. , and forensic pathology Noun 1. forensic pathology - the branch of medical science that uses medical knowledge for legal purposes; "forensic pathology provided the evidence that convicted the murderer" forensic medicine of wildlife. Address for correspondence: Ward B. Stone, NYSDEC--Wildlife Pathology Unit, 108 Game Farm Rd, Delmar, NY 12054, USA; fax: 518-478-3035; email: wbstone@gw.dec.state.ny.us
Table 1. Oral swab RAMP and VecTest sensitivity for real-time
RT-PCR-positive birds. New York, 2004-2005 *
No. positive (%)
Species (presented in taxonomic N
order) ([dagger]) RAMP VecTest
Pelicaniformes
Double Crested Cormorant
(Phalacrocorax auritus) 1 0 0
Falconiformes
Cooper's Hawk (Accipiter
cooperii) 4 0 0
Red-tailed Hawk (Buteo
jamaicensis) 2 0 0
American Kestrel (Falco
sparverius) 1 1 1
Charadriiformes
Ring-billed Gull (Larus
delawarensis) 1 1 0
Strigiformes
Great Horned Owl (Bubo
virginianus) 3 1 0
Passeriformes
Eastern Kingbird (Tyrannus
tyrannus) 1 0 0
Red-eyed Vireo (Vireo
olivaceus) 1 0 0
Blue Jay (Cyanocitta cristata) 27 24 (88.9) 19 (70.4)
American Crow (Corvus
brachyrhynchos) 128 117 (91.4) 108 (84.4)
Fish Crow (Corvus ossifragus) 1 1 1
American Robin (Turdus
migratorius) 7 1 1
Gray Catbird (Dumetella
carolinensis) 1 0 0
Northern Mockingbird (Mimus
polyglottos) 2 1 1
Cedar Waxwing (Bombycilla
cedrorum) 1 1 1
Northern Cardinal (Cardinalis
cardinalis) 1 0 0
Common Grackle (Quiscalus
quiscula) 3 3 2
House Finch (Carpodacus
mexicanus) 2 0 0
House Sparrow (Passer
domesticus) 6 5 3
Total all species 193 156 (80.8) 137 (71.0)
* RAMP, Rapid Analyte Measurement Platform; RT-PCR, reverse
transcription-polymerase chain reaction.
([dagger]) No. of birds real-time RT-PCR-positive.
Table 2. Oral swab RAMP and VecTest specificity for real-time
RT-PCR-negative birds, New York, 2004-2005 *
No. Negative (%)
Species (presented in taxonomic N
order) ([dagger]) RAMP VecTest
Galliformes
Ruffed Grouse (Bonasa umbellus) 12 12 12
Columbiformes
Mourning Dove (Zenaida macroura) 24 24 24
Rock Dove (Columba livia) 12 12 12
Passeriformes
Blue Jay (Cyanocitta cristata) 22 20 (90.9) 22
American Crow (Corvus
brachyrhynchos) 198 195 (98.5) 198
Fish Crow (Corvus ossifragus) 2 2 2
American Robin (Turdus
migratorius) 22 20 (90.9) 22
Gray Catbird (Dumetella
carolinensis) 14 13 (92.9) 14
European Starling (Stumus
vulgaris) 11 11 11
Brown-headed Cowbird (Molothrus
ater) 10 10 10
Common Grackle (Quiscalus
quiscula) 19 19 19
House Sparrow (Passer domesticus) 22 22 22
Other species ([double dagger]) 118 118 118
Total all species 486 478 (98.4) 486
* RAMP, Rapid Analyte Measurement Platform, RT-PCR, reverse
transcription-polymerase chain reaction.
([dagger]) No. real-time RT-PCR-negative birds.
([double dagger]) 52 species, representing 14 orders.
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