Rapid West Nile virus antigen detection.We compared the VecTest WNV WNV West Nile Virus WNV World Net Visions antigen assay with standard methods of 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. (WNV) detection in swabs from American Crows (Corvus brachyrhynchos) and House Sparrows (Passer domesticus). The VecTest detected WNV more frequently than the plaque assay and was comparable to a TaqMan reverse transcription-polymerase chain reaction. ********** Dead bird surveillance is an effective way to monitor the presence and spread of West Nile virus (WNV) in North America North America, third largest continent (1990 est. pop. 365,000,000), c.9,400,000 sq mi (24,346,000 sq km), the northern of the two continents of the Western Hemisphere. (1), and assays to detect infectious WNV virions, antigen, and 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 in tissues from infected birds are reliable techniques (2-4). More than 28,000 bird carcasses were tested for WNV in the United States United States, officially United States of America, republic (2005 est. pop. 295,734,000), 3,539,227 sq mi (9,166,598 sq km), North America. The United States is the world's third largest country in population and the fourth largest country in area. from 1999 to 2002 (5). Processing and testing these carcasses require a substantial commitment of resources from federal, state, and local health departments. Simplifying diagnostic procedures by implementing rapid antigen-capture assays would permit increased specimen processing and, ultimately, improved surveillance. 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. and oral (nasopharyngeal nasopharyngeal pertaining to the nasal and pharyngeal cavities. nasopharyngeal meatus see nasopharyngeal meatus. nasopharyngeal spasm see reverse sneeze. ) swabs from dead corvids (crows and jays) are reliable sources of WNV RNA and infectious virions (6). Three field evaluations of an antigen detection assay applied to corvid carcasses collected shortly after death found that oral swabs were more sensitive than cloacal swabs for detecting WNV antigen, and that sensitivity of the VecTest WNV antigen assay (Medical Analysis Systems, Camarillo, CA, USA) applied to oral swabs was >80% for American Crows, lower for other corvids, and variable for a variety of other species (7-9). Several questions remain unanswered regarding the usefulness of swab specimens for WNV surveillance. How long after death of a bird can WNV be detected in swab specimens? Can swabs from noncorvid birds be used to detect WNV? Can reverse transcription-polymerase chain reaction (RT-PCR RT-PCR reverse transcriptase-polymerase chain reaction. See PCR1. ) or VecTest detect WNV in oral swab samples that have remained dry and at room temperature? To address these questions, we compared the VecTest WNV antigen assay with standard methods of virus detection from oral and cloacal swabs taken 1-4 days postmortem postmortem /post·mor·tem/ (post-mort´im) performed or occurring after death. post·mor·tem adj. Relating to or occurring during the period after death. n. See autopsy. from experimentally infected American Crows (Corvus brachyrhynchos) and House Sparrows (Passer domesticus). The VecTest, which was originally developed for mosquito pools as a simple, 1-step wicking assay available in a kit, requires no specialized equipment, storage conditions, or highly trained personnel and provides results in 15 minutes (10,11). The Study Oral and cloaca cloaca (klōā`kə), in biology, enlarged posterior end of the digestive tract of some animals. The cloaca, from the Latin word for sewer, 1 swab samples were collected daily (for 4 days) from carcasses of crows and sparrows Crows and Sparrows (Simplified Chinese: 乌鸦与麻雀; Traditional Chinese: 烏鴉与麻雀 that had been experimentally infected with either the NY99-4132 strain (30 crows and 6 sparrows) or the Kenyan KN-3829 strain (1 crow and 5 sparrows) of WNV. Carcasses were stored at ambient temperature Outside temperature at any given altitude, preferably expressed in degrees centigrade. ([approximately equal to] 20[degrees]C) during this period. The samples were collected with standard, cotton-tipped applicators by inserting them into the cloaca or 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 then placing them directly into 1 mL VecTest grinding solution A, a physiologic buffer similar to phosphate-buffered saline. Samples were subsequently frozen at -70[degrees]C until tested by a variety of methods for detecting WNV. Some oral swabs from infected crows were left at room temperature without diluent diluent /dil·u·ent/ (dil´oo-int) 1. causing dilution. 2. an agent that dilutes or renders less potent or irritant. dil·u·ent adj. Serving to dilute. n. for 24 or 48 hours before testing. Negative control swab samples were collected from 25 live, healthy, uninfected crows. All swab specimens collected from crow carcasses were positive by the TaqMan RT-PCR method, using 2 sets of WNV-specific primers (2). Several TaqMan RT-PCR-negative swabs for the sparrows were also negative by the other assays; these were disregarded in summarizing the results. Results of VecTest and Vero plaque assay (11) of the RT-PCR-positive swab specimens are shown as sensitivities (using RT-PCR as the standard for detecting WNV RNA) in Table 1. A logistic regression model accounting for anticipated correlation induced by multiple and repeated observations on each bird was used to compare sensitivities for each day postmortem, with significance determined using [alpha] = 0.05 (12). For crows evaluated 1 day postmortem, no significant difference between swab types (oral versus cloacal) (p = 0.63) and no significant difference between the 2 assays (p = 0.10) were detected. At 2 days postmortem, the effect due to swab type was not significant (p = 0.07), but a significant difference was seen in the sensitivities of the 2 assays (p = 0.004), excluding the nonsignificant non·sig·nif·i·cant adj. 1. Not significant. 2. Having, producing, or being a value obtained from a statistical test that lies within the limits for being of random occurrence. effect of swab type from the logistic regression model. At 3 days postmortem, both swab type and assay differences were significant (p<0.01), with oral swabs more likely to yield a positive finding (compared with cloacal swabs) and VecTest more sensitive than plaque assay. For sparrows, no significant differences were seen between the sensitivities of the VecTest and plaque assay for either swab type on any of the 3 days (McNemar test). VecTest detected WNV in 90% of 22 crow oral swabs that were tested after remaining dry and at room temperature for 24 hours Adv. 1. for 24 hours - without stopping; "she worked around the clock" around the clock, round the clock and in 70% of 13 crow oral swabs assayed after 48 hours. By comparison, TaqMan RT-PCR detected WNV in 86% and 70% of these oral swabs, respectively, at the same time points. Over the 4-day sampling period, geometric mean (mathematics) geometric mean - The Nth root of the product of N numbers. If each number in a list of numbers was replaced with their geometric mean, then multiplying them all together would still give the same result. viral titers in crow oral swabs, determined by Vero cell plaque assay, decreased from [10.sup.3.6] to [10.sup.2.2] PFU/swab (Table 2). In contrast, the geometric mean viral titer titer /ti·ter/ (ti´ter) the quantity of a substance required to react with or to correspond to a given amount of another substance. in crow cloacal swabs decreased from [10.sup.3.0] PFU/swab at 1 day postmortem to undetectable by 4 days postmortem. RNA levels, as detected by the TaqMan assay, also decreased over time. Conclusions VecTest has the potential to simplify dead bird surveillance for WNV by reducing required resources such as specialized equipment and costly reagent kits needed to achieve a rapid and accurate result. With appropriate biosafety measures, the assay can be conducted in the field, or in centralized regional laboratories, obviating ob·vi·ate tr.v. ob·vi·at·ed, ob·vi·at·ing, ob·vi·ates To anticipate and dispose of effectively; render unnecessary. See Synonyms at prevent. the need for expensive shipping of bird carcasses to remote reference laboratories. One objective of our study was to determine whether oral or cloacal swabs were preferable for WNV testing of dead birds. To answer this question, several criteria were evaluated, including the ability of 3 different assays to detect WNV, the feasibility of collecting specimens postmortem, and postmortem duration of WNV positivity. TaqMan RT-PCR detected WNV RNA and antigen in similar proportions in all cloacal and oral specimens collected from crows. However, virus isolation by Vero plaque assay was more successful when oral swabs were tested. Virus appears to be more rapidly inactivated inactivated rendered inactive; the activity is destroyed. inactivated viruses treated so that they are no longer able to produce evidence of growth or damaging effect on tissue. in the cloaca compared with the oral cavity. This phenomenon was consistent for both sparrows and crows. Fewer postmortem swab samples were available from sparrows compared with those from crows because fewer sparrow carcasses were available (sparrows are less susceptible to fatal WNV infection than crows) (13). Collecting cloacal swabs from the smaller sparrows was also more difficult after 1 day postmortem because they tended to desiccate des·ic·cate v. To dry thoroughly; render free from moisture. desiccate (des´ikāt), n to dry by chemical or physical means; e.g. quickly. RT-PCR detected WNV RNA in sparrows from 24/24 oral swabs, but only 11/13 cloacal swabs. Antigen was detected by VecTest from 18/24 oral and 8/13 cloacal swabs. Infectious virus was detected by plaque assay in 20/24 oral swabs but in only 6/13 cloacal swabs. Virus titers and RNA concentrations in the carcasses decayed over the 4-day period of observation and this decay was most pronounced in the cloacal swabs. Thus, oral swabs were more effective than cloacal swabs to detect WNV in both crows and sparrows. VecTest consistently detected WNV antigen in a greater proportion of samples than Vero plaque assay detected virions. Thus, although the detection of infectious virus was inconsistent, carcasses contained sufficient quantity of viral components, both RNA and protein, to permit detection for [greater than or equal to] 4 days after death. In a natural setting, carcasses most likely would decay more rapidly than in these experiments, given exposure to temperature fluctuations, microbial microbial pertaining to or emanating from a microbe. microbial digestion the breakdown of organic material, especially feedstuffs, by microbial organisms. attack, and predation predation Form of food getting in which one animal, the predator, eats an animal of another species, the prey, immediately after killing it or, in some cases, while it is still alive. Most predators are generalists; they eat a variety of prey species. . Guidelines for WNV surveillance recommend sampling carcasses <24 hours old (14). These results suggest that older carcasses may have detectable WNV RNA and antigen that still are readily detectable with the TaqMan and VecTest assays. Thus, carcasses should be tested regardless of age, as long as they are not in a condition where sampling is impossible. In addition, swabs collected in the field can be stored at room temperature in empty cryovials for up to 48 hours and then reliably assayed for WNV antigen by VecTest. Detecting WNV from sparrow carcasses demonstrates that swabs are useful to test species other than corvids. House sparrows, like corvids, are 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. birds that develop high levels of WNV in blood and tissues (13). Stone et al. showed that the VecTest had a sensitivity of 76% in detecting WNV in oral swabs of field-collected carcasses of house sparrows (9). In summary, oral swabs are more useful than cloacal swabs for obtaining a reliable result with the diagnostic assays described in our study. Moreover, swabs from non-corvid birds may also be effectively assayed for WNV. Our findings suggest that large numbers of dead corvids of any age, and possibly other passerine birds, could be screened by cautiously collecting dry oral swabs in the field, storing them properly, and then testing them within 48 hours by rapid antigen detection assay or RT-PCR. Acknowledgments We thank Jason Velez for technical assistance, Richard Bowen for providing space for laboratory animals at Colorado State University Colorado State University, at Fort Collins; land-grant with state and federal support; chartered 1870, opened 1879 as an agricultural college, assumed present name in 1957. There is a veterinary teaching hospital, an agricultural campus, and a research campus. , and Thomas Janousek and Charles Cope for providing crows for this study. The authors have no financial interest with the manufacturers or developers of VecTest. References (1.) Eidson M, Komar N, Sorhage F, Nelson R, Talbot T, Mostashari F, et al. Crow deaths a sentinel surveillance system for West Nile virus in the northeastern United States, 1999. Emerg Infect Dis. 2001;7:615-20. (2.) Lanciotti RS, Kerst AJ, Nasci RS, Godsey MS, Mitchell C J, Savage HM, et al. Rapid detection of West Nile virus from human clinical specimens, field-collected mosquitoes, and avian samples by a TaqMan reverse transcriptase-PCR assay. J Clin Microbiol. 2000;38:4066-71. (3.) Hunt A, Hall RA, Kerst AJ, Nasci RS, Savage HM, Panella NA, et al. Detection of West Nile virus antigen in mosquitoes and avian tissues by a monoclonal antibody-based capture enzyme immunoassay Immunoassay An assay that quantifies antigen or antibody by immunochemical means. The antigen can be a relatively simple substance such as a drug, or a complex one such as a protein or a virus. . J Clin Microbiol. 2002;40:2023-30. (4.) Steele KE, Linn linn n. Scots 1. A waterfall. 2. A steep ravine. [Scottish Gaelic linne, pool, waterfall.] M J, Schoepp RJ, Komar N, Geisbert TW, Manduca RM, et al. Pathology of fatal West Nile virus infections in native and exotic birds during the 1999 outbreak in 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. , 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 . J Vet Pathol. 2000;37:208-24. (5.) Komar N. West Nile virus: epidemiology and ecology in North America. Adv Virus Res. 2003;61:185-234. (6.) Komar N, Lanciotti R, Bowen R, Langevin S, Bunning M. Detection of West Nile virus in oral and cloacal swabs collected from bird carcasses. Emerg Infect Dis. 2002;8:741-2. (7.) 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. (8.) 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. (9.) Stone WB, Okoniewski JC, Therrien JE, Kramer LD, Kauffman EB, Eidson M. VecTest as diagnostic and surveillance tool for West Nile virus in dead birds. Emerg Infect Dis. 2004; 10:2175-81. (10.) Ryan J, Dave K, Emmerich E, Fernandez B, Turell M, Johnson J, et al. Wicking assays for the rapid detection of West Nile and St. Louis encephalitis St. Louis encephalitis see St. Louis encephalitis. viral antigens in mosquitoes. J Med Entomol. 2003 ;40:95-9. (11.) Nasci RS, Gottfried KL, Burkhalter KL, Kulasekera VL, Lambert A J, Lanciotti RS, et al. Comparison of Vero cell plaque assay, TaqMan reverse transcriptase Reverse transcriptase Any of the deoxyribonucleic acid (DNA) polymerases present in particles of retroviruses which are able to carry out DNA synthesis using an RNA template. polymerase chain reaction polymerase chain reaction (pŏl`ĭmərās') (PCR), laboratory process in which a particular DNA segment from a mixture of DNA chains is rapidly replicated, producing a large, readily analyzed sample of a piece of DNA; the process is RNA assay, and VecTest antigen assay for detection of West Nile virus in field-collected mosquitoes. J Am Mosq Control Assoc. 2002; 18:294-300. (12.) Diggle P, Heagerty P, Liang K-Y, Zeger S. Analysis of longitudinal data. 2nd ed. Oxford (UK): Oxford University Press; 2002. (13.) Komar N, Langevin S, Hinten S, Nemeth N, Edwards E, Hettler D, et al. Experimental infection of North American birds <onlyinclude> This list of North American birds is a comprehensive listing of all the bird species known from the North American continent north of Mexico. </onlyinclude> with the New York 1999 strain of West Nile virus. Emerg Infect Dis. 2003;9:311-22. (14.) Gubler DJ, Campbell GL, Nasci R, Komar N, Petersen L, Roehrig JT. West Nile virus in the United States: guidelines for detection, prevention, and control. Viral Immunol. 2000; 13:469-75. Nicholas A. Panella, * Kristen L. Burkhalter, * Stanley A. Langevin, * Aaron C. Brault, * Lynn M. Schooley, * Brad J. Biggerstaff, * Roger S. Nasci, * and Nicholas Komar * * 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. , Fort Collins, Colorado The City of Fort Collins, a home rule municipality situated on the Cache la Poudre River along the Colorado Front Range, is the county seat and most populous city in Larimer County, Colorado. , USA Mr Panella is a biologist in 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 Diseases Branch, Division of Vector-Borne Infectious Diseases, Centers for Disease Control and Prevention, Fort Collins, Colorado. He has participated in WNV outbreak investigations and has contributed to studies of WNV ecology in the United States. Address for correspondence: Nicholas A. Panella, Arbovirus Diseases Branch, Centers for Disease Control and Prevention, PO Box 2087, Fort Collins, CO 80522, USA: fax: 970-221-6476; email: nap4@cdc.gov
Table 1. Sensitivities of the VecTest and plaque assay in detecting
West Nile virus in American Crows and House Sparrows, by swab type
and day postmortem *
Swab type
Cloacal,
Day Oral, % positive % positive
Species postmortem Assay (no. tested) (no. tested)
American Crow 1 VecTest 90 (30) 90 (29)
Plaque 83 (30) 69 (29)
2 VecTest 93 (30) 100 (29)
Plaque 90 (30) 55 (29)
3 VecTest 97 (30) 83 (30)
Plaque 83 (30) 13 (30)
4 VecTest 100 (5) 80 (5)
Plaque 80 (5) 0 (5)
House Sparrow 1 VecTest 70 (10) 67 (9)
Plaque 90 (10) 56 (9)
2 VecTest 75 (8) 100 (2)
Plaque 75 (8) 50 (2)
3 VecTest 83 (6) NA
Plaque 83 (6) NA
* NA, not available (valid sampling was not possible given the physical
state of the bird).
Table 2. Log geometric mean titer (SD) of West Nile virus PFU
equivalents (TaqMan RT-PCR) and PFU (plaque assay) for American
Crows and House Sparrows, by swab type and day postmortem *
Day Postmortem
Species Swab type Assay 1 2
American Crow Oral TaqMan 4.8 (1.1) 4.7 (0.6)
Plaque 3.6 (1.9) 3.2 (1.5)
Cloacal TaqMan 5.2 (1.2) 5.5 (1.3)
Plaque 3.0 (2.4) 1.4 (1.5)
House Sparrow Oral TaqMan 4.3 (l.4) 5.0 (1.7)
Plaque 3.8 (2.0) 3.0 (1.9)
Cloacal TaqMan 3.8 (1.6) 4.3 (3.3)
Plaque 1.9 (2.9) 0.7 (0.9)
Day Postmortem
Species Swab type Assay 3 4
American Crow Oral TaqMan 4.3 (0.8) 3.5 (l.5)
Plaque 2.4 (1.6) 2.2 (2.0)
Cloacal TaqMan 4.6 (l.9) 3.5 (0.7)
Plaque 0.3 (0.7) 0.0 (0.0)
House Sparrow Oral TaqMan 4.6 (1.7) NA
Plaque 2.9 (2.5) NA
Cloacal TaqMan NA NA
Plaque NA NA
* RT-PCR, reverse transcription-polymerase chain reaction; NA, not
available (valid sampling was not possible given the physical state
of the bird).
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