Journal of the American Mosquito Control Association, 2l(4):474-476,2OO5 Copyright @ 2005 by the American Mosquito Control Association, Inc.

SCIENTIFIC NOTE

DETECTION OF WEST NILE VIRUS RNA FROM THE LOUSE ICO STA AMERICANA (DIPTERA: )

ARY FARAJOLLAHI,'.' WAYNE J. CRANS,I DIANE NICKERSON,3 PATRICIA BRYANT4 BRUCE WOLE4 AMY GLASER,5 INn THEODORE G. ANDREADIS6

ABSTRACT West Nile virus (WNV) was detected by Taqman reverse transcription-polymerase chain reaction in 4 of 85 (4.7Vo) blood-engorged (n : 2) and unengorged (n : 2) Icosta americana (Leach) hippoboscid that were collected from wild raptors submitted to a wildlife rehabilitation center in Mercer County, NJ, in 2003. This report represents an additional detection of WNV in a nonculicine in North America and the first documented detection of the virus in unengorged hippoboscid flies, further suggesting a possible role that this species may play in the transmission of WNV in North America.

KEY WORDS West Nile vrrns, Icosta americana, Hippoboscidae, raptors

West Nile virus (WNV) is a flavivirus that rs blood feeders that are commonly associated with principally maintained in an enzootic cycle between of prey. Both sexes readily take blood (Be- Culex mosquitoes and avian amplifying hosts. To quaert 1952, 1953; Maa and Peterson 1987) and date, the virus has been detected in or isolated from females are viviparous, exhibiting a high frequency 59 species of mosquitoes in the United States and of blood feeding due to nutritional demands of de- Canada (CDC 2004) and the importance of mos- veloping larvae (Bequaert 1952). Host speciflcity quitoes as enzootic and epidemic vectors of WNV varies greatly among -feeding species (Lloyd in North America is well established (Andreadis et 2OO2),bult most species remain and feed on the host al. 2001.20O4: Nasci etal.2OO2:.Ilribar 2OO3:Rut- throughout the year, thus increasing their potential ledge et al. 2OO3;Anderson et al. 2OO4; Mans et al. to locally acquire and transmit the virus as well as 2004). harbor it through the winter months. Winged hip- Although WNV isolations have been occasion- poboscids are also quite mobile (Lloyd 2OO2) and ally reported from bird-feeding argasid and ixodid they may stray from a live or dead host (Bequaert ticks in the Eastern Hemisphere (Hubalek and Hal- 1952), thus increasing their vectorial capacity. ouzka 1999), the role that nonculicine hematopha- During the active mosquito-breeding season, gous may play in the ecology of WNV wildlife rehabilitators regularly accept birds of prey in North America is unknown. Other than mosgui- that exhibit clinical signs of WNV infection, in- toes, the only other potential arthropod vectors cluding lethargy, neurological abnormalities, and from which WNV has been detected in nature has emaciation. However, because rehabilitation centers been a few blood-engorged hippoboscid louse flies focus on healing , only a small portion of (Icosta sp., Diptera: Hippoboscidae) collected from these birds are ever assayed for viral infection or a symptomatic WNV-positive gteat horned owl in examined for ectoparasites. Preliminary findings in- Pennsylvania (Komar 2003); from 16 lcosta amer- dicate that a high percentage of birds are infested icana (Leaclr) louse flies collected from dead or with louse flies. The current investigation was un- sick owls in Ontario, Canada (Gancz et al.2OO4)i dertaken to more fully assess the possible involve- and from 2 pools of the biting midge Culicoides ment of hippoboscids in the ecology of WNV in sonorensis (Wirth and Jones) (Diptera: Ceraptogon- the northeastern USA. idae) collected in Wyoming (Naugle et al.2OO4). Louse flies were collected from sick or injured Hippoboscid louse flies are obligate ectoparasitic raptors being held at a Wildlife Rehabilitation Cen- ter in Hopewell, NJ. Birds were inspected for ec- rMosquito Research and Control, Department of En- toparasites by trained wildlife rehabilitators, and tomology, Rutgers University, New Brunswick, NJ 08901. hippoboscids were removed and initially stored up 2 Present address: Mercer County Mosquito Control, to 3 wk at 4"C. Samples were later held at -70'C west Tfenton. NJ 08628. until identification and pooling could be conducted 3 Mercer County Wildlife Center, Titusville, NJ 08560. on a chill table by using the keys of Bequaert a New Jersey Department of Health and Senior Servic- (1954, 1955), Maa (1969), and Maa and Peterson Virology es, Deparffnent, Tlenton, NJ 08625. (1987). Individual louse flies were pooled accord- 5 Population Medicine and Diagnostic Sciences, College ing to date, location, species, gonotrophic status of Veterinary Medicine, Cornell University, New York, NY 14850. (empty or blooded), and host species. Specimens 6 Connecticut Agricultural Experiment Station, New were submitted to the New Jersey Department of Haven, CT 065O4. Health and Senior Services (NJDHSS), Thenton,

474 DBcrN4esn2005 ScENTrFrc NorE 475

Table 1. Distribution of West Nile virus (WNV) antibodies in wild raptors collected at a rehabilitation center in New Jersey, and summary of WNV detection by Taqman RT-PCR from the hippoboscid lcosta americana,2OO3. CT values Bird Collection .Bird Hippoboscid no. WNV* no. NJDHSS Bird ID no. soeciesr date Bird status serology2 (status)3 (status)4 (CAES)s MC-l163 RTH 26-Jul-03 Died < 24 h NT 4(u), l(E) l(E) 27.9 (29.19) MC-1214 BWH 29-Jul-03 Died < 24 h NT l(u), 1(E) 0 MC-1238 CPH 3 l -Jul-03 Euthanized on arrival NT 2(U) 0 MC-1311 GHO I l-Aug-03 Euthanized on arrival wNV 1(U) 0 MC-1318 GHO I l-Aug-03 Released 3l-Oct-03 wNv 1(u) 0 MC-1395 GHO 2l-Aug-O3 Died < 24 h NT 1(u) 0 MC-1494 RTH 29-Aug-03 Euthanized in care NT 1(U) 0 MC-l508 RTH 30-Aug-03 Died < 24 h NT 8(U),4(E) 1(u),l(E) 32.87(3s.38) 29.42(28.19) MC-1580 RTH 8-Sep-03 Euthanized in care NT 3(U) 0 MC-1583 RTH 8-Sep-03 Euthanized in care NT 2(U),2(E) 0 MC-1587 GHO 7-Sep-03 Died < 24 h NT 1(E) 0 MC-1603 GHO 9-Sep-03 Released 25-Oct-O3 wNV 4(E) 0 l,dc-1625 GHO I l-Sep-03 Died < 48 h NT 4(u),7(E) 0 l"dc-1629 RTH I I -Sep-03 Died < 24 h NT 1(U), l(E) 0 MC.1630 RTH I 2-Sep-03 Died < 24 h NT 1(E) 0 MC-I632 GHO l2-Sep-03 Released 25-Oct-03 wNV 4(E) 0 MC-1633 GHO 12-Sep-03 Died < 24 h NT 1(u), 1(E) 0 MC-I641 RTH 13-Sep-03 Died < 72 h NT 8(E) 0 MC-1646 GHO 13-Sep-03 Died < 24 h Nr 3(U),3(E) 0 MC-1690 cHO I 8-Sep-03 Released 29-Oct-03 NT 4(U) 0 MC-1766 GHO 26-Sep-03 Euthanized on arrival NT l(U),3(E) 0 MC-1788 GHO 30-Sep-03 Released 26-Feb-O4 wNv 2(u)6,4(E) 1(u) 30.97(27.9r) MC-1892 RTH 6-Nov-03 Released l5-Dec-03 NT 1(E) 0 Total 40(u),46(E) 2(U),2(E) rRTH, Red-tailed hawk; BWH, broad-winged hawk; CPH, Cooper's hawk; GHO, great homed owl. 'Birds serologically tested for antibodies by plaque reduction neutralization test; NT not tested. 3 U, unengorged; E, engorged. aHippoboscids individually tested by Thqman RT-PCR. 5 Cl cycle threshold based on a 45-cycle mplification threshold; NJDHSS, New Jersey Depaxtment of Health and Senior Services; CAES, Connecticut Agricultural Experiment Station. 6 lncf udes one Omithoica viccina.

NJ, for WNV testing via Taqman reverse transcrip- center, only a small portion was serologically tested tion-polymerase chain reaction (RT-PCR) proce- for WNV. Of the 6 raptors that were rehabilitated dures as outlined in Farajollahi et al. (2005). Virus and released, 4 tested positive for WNV-neutraliz- isolation attempts in Vero cell culture and confir- ing antibodies, while the remaining 2 were not test- mational testing by Taqman RT-PCR was addition- ed. Only 1 bird was tested of the 17 birds that either ally conducted on all positive pools at the Con- died in care or were euthanized, and that bird was necticut Agricultural Experiment Station (CAES), also positive for WNV-neutralizing antibodies. New Haven, Cl using techniques described in An- Of the 86 collected hippoboscids, 40 were unen- dreadis et al. (2OO4). Additionally, a portion of the gorged (empty), and 46 contained visible traces of birds were assayed at Cornell University, Ithaca, blood and were recorded as engorged (Thble 1). NY, for neutralizing antibodies to WNV by the Only a single specimen of Ornithoica viccina plaque-reduction neutralization test (Beaty et al. (Walker) was found, whereas all the remaining spe- 1989) essentially as described. cies consisted of lcosta americana. Infestations A total of 86 hippoboscids were collected from ranged from a single individual on a host to more Ju.ly 26, 2003 through November 6, 2OO3 frorn 23 than twelve, with a median of 3.7 hippoboscids per birds comprising 4 species (Table l). Of the 23 rap- bird. Four L americana tested positive for WNV tors that were sampled, 12 (52.2Vo) were great viral RNA during the initial RT-PCR testing at horned owls, 9 (39.l%o) were red-tailed hawks, 1 NJDHSS. These positives were also confirmed at (4.4Vo) was a broad-winged hawk, and | (4.4Vo) CAES by Taqman RT-PCR; however, live virus was was a Cooper's hawk. Eleven (47.8Eo) of the ad- not isolated by using the Vero cell culture assay. mitted birds died in care within i2 h or less, 6 Two of the positives were unengorged, whereas the (26.lVo) were either euthanized on arrival or during other two had traces of blood in the abdomen. care, and 6 (26.1Vo) were rehabilitated and released This investigation conflrms the earlier observa- back into the wild. Because most of the birds died tions from Pennsylvania and Ontario and demon- within a short time after submission to the wildlife strates that the hippoboscid I. americana is likely 476 JounNu- oF THE AMERTcANMosQUITo CoNrnol AssocIATIoN VoL. 21, No. 4 acquiring WNV from its host during the summer (Diptera) of mammals and birds. Part I. Structure, phys- season. Although we have no data on the vector iology and natural history. Entomol Am 33:217-442. JC. 1954. The Hippoboscidae or louse-flies competence of these arthropods, it is particularly Bequaert (Diptera) of mammals and birds. Part II. , that viral RNA was detected in unen- significant evolution and revision of American genera and species. gorged specimens as well as those with imbibed Entomol Am 34:-l-232. blood. This suggests that virus may be disseminated Bequaert JC. 1955. The Hippoboscidae or louse-flies from the gut into the hemocoel and host tissues. (Diptera) of mammals and birds. Part II. Taxonomy, Therefore, vector competency studies are clearly evolution and revision of American genera and species. walranted. Our inability to recover live WNV from Entomol Am 35:233-416. these PCR-positive specimens may have been due CDC [Centers for Disease Control and Prevention].2004. West Nile virus: entomology. Atlanta, GA: CDC. http:// to virus inactivation during our initial collection www.cdc.gov/ncidod/dvbid/westnile/mosquitospecies. and storage procedures. The cold chain was not htm. maintained properly, as specimens were placed in Farajollahi A, Crans WJ, Bryant R Wolf B, Burkhalter an ordinary refrigerator for up to 3 wk before pick- KL, Godsey MS, Aspen SA, Nasci RS. 2005. Detection up and appropriate storage. We also cannot rule out of West Nile viral RNA from an overwintering pool of the possibility that the level of virus in these spec- Culex pipiens pipiens (Diptera: Culicidae) in New Jer- imens was below the detection methods of the Vero sey, 2003. J Med Entomol 42:49O-494. Barker IK, Lindsay R, Dibemardo A, Mc- cell assay. However, we believe that further inves- Gancz AY, Keever K, Hunter B. 20M. West Nile virus outbreak in to more fully assess tigations should be conducted North American owls, Ontario, 2N2. 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