Rift Valley fever (RVF), a disease of ruminants and humans, has been responsible for large outbreaks in Africa that have resulted in hundreds of thousands of human infections and major economic disruption due to loss of livestock and to trade restrictions. As indicated by the rapid spread of West Nile viral activity across North America since its discovery in 1999 and the rapid and widespread movement of chikungunya virus from Africa throughout the Indian Ocean Islands to Asia and Europe, an introduced exotic arbovirus can be rapidly and widely established across wide geographical regions. Although RVF virus (RVFV) is normally transmitted by mosquitoes, we wanted to determine the potential for this virus to replicate in 2 of the most globally distributed and common higher flies: house flies, Musca domestica, and stable flies, Stomoxys calcitrans. Neither species supported the replication of RVFV, even after intrathoracic inoculation. However, S. calcitrans was able to mechanically transmit RVFV to susceptible hamsters (Mesocricetus auratus) after probing on infected hamsters with high viral titers. Therefore, S. calcitrans, because of its close association with domestic animals that serve as amplifying hosts of RVFV, should be considered a possible mechanical vector of RVFV, and it may contribute to the rapid spread of a RVF outbreak. Other Stomoxys species present in Africa and elsewhere may also play similar roles

Dohm, David J.

Geden, Christopher J.

Hogsette, Jerome A.

Linthicum, Kenneth

Turell, Michael J.

English

DigitalCommons@University of Nebraska

University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Publications from USDA-ARS / UNL Faculty U.S. Department of Agriculture: Agricultural Research Service, Lincoln, Nebraska 2010 Potential For Stable Flies And House Flies (Diptera: Muscidae) To Transmit Rift Valley Fever Virus1 Michael J. Turell US Army Medical Research Institute of Infectious Diseases David J. Dohm US Army Medical Research Institute of Infectious Diseases Christopher J. Geden United States Department of Agriculture, Chris.geden@ars.usda.gov Jerome A. Hogsette United States Department of Agriculture Kenneth Linthicum United States Department of Agriculture Follow this and additional works at: https://digitalcommons.unl.edu/usdaarsfacpub  Part of the Agricultural Science Commons Turell, Michael J.; Dohm, David J.; Geden, Christopher J.; Hogsette, Jerome A.; and Linthicum, Kenneth, "Potential For Stable Flies And House Flies (Diptera: Muscidae) To Transmit Rift Valley Fever Virus1" (2010). Publications from USDA-ARS / UNL Faculty. 998. https://digitalcommons.unl.edu/usdaarsfacpub/998 This Article is brought to you for free and open access by the U.S. Department of Agriculture: Agricultural Research Service, Lincoln, Nebraska at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Publications from USDA-ARS / UNL Faculty by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. SCIENTIFIC NOTEPOTENTIAL FOR STABLE FLIES AND HOUSE FLIES (DIPTERA:MUSCIDAE) TO TRANSMIT RIFT VALLEY FEVER VIRUS1MICHAEL J. TURELL,2 DAVID J. DOHM,2 CHRISTOPHER J. GEDEN,3JEROME A. HOGSETTE3 AND KENNETH J. LINTHICUM3ABSTRACT. Rift Valley fever (RVF), a disease of ruminants and humans, has been responsible for largeoutbreaks in Africa that have resulted in hundreds of thousands of human infections and major economicdisruption due to loss of livestock and to trade restrictions. As indicated by the rapid spread of West Nileviral activity across North America since its discovery in 1999 and the rapid and widespread movement ofchikungunya virus from Africa throughout the Indian Ocean Islands to Asia and Europe, an introducedexotic arbovirus can be rapidly and widely established across wide geographical regions. Although RVF virus(RVFV) is normally transmitted by mosquitoes, we wanted to determine the potential for this virus toreplicate in 2 of the most globally distributed and common higher flies: house flies, Musca domestica, andstable flies, Stomoxys calcitrans. Neither species supported the replication of RVFV, even after intrathoracicinoculation. However, S. calcitrans was able to mechanically transmit RVFV to susceptible hamsters(Mesocricetus auratus) after probing on infected hamsters with high viral titers. Therefore, S. calcitrans,because of its close association with domestic animals that serve as amplifying hosts of RVFV, should beconsidered a possible mechanical vector of RVFV, and it may contribute to the rapid spread of a RVFoutbreak. Other Stomoxys species present in Africa and elsewhere may also play similar roles.KEY WORDS RVF, vector, mechanical transmission, North America, emerging disease potentialThe introduction of West Nile virus into theUnited States in 1999 and its subsequent spreadacross North America, as well as the emergenceof chikungunya virus in Africa, the Indian OceanIslands, and much of southern Asia and Europe,illustrate the potential for exotic arboviruses to beintroduced and become established in newregions of the world and to cause significantdisease and economic disruption. Of particularconcern is Rift Valley fever virus (RVFV) (genusPhlebovirus, family Bunyaviridae), which has beenresponsible for numerous outbreaks of severedisease in ruminants and humans in Africa overthe past 80 years (Meegan and Bailey 1988,Gerdes 2004). Although originally limited to sub-Saharan Africa, an outbreak in Egypt in 1977caused an estimated 200,000 human cases as wellas having devastating effects on the sheep andcattle industries (Laughlin et al. 1979, Meegan1979). The detection of RVFV on the ArabianPeninsula (Jupp et al. 2002, Shoemaker et al.2002, Balkhy and Memish 2003, Madani et al.2003) has raised substantial concerns regardingthe agricultural and medical impact that thiszoonotic disease agent might have if it were tocontinue to spread (House et al. 1992). RecentRVFV activity has continued from 2006 to thepresent in various countries in Africa (Anyambaet al. 2009).Although most other members of the genusPhlebovirus are associated with sand flies innature, RVFV has been associated almost exclu-sively with mosquitoes, with the virus isolatedfrom at least 40 species in 8 genera (Meegan andBailey 1988, Fontenille et al. 1998). Laboratorystudies indicate that numerous species of mos-quitoes are susceptible to oral infection and areable to transmit RVFV by bite (McIntosh et al.1973b, 1980, 1983; Meegan and Bailey 1988,Turell et al. 1996, 2007, 2008b), including somepresent in the United States (Gargan et al. 1988,Turell et al. 2008a, 2010). However, the potentialfor higher Diptera, e.g., house flies, Muscadomestica L., and stable flies, Stomoxys calcitrans(L.), both of which are cosmopolitan in theirdistribution, has not been examined. Therefore,we evaluated the potential for these flies tobecome infected with and transmit RVFV afterintrathoracic inoculation and for S. calcitrans tomechanically transmit RVFV after taking apartial blood meal on a viremic hamster (Meso-cricetus auratus Waterhouse). RVFV is a select1 Research was conducted in compliance with theAnimal Welfare Act and other federal statutes andregulations relating to animals and experiments involv-ing animals and adheres to principles stated in theGuide for the Care and Use of Laboratory Animals,National Research Council, 1996. The facility wherethis research was conducted is fully accredited by theAssociation for Assessment and Accreditation ofLaboratory Animal Care International.The views of the authors do not necessarily reflect theposition of the Department of Defense or the Depart-ment of the Army.2 Virology Division, US Army Medical ResearchInstitute of Infectious Diseases, 1425 Porter Street, FortDetrick, MD 21702-5011.3 US Department of Agriculture—Agricultural Re-search Service (USDA-ARS) Center for Medical,Agricultural and Veterinary Entomology, 1600/1700SW 23rd Drive, Gainesville, FL 32608.Journal of the American Mosquito Control Association, 26(4):445–448, 2010Copyright E 2010 by The American Mosquito Control Association, Inc.445agent, and work with animals requires anagricultural biological safety level (ABSL)-3facility with vaccination or a biological safetylevel (BSL)-4 facility.Flies were provided as pupae from the USDepartment of Agriculture—Center for MedicalAgricultural and Veterinary Entomology(CMAVE) and were from long-established colo-nies that were maintained by standard methodsand rearing media (Hogsette 1992). Flies wereallowed to emerge as adults at the United StatesArmy Medical Research Institute of InfectiousDiseases (USAMRIID) and provided a mixtureof milk, sugar, and powdered egg (M. domestica)or a gauze pledget soaked in Gatorade (S.calcitrans) as a food source. Emerged adults weretransferred to an ABSL-3 laboratory at USAM-RIID and then exposed to RVFV.To determine whether RVFV could replicate ineither of these species, we inoculated individualflies intrathoracically (Rosen and Gubler 1974)with about 1 ml of the ZH501strain of RVFV.This strain was isolated in 1977 from the blood ofa 10-year-old Egyptian girl who had a fatalRVFV infection (Meagan 1979) and passed twicein fetal rhesus monkey (Macaca mulatta Zimmer-man) lung cells and once in Vero (African greenmonkey [Chlorocebus sabaeus L.] kidney) cellsbefore use in this study. The inoculum containedabout 105.5 plaque-forming units (PFU)/ml (102.5PFU/fly) of virus, and flies were placed in 0.9-litercardboard cages in an incubator maintained at27uC immediately after inoculation. House flieswere triturated individually 7 days later in 1 ml ofdiluent (10% heat-inactivated fetal bovine serumin Medium 199 with Earle’s salts [Invitrogen Inc.,Carlsbad, CA] and antibiotics) and then frozen at270uC until tested for infectious virus by aplaque assay on Vero cell monolayers. Serial 10-fold dilutions of each specimen were tested on 6-or 12-well plates as described by Gargan et al.(1983). Viral titers were expressed as log10 PFUper specimen. Stable flies were allowed to feedeither individually, or in small groups, on naı¨veadult female Syrian hamsters (Harlan SpragueDawley, Indianapolis, IN) 7 days after inocula-tion. Immediately after the feeding attempt, theflies were killed by freezing and trituratedindividually in 1 ml of diluent and then frozenat 270uC until tested for infectious virus asdescribed above. Hamsters were observed for21 days for evidence of infection.In addition to the studies to determine whetherRVFV could replicate in house or stable flies, wealso determined the ability of a group ofuninoculated stable flies to mechanically transmitRVFV from a viremic hamster to naı¨ve ones.Hamsters were inoculated intraperitoneally with104.2 PFU in 0.1 ml of diluent and anesthetizedwith a ketamine, xylazine, and acepromazinesuspension either 28 or 40 h later, when virustiters were 106.9 and 109.7 PFU/ml. Groups ofuninfected stable flies were allowed to feed on theanesthetized hamsters. To simulate an interruptedfeeding scenario, when we observed a stable flythat had probed for 15–30 sec, we used anaspirator to capture it and placed it in anothercage. About 20 min later, we placed an anesthe-tized, naı¨ve hamster in a 3.8-liter cage withscreening over the top and added one of the‘‘virus-exposed’’ stable flies to that cage andallowed it to continue feeding. A total of 13 flieswere tested in this manner using 7 from the high-and 6 from the low-titer donor hamsters,respectively. After the stable flies had fed on thehigh-titered hamster and on the naı¨ve hamsterswe froze them immediately, triturated theirproboscises and bodies separately in 1 ml ofdiluent, and froze these suspensions at 270uCuntil tested for infectious virus as above. Thehamsters were observed daily. Because RVFV isvirtually 100% fatal in hamsters, death, oreuthanasia if moribund, of these hamsters wasconsidered evidence of virus transmission. Pres-ence of virus was verified by isolating virus frombrain tissue from a subset of the dead oreuthanized hamsters (data not shown).None of the flies contained infectious RVFVwhen tested 7 days after intrathoracic inoculation(Table 1), and none of the 17 inoculated stableflies that fed on a hamster 7 days after inoculationtransmitted virus by bite. Therefore, neitherspecies of fly was able to support replication ofRVFV, and neither species would be able totransmit RVFV biologically.When allowed to probe on a hamster with aviremia of 106.9 PFU/ml, none of the S. calcitranstransmitted virus mechanically; however, wheninitially exposed to a hamster with a viremia of109.7 PFU/ml, 4 of 7 (57%) flies transmitted thevirus mechanically (Table 2). This confirms anTable 1. Lack of replication in stable flies and houseflies inoculated with 102.2 plaque-forming units of RiftValley fever virus.Species Number tested Infection rate1Stomoxys calcitrans 13 0Musca domestica 8 01 Percentage of the inoculated flies that contained infectiousvirus 7 days after inoculation.Table 2. Mechanical transmission of Rift Valley fevervirus replication by stable flies.Viremia indonor hamsterTransmission to recipient hamstersNumber tested Transmission rate1106.9 PFU/ml 6 0109.7 PFU/ml 7 571 Percentage of the exposed flies that mechanically transmittedRift Valley fever virus.446 JOURNAL OF THE AMERICAN MOSQUITO CONTROL ASSOCIATION VOL. 26, NO. 4earlier study by Hoch et al. (1985) that haddemonstrated that stable flies and mosquitoeswere able to mechanically transmit RVFV andthat this transmission was dependant on theviremia of the initial infected animal. Becauseviremias in lambs and calves are as high as 1010.2and 109.2 mouse intracranial LD50, respectively(McIntosh et al. 1973a), stable flies should be ableto mechanically transmit virus if exposed toRVFV-infected lambs or calves in a naturaloutbreak of RVF. When tested after feeding ona naı¨ve hamster about 20 min after they hadprobed on the hamster with a viremia of 109.7PFU/ml, we recovered RVFV from the bodies ofabout half of the flies. However there was nocorrelation between the amounts of virus detect-ed, or even the presence of virus in the fly’s bodyand mechanical transmission. Similarly, we onlydetected RVFV (1 PFU detected) from a singleproboscis sample, and that was from a nontrans-mitting fly.Even though they are not susceptible toinfection and thus unable to transmit virusbiologically, stable flies may play an importantrole in the explosive amplification of a RVFVoutbreak. Rift Valley fever virus is extremelystable, and infectious titers remained nearlyconstant, even in samples held at ambienttemperature (about 22uC) for 24 h (MJT,unpublished data). Similarly, stable fly bites arepainful and often lead to the fly being unable toobtain a complete blood meal. This leads tointerrupted feedings and greatly increases thelikelihood of mechanical transmission. Therehave been 14 species of stable flies identified inAfrica (Mihok et al. 1995), and populations of200 Stomoxys nigra Macquart per cow have beenrecorded in Mauritius (Kunz and Monty 1976);populations of between 50 and 200 S. calcitransper cow have been observed in the United States,Europe, South America, and Australia (JAH,unpublished data). Therefore, considerationshould be given to control of these mechanicalvectors of RVFV.We thank Melissa Doyle and Heather Furlongfor rearing and providing house and stable flies,respectively (from CMAVE); J. Williams forcaring for the hamsters; and K. Kenyon forcritically reading the manuscript.The work was supported in part by a grantfrom the Deployed War-Fighter Protection Re-search Program funded by the US Department ofDefense through the Armed Forces Pest Man-agement Board.REFERENCES CITEDAnyamba A, Chretien JP, Small J, Tucker CJ,Formenty P, Richardson JH, Britch SC, SchnabelDC, Erickson RL, Linthicum KJ. 2009. Prediction ofthe Rift Valley fever outbreak in the Horn of Africa2006–2007. Proc Natl Acad Sci 106:955–959.Balkhy HH, Memish ZA. 2003. Rift Valley fever: anuninvited zoonosis in the Arabian Peninsula.Int J Antimicrob Agents 21:153–157.Fontenille D, Traore-Lamizana M, Diallo M, ThonnonJ, Digoutte JP, Zeller HG. 1998. New vectors of RiftValley fever in West Africa. Emerg Infect Dis 4:289–293.Gargan TP 2nd, Bailey CL, Higbee GA, Gad A, El SaidS. 1983. The effect of laboratory colonization on thevector pathogen interactions of Egyptian Culexpipiens and Rift Valley fever virus. Am J Trop MedHyg 32:1154–1163.Gargan TP 2nd, Clark GG, Dohm DJ, Turell MJ,Bailey CL. 1988. Vector potential of selected NorthAmerican mosquito species for Rift Valley fevervirus. Am J Trop Med Hyg 38:440–446.Gerdes GH. 2004. Rift Valley fever. Rev Sci Tech23:613–623.Hoch AL, Gargan TP 2nd, Bailey CL. 1985. Mechanicaltransmission of Rift Valley fever virus by hematoph-agous Diptera. Am J Trop Med Hyg 34:188–193.Hogsette JA. 1992. New diets for production of houseflies and stable flies (Diptera: Muscidae) in thelaboratory. J Econ Entomol 85:2291–2294.House JA, Turell MJ, Mebus CA. 1992. Rift Valleyfever: present status and risk to the WesternHemisphere. Ann N Y Acad Sci 653:233–342.Jupp PG, Kemp A, Grobbelaar A, Lema P, Burt FJ,Alahmed AM, Al Mujalli D, Al Khamees M,Swanepoel R. 2002. The 2000 epidemic of Rift Valleyfever in Saudi Arabia: mosquito vector studies. MedVet Entomol 16:245–252.Kunz SE, Monty J. 1976. Biology and ecology ofStomoxys nigra Macquart and Stomoxys calcitrans(L.) (Diptera, Muscidae) in Mauritius. Bull EntomolRes 66:745–755.Laughlin LW, Meegan JM, Strausbaugh LJ, MorensDM, Watten RH. 1979. Epidemic Rift Valley fever inEgypt: observations on the spectrum of humanillness. Trans R Soc Trop Med Hyg 73:630–633.Madani TA, Al-Mazrou YY, Al-Jeffri MH, MishkhasAA, Al-Rabeah AM, Turkistani AM, Al-Sayed MO,Abodahish AA, Khan AS, Ksiazek TG, ShobokshiO. 2003. Rift Valley fever epidemic in Saudi Arabia:epidemiological, clinical, and laboratory characteris-tics. Clin Infect Dis 37:1084–1092.McIntosh BM, Dickinson DB, Dos Santos I. 1973a.Rift Valley fever. 3. Viremia in cattle and sheep. 4.The susceptibility of mice and hamsters in relation totransmission of virus by mosquitoes. J S Afr Vet MedAssoc 44:167–169.McIntosh BM, Jupp PG, Anderson D, Dickinson DB.1973b. Rift Valley fever. 2. Attempts to transmit viruswith seven species of mosquito. J S Afr Vet MedAssoc 44:57–60.McIntosh BM, Jupp PG, Dos Santos I, Barnard BJH.1980. Vector studies on Rift Valley fever in SouthAfrica. S Afr Med J 58:127–132.McIntosh BM, Jupp PG, Dos Santos I, Rowe AC.1983. Field and laboratory evidence implicatingCulex zombaensis and Aedes circumluteolus as vectorsof Rift Valley fever virus in coastal South Africa.S Afr J Sci 79:61–64.Meegan JM. 1979. The Rift Valley fever epizootic inEgypt 1977–78. 1. Description of the epizootic andDECEMBER 2010 SCIENTIFIC NOTE 447virological studies. Trans R Soc Trop Med Hyg 73:618–623.Meegan JM, Bailey CL. 1988. Rift Valley fever. In: MonathTP, ed. The arboviruses: epidemiology and ecology.Volume 4. Boca Raton, FL: CRC Press. p 61–76.Mihok S, Kang’ethe EK, Kamau GK. 1995. Trials oftraps and attractants for Stomoxys spp. (Diptera:Muscidae). J Med Entomol 32:283–289.Rosen L, Gubler D. 1974. The use of mosquitoes todetect and propagate dengue viruses. Am J Trop MedHyg 23:1153–1160.Shoemaker T, Boulianne C, Vincent MJ, Pezzanite L,Al-Qahtani MM, Al-Mazrou Y, Khan AS, RollinPE, Swanepoel R, Ksiazek TG, Nichol ST. 2002.Genetic analysis of viruses associated with emergenceof Rift Valley fever in Saudi Arabia and Yemen,2000–01. Emerg Infect Dis 8:1415–1420.Turell MJ, Bennett KA, Wilson WC. 2010. Potential forNorth American mosquitoes (Diptera: Culicidae) to trans-mit Rift Valley fever virus. J Med Entomol 47:884–889.Turell MJ, Dohm DJ, Mores CN, Terracina L, WalletteDL Jr., Hribar LJ, Pecor JE, Blow JA. 2008a.Potential for North American mosquitoes to transmitRift Valley fever virus. J Am Mosq Control Assoc24:502–507.Turell MJ, Lee JS, Richardson JH, Sang RC, KiokoEN, Agawo MO, Pecor J, O’Guinn ML. 2007.Vector competence of Kenyan Culex zombaensisand Culex quinquefasciatus mosquitoes for RiftValley fever virus. J Am Mosq Control Assoc 23:378–382.Turell MJ, Linthicum KJ, Patrican LA, Davies FG,Kairo A, Bailey CL. 2008b. Vector competence ofselected African mosquito (Diptera: Culicidae) spe-cies for Rift Valley fever virus. J Med Entomol 45:102–108.Turell MJ, Presley SM, Gad AM, Cope SE, Dohm DJ,Morrill JC, Arthur RR. 1996. Vector competence ofEgyptian mosquitoes for Rift Valley fever virus.Am J Trop Med Hyg 54:136–139.448 JOURNAL OF THE AMERICAN MOSQUITO CONTROL ASSOCIATION VOL. 26, NO. 4

Potential For Stable Flies And House Flies (Diptera:
Muscidae) To Transmit Rift Valley Fever Virus1

UNL | Libraries

University of Nebraska - Lincoln 
DigitalCommons@University of Nebraska - Lincoln 
Publications from USDA-ARS / UNL Faculty U.S. Department of Agriculture: Agricultural Research Service, Lincoln, Nebraska 
2010 
Potential For Stable Flies And House Flies (Diptera: Muscidae) To 
Transmit Rift Valley Fever Virus1 
Michael J. Turell 
US Army Medical Research Institute of Infectious Diseases 
David J. Dohm 
US Army Medical Research Institute of Infectious Diseases 
Christopher J. Geden 
United States Department of Agriculture, Chris.geden@ars.usda.gov 
Jerome A. Hogsette 
United States Department of Agriculture 
Kenneth Linthicum 
United States Department of Agriculture 
Follow this and additional works at: https://digitalcommons.unl.edu/usdaarsfacpub 
 Part of the Agricultural Science Commons 
Turell, Michael J.; Dohm, David J.; Geden, Christopher J.; Hogsette, Jerome A.; and Linthicum, Kenneth, 
"Potential For Stable Flies And House Flies (Diptera: Muscidae) To Transmit Rift Valley Fever Virus1" 
(2010). Publications from USDA-ARS / UNL Faculty. 998. 
https://digitalcommons.unl.edu/usdaarsfacpub/998 
This Article is brought to you for free and open access by the U.S. Department of Agriculture: Agricultural Research 
Service, Lincoln, Nebraska at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in 
Publications from USDA-ARS / UNL Faculty by an authorized administrator of DigitalCommons@University of 
Nebraska - Lincoln. 
SCIENTIFIC NOTE
POTENTIAL FOR STABLE FLIES AND HOUSE FLIES (DIPTERA:
MUSCIDAE) TO TRANSMIT RIFT VALLEY FEVER VIRUS1
MICHAEL J. TURELL,2 DAVID J. DOHM,2 CHRISTOPHER J. GEDEN,3
JEROME A. HOGSETTE3 AND KENNETH J. LINTHICUM3
ABSTRACT. Rift Valley fever (RVF), a disease of ruminants and humans, has been responsible for large
outbreaks in Africa that have resulted in hundreds of thousands of human infections and major economic
disruption due to loss of livestock and to trade restrictions. As indicated by the rapid spread of West Nile
viral activity across North America since its discovery in 1999 and the rapid and widespread movement of
chikungunya virus from Africa throughout the Indian Ocean Islands to Asia and Europe, an introduced
exotic arbovirus can be rapidly and widely established across wide geographical regions. Although RVF virus
(RVFV) is normally transmitted by mosquitoes, we wanted to determine the potential for this virus to
replicate in 2 of the most globally distributed and common higher flies: house flies, Musca domestica, and
stable flies, Stomoxys calcitrans. Neither species supported the replication of RVFV, even after intrathoracic
inoculation. However, S. calcitrans was able to mechanically transmit RVFV to susceptible hamsters
(Mesocricetus auratus) after probing on infected hamsters with high viral titers. Therefore, S. calcitrans,
because of its close association with domestic animals that serve as amplifying hosts of RVFV, should be
considered a possible mechanical vector of RVFV, and it may contribute to the rapid spread of a RVF
outbreak. Other Stomoxys species present in Africa and elsewhere may also play similar roles.
KEY WORDS RVF, vector, mechanical transmission, North America, emerging disease potential
The introduction of West Nile virus into the
United States in 1999 and its subsequent spread
across North America, as well as the emergence
of chikungunya virus in Africa, the Indian Ocean
Islands, and much of southern Asia and Europe,
illustrate the potential for exotic arboviruses to be
introduced and become established in new
regions of the world and to cause significant
disease and economic disruption. Of particular
concern is Rift Valley fever virus (RVFV) (genus
Phlebovirus, family Bunyaviridae), which has been
responsible for numerous outbreaks of severe
disease in ruminants and humans in Africa over
the past 80 years (Meegan and Bailey 1988,
Gerdes 2004). Although originally limited to sub-
Saharan Africa, an outbreak in Egypt in 1977
caused an estimated 200,000 human cases as well
as having devastating effects on the sheep and
cattle industries (Laughlin et al. 1979, Meegan
1979). The detection of RVFV on the Arabian
Peninsula (Jupp et al. 2002, Shoemaker et al.
2002, Balkhy and Memish 2003, Madani et al.
2003) has raised substantial concerns regarding
the agricultural and medical impact that this
zoonotic disease agent might have if it were to
continue to spread (House et al. 1992). Recent
RVFV activity has continued from 2006 to the
present in various countries in Africa (Anyamba
et al. 2009).
Although most other members of the genus
Phlebovirus are associated with sand flies in
nature, RVFV has been associated almost exclu-
sively with mosquitoes, with the virus isolated
from at least 40 species in 8 genera (Meegan and
Bailey 1988, Fontenille et al. 1998). Laboratory
studies indicate that numerous species of mos-
quitoes are susceptible to oral infection and are
able to transmit RVFV by bite (McIntosh et al.
1973b, 1980, 1983; Meegan and Bailey 1988,
Turell et al. 1996, 2007, 2008b), including some
present in the United States (Gargan et al. 1988,
Turell et al. 2008a, 2010). However, the potential
for higher Diptera, e.g., house flies, Musca
domestica L., and stable flies, Stomoxys calcitrans
(L.), both of which are cosmopolitan in their
distribution, has not been examined. Therefore,
we evaluated the potential for these flies to
become infected with and transmit RVFV after
intrathoracic inoculation and for S. calcitrans to
mechanically transmit RVFV after taking a
partial blood meal on a viremic hamster (Meso-
cricetus auratus Waterhouse). RVFV is a select
1 Research was conducted in compliance with the
Animal Welfare Act and other federal statutes and
regulations relating to animals and experiments involv-
ing animals and adheres to principles stated in the
Guide for the Care and Use of Laboratory Animals,
National Research Council, 1996. The facility where
this research was conducted is fully accredited by the
Association for Assessment and Accreditation of
Laboratory Animal Care International.
The views of the authors do not necessarily reflect the
position of the Department of Defense or the Depart-
ment of the Army.
2 Virology Division, US Army Medical Research
Institute of Infectious Diseases, 1425 Porter Street, Fort
Detrick, MD 21702-5011.
3 US Department of Agriculture—Agricultural Re-
search Service (USDA-ARS) Center for Medical,
Agricultural and Veterinary Entomology, 1600/1700
SW 23rd Drive, Gainesville, FL 32608.
Journal of the American Mosquito Control Association, 26(4):445–448, 2010
Copyright E 2010 by The American Mosquito Control Association, Inc.
445
agent, and work with animals requires an
agricultural biological safety level (ABSL)-3
facility with vaccination or a biological safety
level (BSL)-4 facility.
Flies were provided as pupae from the US
Department of Agriculture—Center for Medical
Agricultural and Veterinary Entomology
(CMAVE) and were from long-established colo-
nies that were maintained by standard methods
and rearing media (Hogsette 1992). Flies were
allowed to emerge as adults at the United States
Army Medical Research Institute of Infectious
Diseases (USAMRIID) and provided a mixture
of milk, sugar, and powdered egg (M. domestica)
or a gauze pledget soaked in Gatorade (S.
calcitrans) as a food source. Emerged adults were
transferred to an ABSL-3 laboratory at USAM-
RIID and then exposed to RVFV.
To determine whether RVFV could replicate in
either of these species, we inoculated individual
flies intrathoracically (Rosen and Gubler 1974)
with about 1 ml of the ZH501strain of RVFV.
This strain was isolated in 1977 from the blood of
a 10-year-old Egyptian girl who had a fatal
RVFV infection (Meagan 1979) and passed twice
in fetal rhesus monkey (Macaca mulatta Zimmer-
man) lung cells and once in Vero (African green
monkey [Chlorocebus sabaeus L.] kidney) cells
before use in this study. The inoculum contained
about 105.5 plaque-forming units (PFU)/ml (102.5
PFU/fly) of virus, and flies were placed in 0.9-liter
cardboard cages in an incubator maintained at
27uC immediately after inoculation. House flies
were triturated individually 7 days later in 1 ml of
diluent (10% heat-inactivated fetal bovine serum
in Medium 199 with Earle’s salts [Invitrogen Inc.,
Carlsbad, CA] and antibiotics) and then frozen at
270uC until tested for infectious virus by a
plaque assay on Vero cell monolayers. Serial 10-
fold dilutions of each specimen were tested on 6-
or 12-well plates as described by Gargan et al.
(1983). Viral titers were expressed as log10 PFU
per specimen. Stable flies were allowed to feed
either individually, or in small groups, on naı¨ve
adult female Syrian hamsters (Harlan Sprague
Dawley, Indianapolis, IN) 7 days after inocula-
tion. Immediately after the feeding attempt, the
flies were killed by freezing and triturated
individually in 1 ml of diluent and then frozen
at 270uC until tested for infectious virus as
described above. Hamsters were observed for
21 days for evidence of infection.
In addition to the studies to determine whether
RVFV could replicate in house or stable flies, we
also determined the ability of a group of
uninoculated stable flies to mechanically transmit
RVFV from a viremic hamster to naı¨ve ones.
Hamsters were inoculated intraperitoneally with
104.2 PFU in 0.1 ml of diluent and anesthetized
with a ketamine, xylazine, and acepromazine
suspension either 28 or 40 h later, when virus
titers were 106.9 and 109.7 PFU/ml. Groups of
uninfected stable flies were allowed to feed on the
anesthetized hamsters. To simulate an interrupted
feeding scenario, when we observed a stable fly
that had probed for 15–30 sec, we used an
aspirator to capture it and placed it in another
cage. About 20 min later, we placed an anesthe-
tized, naı¨ve hamster in a 3.8-liter cage with
screening over the top and added one of the
‘‘virus-exposed’’ stable flies to that cage and
allowed it to continue feeding. A total of 13 flies
were tested in this manner using 7 from the high-
and 6 from the low-titer donor hamsters,
respectively. After the stable flies had fed on the
high-titered hamster and on the naı¨ve hamsters
we froze them immediately, triturated their
proboscises and bodies separately in 1 ml of
diluent, and froze these suspensions at 270uC
until tested for infectious virus as above. The
hamsters were observed daily. Because RVFV is
virtually 100% fatal in hamsters, death, or
euthanasia if moribund, of these hamsters was
considered evidence of virus transmission. Pres-
ence of virus was verified by isolating virus from
brain tissue from a subset of the dead or
euthanized hamsters (data not shown).
None of the flies contained infectious RVFV
when tested 7 days after intrathoracic inoculation
(Table 1), and none of the 17 inoculated stable
flies that fed on a hamster 7 days after inoculation
transmitted virus by bite. Therefore, neither
species of fly was able to support replication of
RVFV, and neither species would be able to
transmit RVFV biologically.
When allowed to probe on a hamster with a
viremia of 106.9 PFU/ml, none of the S. calcitrans
transmitted virus mechanically; however, when
initially exposed to a hamster with a viremia of
109.7 PFU/ml, 4 of 7 (57%) flies transmitted the
virus mechanically (Table 2). This confirms an
Table 1. Lack of replication in stable flies and house
flies inoculated with 102.2 plaque-forming units of Rift
Valley fever virus.
Species Number tested Infection rate1
Stomoxys calcitrans 13 0
Musca domestica 8 0
1 Percentage of the inoculated flies that contained infectious
virus 7 days after inoculation.
Table 2. Mechanical transmission of Rift Valley fever
virus replication by stable flies.
Viremia in
donor hamster
Transmission to recipient hamsters
Number tested Transmission rate1
106.9 PFU/ml 6 0
109.7 PFU/ml 7 57
1 Percentage of the exposed flies that mechanically transmitted
Rift Valley fever virus.
446 JOURNAL OF THE AMERICAN MOSQUITO CONTROL ASSOCIATION VOL. 26, NO. 4
earlier study by Hoch et al. (1985) that had
demonstrated that stable flies and mosquitoes
were able to mechanically transmit RVFV and
that this transmission was dependant on the
viremia of the initial infected animal. Because
viremias in lambs and calves are as high as 1010.2
and 109.2 mouse intracranial LD50, respectively
(McIntosh et al. 1973a), stable flies should be able
to mechanically transmit virus if exposed to
RVFV-infected lambs or calves in a natural
outbreak of RVF. When tested after feeding on
a naı¨ve hamster about 20 min after they had
probed on the hamster with a viremia of 109.7
PFU/ml, we recovered RVFV from the bodies of
about half of the flies. However there was no
correlation between the amounts of virus detect-
ed, or even the presence of virus in the fly’s body
and mechanical transmission. Similarly, we only
detected RVFV (1 PFU detected) from a single
proboscis sample, and that was from a nontrans-
mitting fly.
Even though they are not susceptible to
infection and thus unable to transmit virus
biologically, stable flies may play an important
role in the explosive amplification of a RVFV
outbreak. Rift Valley fever virus is extremely
stable, and infectious titers remained nearly
constant, even in samples held at ambient
temperature (about 22uC) for 24 h (MJT,
unpublished data). Similarly, stable fly bites are
painful and often lead to the fly being unable to
obtain a complete blood meal. This leads to
interrupted feedings and greatly increases the
likelihood of mechanical transmission. There
have been 14 species of stable flies identified in
Africa (Mihok et al. 1995), and populations of
200 Stomoxys nigra Macquart per cow have been
recorded in Mauritius (Kunz and Monty 1976);
populations of between 50 and 200 S. calcitrans
per cow have been observed in the United States,
Europe, South America, and Australia (JAH,
unpublished data). Therefore, consideration
should be given to control of these mechanical
vectors of RVFV.
We thank Melissa Doyle and Heather Furlong
for rearing and providing house and stable flies,
respectively (from CMAVE); J. Williams for
caring for the hamsters; and K. Kenyon for
critically reading the manuscript.
The work was supported in part by a grant
from the Deployed War-Fighter Protection Re-
search Program funded by the US Department of
Defense through the Armed Forces Pest Man-
agement Board.
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