Swedish Culex torrentium and Cx. pipiens (Diptera: Culicidae) as Experimental Vectors of Ockelbo Virus JAN O. LUNDSTROM,12 BO NIKLASSON,13 AND D. BRUCE FRANCY4
KEY WORDS
Insecta, Culex spp., Ockelbo virus, virus transmission
OCKELBO DISEASE, characterized by arthralgia, rash, and in some cases fever, is endemic in central Sweden, where it occurs in late summer (Espmark & Niklasson 1984). An alphavirus isolated from Culiseta mosquitoes collected during the 1982 outbreak was associated with this disease (Niklasson et al. 1984). This virus, designated Ockelbo (OCK) virus, is serologically related closely to, but distinguishable from, Sindbis virus. The mosquito fauna of central Sweden is dominated by mammalophilic species of the genus Aedes (Jaenson & Niklasson 1986). However, in a recent study of an OCK disease focus, Francy et al. (1989) reported that 80% of the OCK virus isolates were made from ornithophilic mosquitoes, although these mosquitoes accounted for only 7% of the specimens collected. Also, neutralizing antibodies to OCK virus were prevalent in many of the common passerine bird species. These data indicated that OCK virus was maintained in an enzootic transmission cycle involving ornithophilic mosquitoes and birds. Culex pipiens (L.) and Cx. torrentium (Martini) are considered to be avian-feeding species (Service 1971, Jaenson & Niklasson 1986). Both occur in central Sweden (Dahl 1977, Francy et al. 1989), and OCK virus has been repeatedly isolated from Culex mosquitoes (Francy et al. 1989). Because of the difficulty in distinguishing adult females of these ' Department of Virology, National Bacteriological Laboratory, S-10521 Stockholm, Sweden. 2 Department of Virology, Karolinska Institute, % National Bacteriological Laboratory, S-10521 Stockholm, Sweden. 3 National Defense Research Establishment, FOA-5, Box 27322, S-10254 Stockholm, Sweden. * Vector-Borne Disease Division, Center for Disease Control, P. O. Box 2087, Fort Collins, Colo. 80522.
two species, it has not been possible to determine if both species were infected naturally or if the infection was restricted preferentially or exclusively to one species. Also, it was not known if infected mosquitoes of these species could transmit OCK virus by bite. To elucidate this ecological problem, we experimentally determined OCK virus infection and transmission rates in Cx. pipiens and Cx. torrentium after the ingestion of blood meals from a viremic chicken. Viremia profiles were determined for chickens inoculated with OCK virus, and mosquitoes then were fed on chickens at different intervals after infection to vary the concentration of the virus ingested. Materials and Methods Mosquitoes. Both Cx. pipiens and Cx. torrentium were colonized in the insectary at the National Bacteriological Laboratory from larvae collected in central Sweden during July 1985. Mosquitoes reared from these larve were provided chickens as a blood source. Egg rafts were hatched individually and the larve from individual egg rafts were held separately. A few males from each egg raft were identified to species based on characters of the terminalia as described by Mohrig (1969). Cx. pipiens larvae were collected from water-filled concrete containers in Uppsala (59°52'N, 17°38'E); and Cx. torrentium larvae were collected in a eutrophic, semipermanent pond in Bergsaker, close to Sundsvall (62°23'N, 17°17'E). Mosquitoes were reared at 21°C, 95% RH, and a 20:4 (L:D) photoperiod with 2 each of simulated dawn and dusk. Virus and Viral Assay Procedures. The Edsbyn 82/5 strain of OCK virus, isolated from Culiseta
0022-2585/90/0561-0563$02.00/0 © 1990 Entomological Society of America
Downloaded from http://jme.oxfordjournals.org/ by guest on June 9, 2016
J. Med. Entomol. 27(4): 561-563 (1990) ABSTRACT The ability of the sibling species Culex pipiens (L.) and Culex torrentium (Martini) from central Sweden to transmit Ockelbo (OCK) virus was determined. Both species became infected after ingesting OCK virus from a viremic chicken; they transmitted this virus to chickens after 21-28 d of extrinsic incubation. In Cx. torrentium, infection rates were 90% or higher, and all 10 refeeding mosquitoes transmitted virus after feeding on chickens with a viremia of at least 1030 plaque-forming units (PFU)/ml. In contrast, only 1 of 28 (4%) of the Cx. pipiens that ingested blood containing 1030-39 PFU/ml became infected, and none of 16 refeeding mosquitoes transmitted virus. However, 98 of 184 (53%) of the Cx. pipiens that ingested a blood meal containing at least 1060 PFU/ml became infected. Transmission rates in Cx. pipiens increased with increasing virus concentration in the blood meal to a maximum of 37% in mosquitoes that ingested >10 80 PFU/ml. Based on these data, both Cx. pipiens and Cx. torrentium are capable of transmitting OCK virus in an enzootic cycle involving birds as hosts. However, Cx. torrentium appears to be physiologically a more efficient vector than Cx. pipiens.
562
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Vol. 27, no. 4
JOURNAL OF MEDICAL ENTOMOLOGY
mean of positive tilers standard deviation number of viremic chickens/ total number tested
were tested for virus. Recovery of OCK virus from a chicken 1 d after exposure to a refeeding mosquito was considered evidence of virus transmission. Infection data were analyzed using Fisher's exact test (Siegel 1956). Results
Downloaded from http://jme.oxfordjournals.org/ by guest on June 9, 2016
All chickens tested were viremic from 0.5 to 3.5 d after infection, and the highest average viremia (1070 PFU/ml) was recorded 1 d after infection 3(Fig. 1). The viremia declined below detectable levels in five of eight chickens by 5.5 d, and only one of the chickens had a detectable viremia at 7.5 d after infection. Days post inoculation Mosquitoes fed on viremic chickens in several Fig. 1. Viremia profile in white leghorn chickens experiments. The viremias in individual chickens 31 inoculated subcutaneously with 10 PFU of Ockelbo exposed to Cx. torrentium were
KEY WORDS
Insecta, Culex spp., Ockelbo virus, virus transmission
OCKELBO DISEASE, characterized by arthralgia, rash, and in some cases fever, is endemic in central Sweden, where it occurs in late summer (Espmark & Niklasson 1984). An alphavirus isolated from Culiseta mosquitoes collected during the 1982 outbreak was associated with this disease (Niklasson et al. 1984). This virus, designated Ockelbo (OCK) virus, is serologically related closely to, but distinguishable from, Sindbis virus. The mosquito fauna of central Sweden is dominated by mammalophilic species of the genus Aedes (Jaenson & Niklasson 1986). However, in a recent study of an OCK disease focus, Francy et al. (1989) reported that 80% of the OCK virus isolates were made from ornithophilic mosquitoes, although these mosquitoes accounted for only 7% of the specimens collected. Also, neutralizing antibodies to OCK virus were prevalent in many of the common passerine bird species. These data indicated that OCK virus was maintained in an enzootic transmission cycle involving ornithophilic mosquitoes and birds. Culex pipiens (L.) and Cx. torrentium (Martini) are considered to be avian-feeding species (Service 1971, Jaenson & Niklasson 1986). Both occur in central Sweden (Dahl 1977, Francy et al. 1989), and OCK virus has been repeatedly isolated from Culex mosquitoes (Francy et al. 1989). Because of the difficulty in distinguishing adult females of these ' Department of Virology, National Bacteriological Laboratory, S-10521 Stockholm, Sweden. 2 Department of Virology, Karolinska Institute, % National Bacteriological Laboratory, S-10521 Stockholm, Sweden. 3 National Defense Research Establishment, FOA-5, Box 27322, S-10254 Stockholm, Sweden. * Vector-Borne Disease Division, Center for Disease Control, P. O. Box 2087, Fort Collins, Colo. 80522.
two species, it has not been possible to determine if both species were infected naturally or if the infection was restricted preferentially or exclusively to one species. Also, it was not known if infected mosquitoes of these species could transmit OCK virus by bite. To elucidate this ecological problem, we experimentally determined OCK virus infection and transmission rates in Cx. pipiens and Cx. torrentium after the ingestion of blood meals from a viremic chicken. Viremia profiles were determined for chickens inoculated with OCK virus, and mosquitoes then were fed on chickens at different intervals after infection to vary the concentration of the virus ingested. Materials and Methods Mosquitoes. Both Cx. pipiens and Cx. torrentium were colonized in the insectary at the National Bacteriological Laboratory from larvae collected in central Sweden during July 1985. Mosquitoes reared from these larve were provided chickens as a blood source. Egg rafts were hatched individually and the larve from individual egg rafts were held separately. A few males from each egg raft were identified to species based on characters of the terminalia as described by Mohrig (1969). Cx. pipiens larvae were collected from water-filled concrete containers in Uppsala (59°52'N, 17°38'E); and Cx. torrentium larvae were collected in a eutrophic, semipermanent pond in Bergsaker, close to Sundsvall (62°23'N, 17°17'E). Mosquitoes were reared at 21°C, 95% RH, and a 20:4 (L:D) photoperiod with 2 each of simulated dawn and dusk. Virus and Viral Assay Procedures. The Edsbyn 82/5 strain of OCK virus, isolated from Culiseta
0022-2585/90/0561-0563$02.00/0 © 1990 Entomological Society of America
Downloaded from http://jme.oxfordjournals.org/ by guest on June 9, 2016
J. Med. Entomol. 27(4): 561-563 (1990) ABSTRACT The ability of the sibling species Culex pipiens (L.) and Culex torrentium (Martini) from central Sweden to transmit Ockelbo (OCK) virus was determined. Both species became infected after ingesting OCK virus from a viremic chicken; they transmitted this virus to chickens after 21-28 d of extrinsic incubation. In Cx. torrentium, infection rates were 90% or higher, and all 10 refeeding mosquitoes transmitted virus after feeding on chickens with a viremia of at least 1030 plaque-forming units (PFU)/ml. In contrast, only 1 of 28 (4%) of the Cx. pipiens that ingested blood containing 1030-39 PFU/ml became infected, and none of 16 refeeding mosquitoes transmitted virus. However, 98 of 184 (53%) of the Cx. pipiens that ingested a blood meal containing at least 1060 PFU/ml became infected. Transmission rates in Cx. pipiens increased with increasing virus concentration in the blood meal to a maximum of 37% in mosquitoes that ingested >10 80 PFU/ml. Based on these data, both Cx. pipiens and Cx. torrentium are capable of transmitting OCK virus in an enzootic cycle involving birds as hosts. However, Cx. torrentium appears to be physiologically a more efficient vector than Cx. pipiens.
562
I7 o 6" •S 5 -
Vol. 27, no. 4
JOURNAL OF MEDICAL ENTOMOLOGY
mean of positive tilers standard deviation number of viremic chickens/ total number tested
were tested for virus. Recovery of OCK virus from a chicken 1 d after exposure to a refeeding mosquito was considered evidence of virus transmission. Infection data were analyzed using Fisher's exact test (Siegel 1956). Results
Downloaded from http://jme.oxfordjournals.org/ by guest on June 9, 2016
All chickens tested were viremic from 0.5 to 3.5 d after infection, and the highest average viremia (1070 PFU/ml) was recorded 1 d after infection 3(Fig. 1). The viremia declined below detectable levels in five of eight chickens by 5.5 d, and only one of the chickens had a detectable viremia at 7.5 d after infection. Days post inoculation Mosquitoes fed on viremic chickens in several Fig. 1. Viremia profile in white leghorn chickens experiments. The viremias in individual chickens 31 inoculated subcutaneously with 10 PFU of Ockelbo exposed to Cx. torrentium were
