The potential of the eastern cottontail rabbit (CTR; Sylvilagus floridanus) to contribute to an enzootic West Nile virus (WNV) cycle was demonstrated by characterizing the WNV viremia profile of 15 CTRs and demonstrating that mosquitoes could become infected by feeding on these CTRs. Eight CTRs were infected with a titer of 10^5.0 cell-infectious dose 50% endpoints (CID₅₀s) of WNV (NY99-Crow) by needle and seven CTRs by bite of one or more WNV-infected mosquitoes. There were no marked differences between the WNV viremia profiles of CTRs infected by either method. West Nile virus was detected in serums of all CTRs by 24 h p.i. The daily mean titers of all 15 CTRs on days 1–4 p.i. were 10^4.1±0.4, 10^4.7±0.3, 10^4.1±0.6, and 10^3.7±0.6 respectively, declining to 10^1.2±0.1 CID₅₀s/ml of serum by day 6 p.i. No virus was detected in the blood of any CTR on day 7 p.i. The average duration of WNV titers of ≥10^4.3 and 10^5.0 were 11.5 ± 5.5 and 21 ± 6.0%, respectively. These rates increased to 20.5 ± 6.4% and 25.0 ± 3.0% when CTR serum titers were >10^5.0 CID₅₀s/mL. Neither Aedes aegypti (L.) nor Aedes albopictus (Skuse) were infected by feeding on CTRs with titers of 10^5.0 CID₅₀s/ml. None of the CTRs infected by mosquito bite or by needle showed any symptoms of WNV disease. Vector Borne Zoonotic Dis. 5, 342–350.

Transmission of West Nile virus (WNV) by Ochlerotatus trivittatus, Culex pipiens, and Aedes albopictus were compared 14 days after taking blood meals from viremic chickens with titers ranging from 10(2.5) to 10(9.5) cell infective dose (50)s (CID50s)/mL serum. Transmission occurred in one of four (25%) Oc. trivittatus and one of 25 (4%) Cx. pipiens that fed on chickens with titers of 10(5.5) CID50s/mL. No transmission occurred among two of 16 (13%) Oc. trivittatus or one of 25 (4%) Cx. pipiens that became infected after blood meals with titers of 10(5.0) and 10(4.5) CID50s/mL, the next lowest blood meal titers evaluated. Seventeen of 28 (61%) Ae. albopictus transmitted WNV after blood meals with titers of 10(7.0) CID50s/mL, but no infection or transmission was observed among 21 Ae. albopictus that fed on chickens with titers of 10(5.0) CID50s/mL, the next lowest titer evaluated. Transmission by all three species increased dramatically after blood meals with WNV titers of > or = 10(5.5) CID50s/mL. No significant differences occurred in dissemination and transmission rates of the three species after taking blood meals with titers of > 10(7.0) CID50s/mL. The cumulative mean +/- SE transmission rates of Oc. trivittatus, Cx. pipiens, and Ae. albopictus after blood meals with titers of > or = 10(7.0) CID50s/mL were 45.5 +/- 4.1%, 46.8 +/- 4.5%, and 72.4 +/- 5.5%. The cumulative mean dissemination rates of the three species were 78.3 +/- 6.7%, 74.8 +/- 2.6%, and 88.6 +/- 2.1%. The rates of transmission by the three species that developed disseminated infections after blood meals with titers of > or = 10(7.0) CID50s/mL were 58.8 +/- 4.4%, 62.6 +/- 5.8%, and 81.6 +/- 5.4%, respectively. In a previous study, we found that susceptibility of the three species to WNV was essentially the same when fed on chickens with WNV titers of > 10(7.0) CID50s/mL, but Oc. trivittatus and Cx. pipiens were more susceptible than Ae. albopictus to WNV at lower virus titers. The current study strongly suggests that Ae. albopictus is a more efficient vector than Oc. trivittatus and Cx. pipiens when fed blood meals with titers of > 10(7.0) CID50s/mL. However, Oc. trivittatus and Cx. pipiens might be more efficient as vectors when infected by blood meals with titers of < 10(7.0) CID50s/mL.

In eastern chipmunks (Tamias striatus) inoculated intramuscularly with 101.5 to 105.7 PFU of West Nile virus (WNV), serum titers developed sufficient to infect Aedes triseriatus (Say), Ae. vexans (Meigen), and Culex pipiens (L.). Mean titers (95% confidence interval) of 8 chipmunks were 103.9(3.3–4.5), 106.7(6.4–7.0), and 105.8(4.1–7.5) PFU/mL on days 1–3 postinoculation (p.i.) and 105.8 PFU/mL in 1 chipmunk on day 4 p.i. Mean estimated days that WNV titers were >104.8 and >105.6 were 1.7 (1.1–2.3) and 1.4 (1.0–1.6). The longest period of viremia >104.8 PFU/mL was 3–4 days. WNV antigen was detected in the small intestine of 2 chipmunks and the kidneys of 4 chipmunks by immunohistochemistry. WNV also was detected in urine, saliva, and feces of some chipmunks. These data suggest chipmunks might play a role in enzootic WNV cycles and be an amplifying host for mosquitoes that could infect humans.

Canine filariasis is caused by several nematode species, such as Dirofilaria immitis, Dirofilaria repens, Brugia pahangi, Brugia malayi, and Acanthocheilonema reconditum. Zoonotic filariasis is one of the world’s neglected tropical diseases. Since 2000, the World Health Organization (WHO) has promoted a global filarial eradication program to eliminate filariasis by 2020. Apart from vector control strategies, the infection control of reservoir hosts is necessary for more effective filariasis control. In addition, many studies have reported that Wolbachia is necessary for the development, reproduction, and survival of the filarial nematode. Consequently, the use of antibiotics to kill Wolbachia in nematodes has now become an alternative strategy to control filariasis. Previously, a case of subconjunctival dirofilariasis caused by Dirofilaria spp. has been reported in a woman who resides in the center of Bangkok, Thailand. Therefore, our study aimed to principally demonstrate the presence of filarial nematodes and Wolbachia bacteria in blood collected from domestic dogs from the Bangkok Metropolitan Region, Thailand. A total of 57 blood samples from dogs with suspected dirofilariasis who had visited veterinary clinics in Bangkok were collected. The investigations for the presence of microfilaria were carried out by using both microscopic and molecular examinations. PCR was used as the molecular detection method for the filarial nematodes based on the COI and ITS1 regions. The demonstration of Wolbachia was performed using PCR to amplify the FtsZ gene. All positive samples by PCR were then cloned and sequenced. The results showed that the filarial nematodes were detected in 16 samples (28.07%) using microscopic examinations. The molecular detection of filarial species using COI-PCR revealed that 50 samples (87.72%) were positive; these consisted of 33 (57.89%), 13 (22.81%), and 4 (7.02%) samples for D. immitis, B. pahangi, and B. malayi, respectively. While the ITS1-PCR showed that 41 samples (71.93%) were positive—30 samples (52.63%) were identified as containing D. immitis and 11 samples (19.30%) were identified to have B. pahangi, whereas B. malayi was not detected. Forty-seven samples (82.45%) were positive for Wolbachia DNA and the phylogenetic tree of all positive Wolbachia was classified into the supergroup C clade. This study has established fundamental data on filariasis associated with Wolbachia infection in domestic dogs in the Bangkok Metropolitan Region. An extensive survey of dog blood samples would provide valuable epidemiologic data on potential zoonotic filariasis in Thailand. In addition, this information could be used for the future development of more effective prevention and control strategies for canine filariasis in Thailand.

Haemosporida parasites of even-toed ungulates are diverse and globally distributed, but since their discovery in 1913 their characterization has relied exclusively on microscopy-based descriptions. In order to bring molecular approaches to bear on the identity and evolutionary relationships of ungulate malaria parasites, we conducted Plasmodium cytb-specific nested PCR surveys using blood from water buffalo in Vietnam and Thailand, and goats in Zambia. We found that Plasmodium is readily detectable from water buffalo in these countries, indicating that buffalo Plasmodium is distributed in a wider region than India, which is the only area in which buffalo Plasmodium has been reported. Two types (I and II) of Plasmodium sequences were identified from water buffalo and a third type (III) was isolated from goat. Morphology of the parasite was confirmed in Giemsa-reagent stained blood smears for the Type I sample. Complete mitochondrial DNA sequences were isolated and used to infer a phylogeny in which ungulate malaria parasites form a monophyletic clade within the Haemosporida, and branch prior to the clade containing bird, lizard and other mammalian Plasmodium. Thus it is likely that host switching of Plasmodium from birds to mammals occurred multiple times, with a switch to ungulates independently from other mammalian Plasmodium.

Vector competence of Aedes vexans (Meigen) and Culex pipiens pipiens L. (Diptera: Culicidae) for West Nile virus (family Flaviviridae, genus Flavivirus, WNV) was compared. Infection rates of both species were similar 14 d after feeding on chickens, with WNV titers ranging from 10(4.2) to 10(8.7) plaque-forming units (PFU)/ml. Median infectious doses and 95% confidence intervals (CI) were 10(6.0(5.8, 63)) and 10(5.7(5.4, 5.9)) PFU for Ae. vexans and Cx. p. pipiens, respectively. WNV transmission was not observed in Ae. vexans that fed on chickens with WNV titers < 10(5.0) PFU/ml, in contrast to a mean (95% CI) transmission rate of 7(2,18)% for Cx. p. pipiens. Mean WNV transmission rates for Ae. vexans and Cx. p. pipiens were 13(7,21)% and 10(5,19)%, respectively, after feeding on chickens with WNV titers of 10(5.3 +/- 0.1) and 10(5.7 +/- 0.1) PFU/ml, and 31(25,37)% and 41(30,53)% after feeding on chickens with WNV titers > or = 10(6.1 +/- 0.1) PFU/ml. Time postinfection (p.i.) significantly influenced WNV transmission by Ae. vexans as indicated by a nearly 10-fold increase in transmission rate between days 7 and 14 p.i. Mean WNV load expectorated with saliva ofAe. vexans was 10(2.4(2.1, 2.7)) PFU, and it was not significantly affected by the titer of chickens on which they originally fed or time p.i. These data indicate that vector competence of the primarily mammalophilic Ae. vexans, which also feeds on birds, approaches that of Cx. p. pipiens for WNV. Because peridomestic mammals, such as cottontail rabbits, squirrels, and chipmunks, develop WNV titers infective for Ae. vexans, this species may play a significant role in WNV enzootic cycles.