Higher sika deer density is associated with higher local abundance of Haemaphysalis longicornis nymphs and adults but not larvae in central Japan.

Haemaphysalis longicornis (Acari: Ixodidae) is one of the most common and important arthropod disease vectors in Japan, carrying Japanese spotted fever and bovine theileriosis. The recent expansion of sika deer (Cervus nippon, Artiodactyla: Cervidae) populations, the most common wild host of H. longicornis, has also caused concern about increasing the risk of vector-borne diseases in Japan. We used generalized linear mixed model analysis to determine the relative contribution of deer density and other biological and abiotic factors on the abundance of H. longicornis ticks questing at each developmental stage. A total of 6223 H. longicornis adults, nymphs, and larvae were collected from 70 sites in three regions of central Japan. The abundance of questing adult and nymphal ticks was associated with deer density and other biotic and abiotic factors. However, the abundance of questing larvae showed no association with deer density but did show an association with other biotic and abiotic factors. These findings show that a high density of deer along with other biotic and abiotic factors is associated with increased risk of vector-borne diseases through amplified local abundance of questing nymphal and adult H. longicornis. Further, questing larvae abundance is likely regulated by environmental conditions and is likely correlated with survival potential or the distribution of other host species.

Experimental West Nile virus infection in aigamo ducks, a cross between wild ducks (Anas platyrhynchos) and domestic ducks (Anas platyrhynchos var. domesticus).

Four 2-wk-old and four 4-wk-old aigamo ducks, a cross between wild and domestic ducks (Anas platyrhynchos and Anas platyrhynchos var. domesticus, respectively), were infected with the NY99 strain of West Nile virus (WNV) to investigate WNV's pathogenicity in aigamo ducks and the possibility that they could transmit WNV. In the group of infected 2-wk-old aigamo ducks (2w-infection group), all of the ducks ate and drank less and showed decreased activity, some showed ataxia, and one died. Meanwhile, the group of infected 4 wk olds (4w-infection group) showed no clinical signs during the experimental period. Viremia was observed in all of the ducks in both age groups. Peak viral titers in the three surviving members of the 2w-infection group were 10(3.7)-10(5.3) plaque-forming units (PFU)/ml serum; the peak was 10(7.1) PFU/ml serum in the 2w duck that died from the infection. Peak viral titers in the 4w-infection group were 10(4.1)-10(4.9) PFU/ml serum. Viral shedding in the oral and/or cloacal cavity was observed in all four members of the 2w-infection group and in three of the four members of the 4w-infection group. These results suggest that WNV-infected aigamo ducks can transmit WNV. Although aigamo ducks are reared in East Asia, where WNV is an exotic pathogen, the virus could be introduced and spread there in the future; thus it is important to take precautions against an introduction, and measures to prevent infection to aigamo duck operations should be prepared.

Antibody responses induced by experimental West Nile virus infection with or without previous immunization with inactivated Japanese encephalitis vaccine in horses.

A group of horses immunized with inactivated Japanese encephalitis (JE) vaccine (JE-Immune Group) and a group of non-immunized horses (Non-Immune Group) were infected with West Nile virus (WNV). After WNV infection, neutralizing (Nt) antibody (Ab) titers to WNV were higher than those to JE virus (JEV) in the Non-Immune Group, but the NtAb titers to JEV were higher than those to WNV during most of the post-challenge observation period in the JE-Immune Group. Immunoglobulin M (IgM) Abs to WNV tested positive in the Non-Immune Group but negative in the JE-Immune Group, except for in one horse. These results suggest that diagnosis of WNV infection in JE-immunized horses requires serological tests for NtAb and IgM titers to both WNV and JEV.

The inhibitory effect of a combination of imidacloprid and permethrin on blood feeding by mosquitoes in dogs raised under outdoor conditions.

Combinations of imidacloprid and permethrin were frequently used to control harmful arthropod of companion animals. The inhibitory effects on blood-feeding activity of mosquitoes in dogs raised under outdoor conditions were evaluated by using combination of 10% (w/v) of imidacloprid and 50% (w/v) of permethrin as spot-on form. Dogs in the treated group received the combination imidacloprid/permethrin spot-on. After treatment, dogs in the control and treated groups were kept separately from the evening (17:00) to the morning of the following day (09:00) in two different kennels installed outdoors to mimic realistic dog-raising conditions. Mosquitoes in the kennels were collected by light traps placed in the kennels and a sweep net to determine evidence of blood feeding, and for species identification. Mosquitoes were collected at Days 5, 3 and 1 before agent treatment, and the Day of treatment, and Days 3, 7, 14, 21, 28, 35 and 42 after treatment. The percentages of blood-fed mosquitoes measured at Days 0, 3, 21, 28 and 42 after treatment were statistically significantly lower (p<0.01) in the treated group than in the control group. The most commonly collected mosquito, Culex tritaeniorhynchus, revealed statistically significant lower percentages (p<0.01) of blood-fed mosquitoes in the treated group than in the control group at the Day of treatment, and Days 3, 7, 21, 28 and 42 after treatment. It appeared that the test agent was effective in inhibiting blood feeding by adult female mosquitoes, and the efficacy lasts for 42 days after treatment under outdoor conditions.

Cloning and molecular characterization of a cubilin-related serine proteinase from the hard tick Haemaphysalis longicornis.

Serine proteinases are one of the largest proteolytic families of enzymes, and have diverse cellular activities in mammalian tissues. We report here the cloning and molecular characterization of a cDNA encoding the serine proteinase of the hard tick Haemaphysalis longicornis (HlSP). The HlSP cDNA is 1570 bp long and the deduced precursor protein consists of 464 amino acids with a predicted molecular mass of 50.4 kDa and a pI of 8.2. The preprotein, consisting of 443 amino acids, was predicted to include a complement C1r/C1s, Uegf, and bone morphogenic protein-1 domain, a low-density lipoprotein receptor class A domain, and a catalytic domain. HlSP sequence analysis showed high similarity to serine proteinases reported from arthropods and vertebrate animal species. Two-dimensional immunoblot analysis revealed endogenous HlSP in adult tick extracts at 50 kDa. Endogenous HlSP was also expressed in all lifecycle stages of H. longicornis. Immunohistochemical studies detected the endogenous enzyme in the midgut epithelial cells of an adult tick. The Escherichia coli-expressed recombinant HlSP was demonstrated to degrade bovine serum albumin and hydrolyze the substrate Bz-L-Arg-pNA at the rate of 30.2 micromol/min/mg protein. Further, HlSP expression was up-regulated during a blood-feeding process, indicating its involvement in the digestion of host blood components.

The expression system of biologically active canine interleukin-8 in Leishmania promastigotes.

It has been reported that Leishmania promastigotes have ability to express foreign genes on drug selectable plasmids. To investigate further abilities of the recently described expression vector, P6.5, in the transfection of Leishmania organisms (Chen D-Q, Kolli BK, Yadava N et al. Episomal expression of specific sense and antisense mRNAs in Leishmania amazonensis: modulation of gp63 levels in promastigotes and their infection of macrophages in vitro. Infect Immun 2000;68:80--86), the constructed expression vector, which contains canine interleukin-8 (cIL-8) coding cDNA, was introduced by electroporation to promastigotes of four species of the genus Leishmania: Leishmania amazonensis, L. equatorensis, L. donovani and L. infantum. Extrachromosomal DNAs and total RNAs from the transfected promastigotes were subjected to polymerase chain reaction (PCR) and reverse transcriptase-PCR, respectively, using cIL-8 gene specific primers, and a predicted product of 330 bp was detected. Western blot analysis using a mouse monoclonal antibody raised against cIL-8 demonstrated the successful expression of cIL-8 in the transfectants and culture supernatants. Culture supernatants of the transfected L. amazonensis and L. equatorensis promastigotes showed a high chemotactic activity to both dog and mouse polymorphonuclear leukocytes. These results indicate that Leishmania promastigotes transfected with the expression vector P6.5 containing cIL-8 cDNA are capable of producing biologically active cIL-8. The Leishmania expression system using the P6.5 vector might be a useful alternative for the production of biologically active recombinant cytokines.

Gene silencing of a cubilin-related serine proteinase from the hard tick Haemaphysalis longicornis by RNA interference.

RNA interference (RNAi) has been recently exploited to determine gene function by degrading specific mRNAs in several eukaryotic organisms. We constructed a double stranded RNA (dsRNA) from a previously cloned Haemaphysalis longicornis serine proteinase (HlSP) gene to test the importance of the function of the HlSP gene product during blood-feeding. Growth of unfed ticks treated with HlSP dsRNA was significantly inhibited compared to that of PBS-treated ticks. This inhibition was supported by the level of HlSP mRNA. HlSP may play a crucial role for blood-feeding in these ticks. This is the first report on gene silencing of a functional serine proteinase in hard ticks.

Enzymatic characterization of a cubilin-related serine proteinase from the hard tick Haemaphysalis longicornis.

In the present study, we performed enzymatic characterization of Haemaphysalis longicornis serine proteinase (HlSP) with a view to shed light on the mechanisms of blood digestion in the hard ticks. Escherichia coli-expressed recombinant HlSP (rHlSP) was shown to potently hydrolyze the synthetic substrates Bz-(DL)-Arg-pNA, Z-Ala-Ala-Leu-pNA and Suc-Ala-Ala-Ala-pNA and yielded an activity of 31.5, 88.2 and 18.3 mumol/min/mg protein, respectively at an optimum temperature of 25 degrees C. However, the enzyme showed little activity to hydrolyze the substrates Suc-Arg-Pro-Phe-His-Leu-Leu-Val-Tyr-MCA and Pyr-Phe-Leu-pNA. The optimum pH for the enzyme was shown to be 4.0 to 5.0. Several inhibitors such as antipain, leupeptin and phenylmethylsulfonyl fluoride (PMSF), specific for serine proteinase were shown to inhibit enzyme activity by 20-82%, while E-64 (specific for cysteine proteinases) and pepstatinA (specific for aspartic proteinases) had shown only little inhibitory effects on it. This is the first report on enzymatic characterization of a functional serine proteinase from the hard ticks.

Comparison of macrophage scavenger receptor-A knockout mice with wild type ones in the immune response against repeated infestation with Haemaphysalis longicornis.

Using macrophage scavenger receptor-A knockout (SRKO) mice, we examined the role of macrophage class A scavenger receptors (MRS-A) on the immune response and acquisition of host resistance against repeated infestation with Haemaphysalis longicornis. Except for one batch of nymphs that infested one of the SRKO (SR-/-) mice and showed no appreciable reduction in body weight, all the other groups of nymphs manifested significant decrease in body weight. Both SR-/- and wild type (SR+/+) mice showed a sustained increase in anti-tick antibody titers, but SR+/+ mice showed significantly higher titers. The IFN-gamma assayed in SR-/- mouse immune sera was substantially less compared with that in SR+/+ mice. Immune sera from SR-/- and SR+/+ mice recognized the 51 and 44 kDa, and 44 kDa proteins, respectively, of the salivary gland antigen. The difference in the level of anti-tick resistance manifested by both groups of mice may be influenced by less efficient trapping and processing of tick antigens by macrophages in mice lacking for the macrophage scavenger receptors, and consequently affected the cascade of Th1 and Th2 responses. We have thus obtained valuable data that strongly infer the role of MSR-A in enhancing host defense against repeated infestation with H. longicornis.

Specific antibody responses to West Nile virus infections in horses preimmunized with inactivated Japanese encephalitis vaccine: evaluation of blocking enzyme-linked immunosorbent assay and complement-dependent cytotoxicity assay.

West Nile virus (WNV) and Japanese encephalitis (JE) virus are distributed separately in the world with some exceptions. There is a concern that WNV may invade into Asia where JE virus exists. On and after such invasion, any differential diagnosis could be complicated by serological crossreactivities. We previously demonstrated experimentally using horses infected with WNV that preimmunization with inactivated JE vaccine considerably affected the ability of neutralization tests and immunoglobulin M (IgM) antibody-capture enzyme-linked immunosorbent assay (ELISA) to diagnose WNV infection. Here, we investigated WNV-specific antibody responses in vaccinated horses using a blocking ELISA and complement-dependent cytotoxicity (CDC) assay to evaluate these two newly developed serodiagnostic methods for WNV infection. Sera previously collected from six experimentally infected horses were used: Three were vaccinated before the infection, whereas the other three remained unvaccinated. WNV-specific antibody responses were successfully detected in the vaccinated and unvaccinated horses using both new methods, except for one vaccinated horse in which responses were not induced, probably as a result of crossprotection induced by JE vaccination. Specific antibody responses were at earliest detected from days 9 to 10 postinfection in the blocking ELISA, whereas the CDC assay provided earlier detection (at days 7-8) in all horses. The time courses of antibody levels were similar between vaccinated and unvaccinated horses in either method, indicating no notable effect of vaccination on detection of specific antibody responses, as far as antibodies were induced. These results indicated that blocking ELISA, but preferably the CDC assay, can be useful for detecting WNV infection in JE-vaccinated horses.

Experimental West Nile virus infection in jungle crows (Corvus macrorhynchos).

We experimentally infected jungle crows (Corvus macrorhynchos), which are representative corvids in East Asia, with West Nile virus (WNV) to study their susceptibility toward WNV infection. Six jungle crows were subcutaneously inoculated with 1,000 plaque-forming units (PFU) of the WNV NY99 strain. Within 7 days after inoculation, five of the six infected crows died, and peak viremias ranged from 10(6.5) to 10(10.9) PFU/mL serum. In addition, infected crows shed WNV in the oral cavity and cloaca, and the virus was widely disseminated in the organs of the crows. Based on these findings, we conclude that jungle crows are highly susceptible to WNV infection, and they could serve as amplifying hosts in the transmission of WNV. Although WNV has not been detected in East Asia, the virus could spread rapidly on introduction into this region because of the large number of potential amplifying hosts and vector mosquitoes that inhabit this region.

Identification and molecular characterization of a chitinase from the hard tick Haemaphysalis longicornis.

A cDNA encoding tick chitinase was cloned from a cDNA library of mRNA from Haemaphysalis longicornis eggs and designated as CHT1 cDNA. The CHT1 cDNA contains an open reading frame of 2790 bp that codes for 930 amino acid residues with a coding capacity of 104 kDa. The deduced amino acid sequence shows a 31% amino acid homology to Aedes aegypti chitinase and a multidomain structure containing one chitin binding peritrophin A domain and two glycosyl hydrolase family 18 chitin binding domains. The endogenous chitinase of H. longicornis was identified by a two-dimensional immunoblot analysis with mouse anti-rCHT1 serum and shown to have a molecular mass of 108 kDa with a pI of 5.0. A recombinant baculovirus AcMNPV.CHT1-expressed rCHT1 is glycosylated and able to degrade chitin. Chitin degradation was ablated by allosamidin in a dose-dependent manner. The optimal temperature and pH for activity of the purified chitinase were 45 degrees C and pH 5-7. The CHT1 cDNA has an ELR motif for chemokine-mediated angiogenesis and appears to be a chitinase of the chemokine family. Localization analysis using mouse anti-rCHT1 serum revealed that native chitinase is highly expressed in the epidermis and midgut of the tick. AcMNPV.CHT1 topically applied to H. longicornis ticks exhibited replication. This is the first report of insect baculovirus infection of ticks. The importance of AcMNPV.CHT1 as a novel bio-acaricide for tick control is discussed.