Maturation of Theileria sergenti in salivary glands of Haemaphysalis longicornis induced by temperature stimulation

The parthenogenetic Haemaphysalis longicornis larvae engorged on cattle naturally infected with Theileria sergenti were reared at 24 degrees C. The resultant nymphal ticks were incubated at 37 degrees C to clear the effect of incubation on the development and maturation of sporozoites. The sporozoites in the salivary glands of the nymphal ticks exposed to 37 degrees C for 16 days were observed by the methyl green pyronin staining method. The ticks exposed to 37 degrees C were ground up in a mortar and the supernatant of the tick suspension in PBS was inoculated into cattle. The cattle showed parasitemia and specific antibody response 18 days after inoculation. Consequently, the parasites in the tick salivary glands became infective to cattle by incubating infected. H. longicornis nymphs at 37 degrees C.     

Infection rates of Theileria sergenti in Haemaphysalis longicornis ticks collected from the field in Japan

For a period of one year Haemaphysalis longicornis ticks were collected in a pasture from vegetation by dragging flannel cloth and from soil samples at monthly intervals. Nymphal ticks were assessed for Theileria infection. Salivary glands were stained with methylgreen-pyronin and examined for parasite masses. Nymphal H. longicornis infected with Theileria sergenti were found in all samples during 12 months including the winter. After shortening the prefed period on rabbits to 24 hours, the parasite masses could be detected in the salivary glands of nymphs collected in the grazing season, from May to October, while no parasite masses were detected in other season. It was suggested that the environmental factors in the grazing season might influence on the maturation of parasites in the salivary glands of ticks.     

Reduction in tick numbers (Haemaphysalis longicornis), mortality and incidence of Theileria sergenti infection in field-grazed calves treated with flumethrin pour-on

The effectiveness of the pour-on formulation of flumethrin was tested on grazing cattle. Flumethrin was applied once a month from April to October from 1990 to 1995 to cattle grazing in the Aso area of Kumamoto Prefecture in Japan. Both the number of ticks in the field and the number of ticks feeding on cattle decreased remarkably in relation to the number of years flumethrin was applied. Ticks in the field were not detected in 1994 and 1995, and ticks feeding on cattle decreased to 4% in 1995. Mortality due to Theileria sergenti infection also decreased significantly after more than 3 years of flumethrin pour-on application, although overall mortality did not change. At the end of the trial the incidence of T. sergenti had decreased to one-fifth of the pretrial value, although total incidence of disease had not changed. These results indicated that multiple-year seasonal application of flumethrin pour-on to grazing cattle effectively decreased the number of ticks and decreased both mortality and incidence of T. sergenti.     

Identification of genes encoding cement-like antigens expressed in the salivary glands of Haemaphysalis longicornis

A cDNA expression library from the salivary glands of hard tick, Haemaphysalis longicornis, was constructed. Immunoscreening was performed using sera of the rabbit repeatedly infested with ticks and seventeen positive clones were obtained. A BLASTP search suggested that 8 sequences matched with that of hypothetical H. longicornis sequence and one clone encoded HL35 antigen U from the same tick species. Eight of 17 gave no match to any sequence reported in the database. The proteins expected from these novel sequences possess common characteristics with cement proteins which assist ticks in their attachment to the host during blood feeding. The expression of these genes in salivary glands was confirmed by RT-PCR. Four of the 8 sequences showed to be upregulated upon blood feeding. These immunodominant antigens are of particular interest as candidates for future cement protein based-tick vaccine.     

Development of Babesia ovata in the midgut of the tick, Haemaphysalis longicornis

Studies were made on the development of Babesia ovata in the midgut of the nymphal tick vector, Haemaphysalis longicornis. In 12 hr post-repletion, merozoites were observed outside of erythrocytes infected with B. ovata in the contents of the midgut of the tick. After that, these merozoites were transformed into ring-forms which were comparatively large ring 2-3 microns in diameter. Within 48-72 hr post-repletion, ring-form protozoa developed into spherical form 4-5 microns in diameter. Within 3-4 days post-repletion, fission-forms were transformed into fission-bodies 2-3 microns in diameter. Within 4-6 days post-repletion, fission-bodies developed into bizarre-forms 6-7 microns in diameter. At this time, elongated form protozoa which were considered as microgametes, 6-8 microns in length, are also seen. Within 6-8 days post-repletion, round-formed protozoa which were considered as zygotes in 9-10 microns in diameter were observed in the gut. About 10 days after repletion, those round-formed protozoa were transformed into vermicule-formed and round-formed protozoa, 13-15 microns in length, appeared again in the gut epithelial cells.     

Infection rates of Theileria annulata in the salivary glands of the tick Hyalomma marginatum rufipes

Theileria annulata was experimentally transmitted to cattle on two occasions by the two-host tick Hyalomma marginatum rufipes. Transmission was transstadial; engorged nymphs fed on Theileria annulata-infected calves transmitted the disease as adults. Salivary glands of all partially fed and incubated adult ticks were heavily infected with Theileria parasites. Immatures attached rapidly and fed successfully on cattle. However, since the immature stages of this species normally feed on birds, this tick is unlikely to be an important vector in the field.     

延边某地长角血蜱和全沟硬蜱体内东方泰勒虫的分子生物学检测

为调查吉林省延边地区自然生境内游离蜱携带东方泰勒虫的情况,以东方泰勒虫主要表面蛋白基因(MPSP)基因为靶基因,采用PCR方法对延边地区自然生境内的游离蜱DNA进行扩增。在长角血蜱的DNA中检测了东方泰勒虫,检出率为29%;而全沟硬蜱中没有检测到东方泰勒虫。结果表明,自然生境中的游离长角血蜱体内携带着东方泰勒虫。     

Molecular cloning of two caspase-like genes from the hard tick Haemaphysalis longicornis

We identified two caspase-like genes from the midgut cDNA library of the hard tick, Haemaphysalis longicornis. Sequence analysis showed that these cDNAs encoded homologues of caspase-2 and caspase-8 that were categorized as apoptosis initiators. The H. longicornis caspase-2 (Hlcaspase-2) cDNA encodes 340 amino acid residues with a predicted molecular weight (Mw) of 38.5 kDa. Another cDNA identified as the H. longicornis caspase-8 (Hlcaspase-8) encodes 306 amino acid residues with an estimated Mw of 35.3 kDa. A catalytic active site was highly conserved in Hlcaspase-8 but not in Hlcaspase-2. RT-PCR analysis showed that both Hlcaspase-2 and Hlcaspase-8 were expressed in tick midgut and salivary glands. This is the first report of the molecular cloning of apoptosis-related genes in the tick.     

Production and characterization of monoclonal antibodies against midgut of ixodid tick,Haemaphysalis longicornis

There are concerted efforts toward development of tick vaccines to replace current chemical control strategies that have serious limitations [Parasitologia 32 (1990) 145; Infectious Disease Clinics of North America (1999) 209-226]. In this study, monoclonal antibodies (mAbs) specific to Haemaphysalis longicornis midgut proteins were produced and characterized. Eight antibody-secreting hybridomas were cloned and the mAbs typed as IgG1, IgG2a and IgG2b. On immunoblots, all mAbs reacted with a midgut protein band of about 76 kDa. All mAbs uniformly immunogold-stained the surface or epithelial layers of H. longicornis midgut and endosomes. Adult ticks (50%) that fed on an ascitic mouse producing the IgGs developed a red coloration and did not oviposit. As such, the 76 kDa protein that reacted with the mAbs could, therefore, be a potential candidate for tick vaccine development.     

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.