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Background: Daclatasvir (DCV) and asunaprevir (ASV) combination therapy has been primarily used in patients without NS5A L31 or Y93 resistance-associated substitutions (RASs) before treatment. We examined the characteristics of patients without these baseline RASs who did not achieve hepatitis C virus eradication with DCV and ASV combination therapy and identified new baseline NS5A RASs that are closely associated with failure of combination therapy., Methods: Three hundred thirty-five patients with hepatitis C virus genotype 1 infection with no NS5A L31, NS5A Y93, and NS3 D168 RASs before DCV and ASV combination therapy and no history of protease inhibitor therapy were enrolled. All RASs were evaluated by direct sequencing., Results: Sustained virologic response at 12 weeks (SVR12) was achieved in 297 patients (89%). Patients with NS5A Q24, L28, and/or R30 RASs or concomitant NS5A F37 and Q54 RASs had a significantly lower SVR12 rate than patients without these RASs (70% vs 92%, p < 0.001 and 79% vs 92%, p = 0.002 respectively). Multivariate analysis showed that NS5A Q24, L28, and/or R30 RASs and concomitant NS5A F37 and Q54 RASs were significantly associated with virologic failure. The SVR12 rate in patients without NS5A Q24, L28, and/or R30 RASs and concomitant NS5A F37 and Q54 RASs was 96.2% (202/210)., Conclusions: In patients without NS5A L31 or Y93 RASs, the presence of NS5A Q24, L28, and/or R30 RASs and concomitant NS5A F37 and Q54 RASs at the baseline was associated with failure of DCV and ASV combination therapy. The coexistence of baseline RASs other than NS5A L31 and Y93 may affect the therapeutic effectiveness of DCV and ASV combination therapy.

Haemaphysalis longicornis is a tick known for transmitting Babesia parasites, including Babesia gibsoni, in East Asian countries. The vector tick must have strategies to control Babesia parasites, while Babesia parasites are also considered to establish an evasive mechanism from the tick's innate immunity. Due to this mutual tolerance, H. longicornis is considered to be a vector of Babesia parasites. Recent studies have shown the important roles of leucine-rich repeat (LRR) domain-containing proteins in innate immunity in many living organisms. Some LRR domain-containing proteins were identified in ticks; however, their functions are still unknown. In this study, a novel LRR domain-containing protein was identified from H. longicornis (HlLRR). HlLRR contains two LRR domains, and the expression levels of mRNA and proteins were upregulated during blood feeding, particularly in the salivary glands and midgut. In addition, recombinant HlLRR (rHlLRR) demonstrated growth inhibition activity against B. gibsoni in vitro without a hemolytic effect at any concentration used. Moreover, the diameters of Babesia merozoites treated with rHlLRR were significantly larger than those of the control group. These results strongly indicate the key roles of HlLRR in the tick's innate immunity against Babesia parasites. Furthermore, HlLRR might be a potential alternative drug to treat babesiosis.

Haemaphysalis longicornis is known as one of the most important ticks transmitting Babesia parasites in East Asian countries, including Babesia ovata and Babesia gibsoni, as well as Theileria parasites. H. longicornis is not the natural vector of Babesia bovis and Babesia bigemina. Vector ticks and transmitted parasites are thought to have established unique host-parasite interaction for their survival, meaning that vector ticks may have defensive molecules for the growth control of parasites in their bodies. However, the precise adaptation mechanism of tick-Babesia parasites is still unknown. Recently, cyclophilin A (CyPA) was reported to be important for the development of Babesia parasites in ticks. To reveal a part of their adaptation mechanism, the current study was conducted. An injection of B. bovis-infected RBCs into adult female H. longicornis ticks was found to upregulate the expression profiles of the gene and protein of CyPA in H. longicornis (HlCyPA). In addition, recombinant HlCyPA (rHlCyPA) purified from Escherichia coli exhibited significant inhibitory growth effects on B. bovis and B. bigemina cultivated in vitro, without any hemolytic effect on bovine RBCs at all concentrations used. In conclusion, our results suggest that HlCyPA might play an important role in the growth regulation of Babesia parasites in H. longicornis ticks, during natural acquisition from an infected host. Furthermore, rHlCyPA may be a potential alternative chemotherapeutic agent against babesiosis.

4E-BP, an eIF4E-binding protein, is well known as a cap-dependent translation inhibitor. Here, the 4E-BP homolog, Hl4E-BP, was isolated and identified from the hard tick Haemaphysalis longicornis. Hl4E-BP transcripts were ubiquitously expressed in the active stages, including the larvae, nymphs, and female adults, and the transcription levels were found to be higher in unfed than engorged ticks. In contrast, the expression levels of non-phosphorylated Hl4E-BP, which is a 13.4-kDa protein detected by the anti-recombinant Hl4E-BP antibody, were the highest in engorged ticks and significantly decreased progressively during the unfed starvation period of ticks. The functional role of Hl4E-BP as a metabolic brake was verified by histochemical observations on the lipid storage in midguts and fat bodies during the starvation period using ticks injected with dsHl4E-BP. The results indicate that Hl4E-BP is highly relevant to the lipid storage of ticks during the non-feeding starvation period. Our results suggest, for the first time, that Hl4E-BP may have a crucial role in the starvation resistance of ticks in an off-host condition via lipid metabolism control, although it was unclear whether Hl4E-BP might be involved in lipid synthesis regulation and/or lipid consumption inhibition.

Autophagy is the intracellular protein degradation process which is induced by starvation. Ticks have a unique tolerance for starvation, and it is possible that this tolerance is associated with their longevity. Previously, we isolated the homologues of four autophagy-related (ATG) genes in the hard tick, Haemaphysalis longicornis, suggesting that autophagy appeared to play an important role in tolerance for starvation as well as the development of ticks. In this study, the homologue of ATG6 was isolated from H. longicornis (HlATG6). HlATG6 mRNA expression was higher in the egg and unfed larval stages than in other stages and upregulated in ovaries during the blood-feeding period. Moreover, HlATG6-knockdowned ticks laid a few and poorly developed eggs that were white brown in color and not well surface-coated with wax. However, the expression of vitellogenin (Vg)-2, HlVg-2, in the fat body of HlATG6-knockdowned ticks was significantly upregulated. In addition, hemolymph had a deep brown color in HlATG6-knockdowned ticks on day 21 after engorgement and drop-off, indicating that the Vgs synthesized by the fat body and midgut are retained and accumulated in the hemolymph of HlATG6-knockdowned ticks, probably due to the downregulation of the Vg uptake capability of oocytes. Interestingly, HlATG6 knockdown provided non-significant influences on the expression of the Vg receptor (HlVgR) at oocytes, suggesting a non-significant depression of VgR-mediated endocytosis in the oocytes of HlATG6-knockdowned ticks. Therefore, it was interpreted that the repression of Vg uptake in the oocytes of HlATG6-knockdowned ticks may be involved in endocytic processes other than the receptor recognition of Vgs in oocytes.

The characterization of the cross-reactive and species-specific antigens of Neospora caninum and Toxoplasma gondii is important in the exploration to determine the common mechanisms of parasite-host interaction and to improve the serological diagnosis; it is also useful for the selection of the cross-reactive antigens that could be used in the development of vaccines or drugs for controlling the diseases caused by these two parasites. In this study, cross-reactive and species-specific antigens between N. caninum and T. gondii tachyzoites were comprehensively investigated using a proteomics approach with the application of two-dimensional gel electrophoresis, immunoblot analysis, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS), and MALDI-TOF/TOF-MS analysis. Immunoblotting and mass spectrometry analysis revealed that at least 42 individual protein spots of N. caninum were reacted with the anti-N. caninum serum, among which at least 18 protein spots were cross-reacted with the anti-T. gondii serum. Moreover, at least 31 protein spots of T. gondii were reacted with the anti-T. gondii serum, among which at least 19 protein spots were cross-reacted with the anti-N. caninum serum. Furthermore, some new specific proteins were also identified in the N. caninum protein profile by searching Toxoplasma sequences or sequences from other organisms. This study substantiates the usefulness of proteomics in the immunoscreening of the cross-reactive or species-specific antigens of both parasites. In addition, the present study showed that there was significant homology in the antigenic proteome profiles between the two parasites. These observations have implications for the design of multicomponent common vaccines against both parasite infections.

Scavenger receptors (SRs) are cell-surface proteins and exhibit distinctive ligand-binding properties, recognizing a wide range of ligands that include microbial surface constituents and intact microbes. The class B scavenger receptor CD36 (SRB) is predominantly expressed by macrophages and is considered important in innate immunity. We here show the identification and characterization of SRB from the hard ixodid tick, Haemaphysalis longicornis (HlSRB). The full-length cDNA was 2,908 bp, including an ORF encoding of 1,518 amino acids with a pI value of 5.83. H. longicornis SRB contains a hydrophobic SRB domain and four centrally clustered cysteine residues for arrangement of disulfide bridges. Deduced amino acid sequence has an identity of 30-38% with the SRB of other organisms. RT-PCR analysis showed that mRNA transcripts were expressed in multiple organs of adult ticks but with a different transcript level in the developmental stages of H. longicornis ticks. His-tagged recombinant HlSRB was expressed in Escherichia coli with an expected molecular mass of 50 kDa. In Western blot analysis, mouse anti-rHlSRB serum recognized a strong reaction with a 50 kDa protein band in lysates prepared from egg and adult tick but showed a weak reaction with lysates of larva and nymph. In an indirect immunofluorescent antibody test, HlSRB antiserum recognized the protein located on the midgut, salivary glands, and ovary of partially fed H. longicornis females. Silencing of the HlSRB gene by RNAi led to a significant reduction in the engorged female body weight. It is noteworthy that more than a dozen SRB orthologs have been identified in the genomes of insect species with functions related to pheromone signaling, innate immunity, phagocytic clearance of apoptotic cells, and various aspects of the fatty acid metabolism. This is the first report of the identification and characterization of the SRB homologue in Chelicerata, including ticks, horseshoe crabs, scorpions, spiders, and mites.

Ticks feed exclusively on blood to obtain their nutrients, but the gene products that mediate blood-sucking processes in ticks are still unknown. We report here the molecular characterization and possible biological function of a cysteine protease inhibitor (HlSC-1) identified in the salivary gland of the ixodid tick Haemaphysalis longicornis. The HlSC-1 cDNA contains 423 bp that code for 140 amino acids with a predictable molecular weight of 12 kDa. The recombinant HlSC-1 expressed in Escherichia coli was shown to inhibit the activity of papain and cathepsin L, while cathepsin B activity was unaffected. Immunolocalization studies detected the endogenous enzyme in the salivary gland type II acini of an adult tick. Furthermore, quantitative RT-PCR analysis showed that the expression of HlSC-1 transcripts was associated with blood-feeding processes and was highly up-regulated in the early phase of feeding. Our results strongly suggest that HlSC-1 may play pivotal roles in the blood-feeding processes.

A complementary DNA coding a novel kynurenine aminotransferase (KAT) molecule from Haemaphysalis longicornis tick embryo was cloned and characterized. The transcription of the HlKAT occurs at all stages during tick development as well as in the midgut, salivary glands, ovary, and synganglion of adult ticks, and protein expression levels increased during the blood-feeding course. The HlKAT gene without signal peptide was successfully expressed as a glutathione S-transferase fusion protein in soluble form, which is capable of catalyzing the transamination of kynurenine and 3-hydroxykynurenine to kynurenic acid and xanthurenic acid, respectively. The purified recombinant HlKAT showed dose-dependent inhibition effect on the growth of equine babesial parasite, Babesia caballi, in in vitro culture. All results suggested that a specific HlKAT is present in tick and HlKAT may play an important physiological role in H. longicornis. This is the first report of a member enzyme of tryptophan pathway in Chelicerata.

Babesia gibsoni ribosomal phosphoprotein P0 (BgP0) was previously identified as a cross-protective antigen against Babesia microti infection in mice. Interestingly, the same protein showed considerable antigenicity when tested with serum samples collected from Babesia-infected animals. Moreover, the polyclonal antibody raised against the recombinant BgP0 (rBgP0) recognized the P0 homologues from other Babesia species either by immunoblotting or by immunoscreening. The P0 genes from Babesia caballi, Babesia equi, and Babesia bigemina were then cloned and sequenced. The phylogenic analyses based on the amino acid sequences indicated that BgP0 has high identities with B. caballi P0 (88.1%), B. bigemina P0 (85.6%), Babesia bovis P0 (81.4%), and B. equi P0 (64.9%). Western blot analyses revealed that the corresponding native proteins ranged between 31 and 34 kDa, consistent with predicated molecular weight of Babesia P0. Furthermore, the immunogenic property of anti-rBgP0 IgG was evaluated against a B. bovis in vitro culture. The growth of B. bovis parasites was restricted by anti-rBgP0 IgG in a concentration-dependent manner, and significant reductions in parasitemia were observed only at 1 mg/ml in the culture. Taken together, these data suggest that P0 is a conserved protective antigen among Babesia species and might be a potentially universal vaccine candidate for babesiosis.

Ticks are effective vectors of pathogens because of their blood feeding and high fecundity. This high fecundity is related to the size of the blood meal. Therefore, knowledge of how blood proteins are degraded and converted to proteins, including yolk protein, is important for the development of ways to inhibit the utilization of blood proteins by ticks. RNA interference (RNAi) is becoming a powerful post-transcriptional gene silencing technique that provides insight into gene function. We constructed a double-stranded RNA (dsRNA) based on a previously cloned Haemaphysalis longicornis leucine aminopeptidase (HlLAP) gene to reevaluate the biological role in tick blood digestion. Gene specific transcriptional, translational, and functional disruptions were achieved by the introduction of dsRNA into the ticks. Significantly delayed onset of egg-laying and reduced egg oviposition resulted from the RNAi for the HlLAP gene. These results suggest that HlLAP actually works as a blood digestive enzyme and affects tick fecundity via unknown mechanisms. The reduction of egg oviposition may be caused by a decrease in nutrients, especially free amino acids generated by HlLAP, from the blood meal. This is the first report of an impact on tick reproduction caused by gene silencing of a blood digestion-related molecule.

A cDNA coding a glycine-rich protein was identified from the Rhipicephalus haemaphysaloides tick. The cDNA named here as RH50 was 1,823 bp, including a single open reading frame (ORF) of 1,518 nucleotides. The ORF encodes a polypeptide of 506 amino acid residues with a size of 50 kDa, as calculated by a computer. The predicted amino acid sequence of RH50 showed a low homology to sequences of some known extracellular matrix-like proteins. The native protein was identified in both the fed tick salivary gland lysates and extracts of cement material using the serum against the recombinant protein. Reverse transcription polymerase chain reaction results showed that RH50 mRNA was only transcribed in partially fed tick salivary glands, not in unfed tick salivary glands or partially fed tick midgut, fat body, or ovary. The differential expression of RH50 protein in fed tick salivary glands was confirmed by immunofluorescence. The low attachment rate both in the adult and nymphal tick, and the high mortality of immature ticks (nymph) feeding on recombinant RH50-immunized rabbits were found. These results show that the RH50 protein could be a useful candidate for anti-tick vaccine development.

Baculoviruses are specific insect pathogens used as selective biological insecticides on lepidopteran insects. We have tested a recombinant baculovirus expressing a chitinase gene for its efficacy as a tick bioacaricide. The recombinant Autographa californica multiple nuclear polyhedrosis virus expressing a chitinase enzyme (AcMNPV-CHT1) from the hard tick, Haemaphysalis longicornis, was constructed and found to have a novel bioacaricidal effect against ticks. The recombinant baculovirus was used to express the chitinase enzyme in Spodoptera frugiperda (Sf9) insect cells. Topical application of the supernatant harvested from the insect cell culture was found to cause mortality in nymphal ticks of H. longicornis. High temperature (>30 degrees C) and infrared radiation affected the chitinase enzyme activity and recombinant baculovirus infectivity by reducing the speed of tick killing by 60%. A mixture of recombinant virus and chitinase was found to kill ticks faster (p < 0.01) than pure chitinase and recombinant virus alone. Thus, the recombinant virus showed a synergistic effect with the foreign chitinase gene. In order to reduce the excessive use and cost of acaricides, it was found that a mixture of recombinant virus and flumethrin could halve the dose of the chemical acaricide used. These findings are important for the safe use of the recombinant virus expressing chitinase as a bioacaricide against ticks.

Background: Interferon plus ribavirin (IFN/Rib) therapy is currently standard treatment for chronic hepatitis C. Hemolytic anemia, however, is a serious side effect of this treatment, requiring reductions in or complete withdrawal of ribavirin., Methods: We retrospectively investigated the effect of the Kampo medicine Juzen-taiho-to (TJ-48), which contains bone marrow-stimulating compounds, on anemia in 67 patients with chronic hepatitis C, who received IFN/Rib therapy., Results: The reduction in hemoglobin levels was significantly ameliorated in TJ-48-treated patients (P<0.05). Consequently, only 13% (4/32) of TJ-48-treated patients received altered doses of ribavirin, while the ribavirin dose had to be reduced or withdrawn in 43% (15/35) of patients in the absence of TJ-48 administration (P<0.001)., Conclusions: These results indicate the possibility that oral administration of TJ-48 supports IFN/Rib therapy without necessitating ribavirin reduction or withdrawal.

In the present study, morphological observations on the extracellular structures found on the apical surface of the midgut epithelium, known as the peritrophic membrane (PM) or glycocalyx, are described in Haemaphysalis longicornis females and larvae. These structures have been hypothesized to provide protection to the microvilli of epithelial cells of the digestive tract. Our aim was to determine whether the extracellular structures are important in the digestion of the blood meal and/or as a protection against infection or injury. The PM was detectable in the midgut of engorged larvae by electron microscopy, but not in engorged females. However, a PM-like structure, stainable with toluidine blue, was observed in females by light microscopy. From the results of confocal laser scanning and electron microscopic observations with wheat germ agglutinin (WGA lectin) staining for chitin of the PM, however, the structure was clearly recognized. The structure in the female is likely to be PM because staining with WGA lectin in the presence of GlcNAc indicates the presence of chitin and various morphologies of PM have been reported in insects and ticks. These results show morphologically that different types of PM-like structure are formed in larvae and females of H. longicornis.

Phenol oxidase (PO, EC 1.10.3.1) activity was detected in the hemolymph of the fourth instar nymphs of the argasid tick, Ornithodoros moubata, with peak levels corresponding to the days before the majority of the nymphs had molted, suggestive of a protective role of PO during the ecdysial phase. Higher PO activity was detected in plasma relative to the hemolymph and was negligible in hemocytes. The concentration of the hemolymph and plasma assayed clearly influenced the level of PO activity, and was significantly reduced ( P<0.005) after treatment with 1-phenyl-2 thiourea, a specific PO inhibitor. This is the first report of the existence of PO in the hemolymph and plasma of a soft tick species. The regulation of PO activity and its precise role in soft tick immunity, particularly during the ecdysial phase, are interesting and need to be examined further.

In order to investigate whether a recombinant viral vaccine composed of a feline herpesvirus type 1 (FHVI) vector and an immunogenic antigen of Toxoplasma gondii can induce a protective immunity in cats, a recombinant FHV1 expressing ROP2 antigen of T. gondii was prepared. We introduced a DNA fragment encoding 1-538 amino acid residues of ROP2 precursor into FHV1 genome under the control of a cytomegalovirus promoter. The recombinant FHV1 (FHV/ROP2) successfully expressed a 59 kDa antigen that was recognized by anti-ROP2 antibodies. Vaccination of cats with FHV/ROP2 induced serum IgG recognizing the native antigen. Moreover the antibodies inhibited the in vitro invasion of tachyzoites. The vaccination also accelerated the IgG response after T. gondii infection and reduced brain parasite load in cats.

In order to develop a vaccine against Neospora caninum in dogs and cattle, we constructed a recombinant vaccinia virus expressing the N. caninum surface protein, NcSRS2 (Nc-p43). Monoclonal antibodies to NcSRS2 and anti-N. caninum tachyzoite mouse serum recognized the NcSRS2 expressed by the recombinant vaccinia virus. In addition, recombinant NcSRS2 was transported to the cell surface. Mice infected with the recombinant virus predominantly produced IgG1 antibody (Ab) to N. caninum, rather than producing IgG2a Ab. Moreover, splenocytes from mice infected with the recombinant virus proliferated in the presence of the N. caninum antigen. Mice immunized with the recombinant virus gave rise to humoral and cellular immune responses to N. caninum tachyzoites. This study showed that a recombinant vaccinia virus expressing NcSRS2 might be useful for the production of a live vaccine against N. caninum infection.

To clarify the effector pathway of Neospora caninum growth-inhibitory activity induced by interferon-gamma (IFN-gamma) in murine macrophages we examined the relationship between IFN-gamma and nitric oxide (NO). Production of NO was enhanced in cultures of macrophages supplemented with IFN-gamma, and dose-dependent growth inhibition was observed. These findings suggest that the inhibitory activity induced in macrophages by IFN-gamma is mediated NO molecules. A competitive inhibitor of the L-arginine-dependent effector pathway, NG-monomethyl-L-arginine, virtually abolished the inhibitory effects induced by IFN-gamma. From this finding it appears that the inhibitory effects induced by IFN-gamma in macrophages may be mediated by an L-arginine-dependent effector pathway that involves NO production. In vivo, mice with a targeted disruption of the inducible NO synthase gene (iNOS-/-) were more susceptible than wild-type mice to N. caninum. Therefore, the production of NO in macrophages induced by IFN-gamma is an important mechanism for the killing of intracellular N. caninum.

We describe the expression of SAG-1 cDNA in B6C3F1 mice by microinjecting a 3.3 kbp DNA fragment, consisting of the cytomegalovirus enhancer-chicken beta-actin hybrid promoter and SAG-1 into the pronucleus of a fertilized egg at the one-cell stage. Offspring derived from this microinjection were analyzed for the integration and functional expression of the SAG-1 transgene. Steady-state expressions of both the mRNA for SAG-1 and SAG-1 protein product were detected in the brain, thymus, spleen and liver. Approximately 50% of F1 and F2 progeny inherited the SAG-1 transgene from SAG-1 transgenic mice in Mendelian fashion. These results indicated that SAG-1 transgenic lines were established. Transgenic mice harboring the SAG-1 gene will contribute a critical tool of defining the molecular mechanisms of SAG-1 in pathogenesis and host immune response.

Polyarteritis nodosa is a necrotizing angitis that predominantly affects small and medium-sized arteries. The prognosis of untreated polyarteritis nodosa is very poor. Since symptoms are diverse and no serologic test is specific for polyarteritis nodosa, the diagnosis is difficult and often delayed. We describe a patient with polyarteritis nodosa who had gastrointestinal involvement with multiple aneurysms of the inferior mesenteric artery; only abdominal angiography provided a conclusive diagnosis. Alleviation of symptoms and regression of aneurysms were observed after combination therapy of an immunosuppressive agent, cyclophosphamide, and prednisolone. We emphasize the importance of early diagnosis by angiography and aggressive therapy in patients in whom physical signs indicating definite polyarteritis nodosa are not present.

Babesia caballi cultured continuously in equine erythrocytes was examined by transmission electron microscopy. The use of cultured B. caballi permitted examination of a large number of parasitized cells with various stages of intra erythrocytic development. The piriform merozoites of B. caballi were composed of an outer membrane and an inner double-membrane complex. Numerous micronemes and three rhoptries were found in the pellicle of the merozoite, and a spherical body was seen in the anterior part of the merozoite which usually lay adjacent to the nucleus and the pellicle. These findings were similar to those for merozoites of bovine Babesia parasites such as B. bigemina. The trophozoites were surrounded by a single membrane, were continuously changing their body shape with extension and retraction of the pseudopod. A long pseudopod extended far into the host cell cytoplasm, and was finally completely enclosed in a cell, but did not have hemozoin pigment, the breakdown product of hemoglobin digestion.

The in vitro transformation system of Strongyloides venezuelensis has been established which induces free-living infective larvae to transform into the parasitic stage by a temperature shift from 25 to 37 degrees C. Comparison of the profiles of proteins labeled with [35S]-methionine in infective larvae at 25 and 37 degrees C with marked morphological transformation revealed an increase in the 70-kDa protein, thus showing these proteins to be related to the family of heat-shock proteins. In addition, the new appearance of two complexes between 16-kDa and 22-kDa proteins was observed. The 70-kDa protein cross-reacted with monoclonal antibody against human heat-shock protein 70, which was constitutively expressed. These proteins, synthesized during the transformation of S. venezuelensis from the infective larval stage to the parasitic stage, might be crucial for biochemical events that regulate the infectivity of the parasite for the host.

Serological comparisons among piroplasm antigens of the benign Theileria species of cattle from Japan, Australia and Britain, which are frequently referred to as T. sergenti, T. buffeli and T. orientalis, were carried out. The results obtained from comparative enzyme-liked immunosorbent assay (ELISA) using sera from infected cattle suggest that T. sergenti could be differentiated from both T. buffeli and T. orientalis by their serological dissimilarities. Western blotting combined with two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) revealed that both the 33-kDa piroplasm protein of T. sergenti and the similar 34-kDa protein of T. buffeli and T. orientalis corresponded to immuno-dominant antigens against cattle. The other 32-kDa proteins of . T. buffeli and T. orientalis also represented immunodominant antigens. Cross-reactivities of the 32-and 34-kDa proteins were observed between T. buffeli and T. orientalis, whereas the 33-kDa protein of T. sergenti could be differentiated from the similar 34-kDa proteins of T. buffeli/orientalis. The present study suggests that T. sergenti should be separated from T. buffeli and T. orientalis on the basis of their serological dissimilarities.

A simple and rapid method for purifying merozoites of Theileria sergenti from infected bovine erythrocytes was developed. Infected erythrocytes were lysed by the use of a cytolytic toxin produced by Aeromonas hydrophila and the lysate was subjected to ultracentrifugation in a Percoll discontinuous density gradient. Pure and morphologically intact merozoites free of erythrocyte membrane were recovered from a band formed at the interface of 40% and 60% (by vol.) Percoll solutions. In this merozoite fraction, contamination of erythrocyte membrane proteins was not detected as examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.