ABSTRACT Malaria remains one of the most important infectious diseases in the world. In 2017 alone, approximately 219 million people were infected with malaria, and 435,000 people died of this disease. Plasmodium falciparum , which causes falciparum malaria, is becoming resistant to artemisinin (ART) in Southeast Asia; therefore, new antimalarial drugs are urgently needed. Some excellent antimalarial drugs, such as quinine and ART, were originally obtained from plants. Hence, we analyzed the antimalarial effects of marine natural products to find new antimalarial agents. We used a malaria growth inhibition assay to determine the antimalarial ability and half-maximal inhibitory concentration (IC 50 ) values of the marine organism–derived compounds. Three compounds (kapakahine A, kapakahine B, and kulolide-1) showed antimalarial effects, and one (kapakahine F) showed selective antimalarial effects on the Dd2 clone. Although the IC 50 values obtained for these compounds were greater than that of ART, their potency against P. falciparum is sufficient to warrant further investigation of these compounds as possible drug leads.

The M17 leucine aminopeptidase (M17LAP) enzymes of the other apicomplexan parasites have been characterized and shown to be inhibited by bestatin. Though Babesia bovis also belongs to the apicomplexan group, it is not known whether its M17LAP could display similar biochemical properties as well as inhibition profile. To unravel this uncertainty, a B. bovis M17LAP (BbM17LAP) gene was expressed in Escherichia coli , and activity of the recombinant enzyme as well as its inhibition by bestatin were evaluated. The inhibitory effect of the compound on growths of B. bovis and Babesia gibsoni in vitro was also determined. The expression of the gene fused with glutathione S-transferase (GST) yielded approximately 81-kDa recombinant BbM17LAP (rBbM17LAP). On probing with mouse anti-rBbM17LAP serum, a green fluorescence was observed on the parasite cytosol on confocal laser microscopy, and a specific band greater than the predicted molecular mass was seen on Western blotting. The K m and V max values of the recombinant enzyme were 139.3 ± 30.25 and 64.83 ± 4.6 μM, respectively, while the K i was 2210 ± 358 μM after the inhibition. Bestatin was a more potent inhibitor of the growth of B. bovis [IC 50 (50% inhibition concentration) = 131.7 ± 51.43 μM] than B. gibsoni [IC 50 = 460.8 ± 114.45 μM] in vitro. The modest inhibition of both the rBbM17LAP activity and Babesia parasites' growth in vitro suggests that this inhibition may involve the endogenous enzyme in live parasites. Therefore, BbM17LAP may be a target of bestatin, though more studies with other aminopeptidase inhibitors are required to confirm this.

The resistance of Babesia parasites to current anti-babesiosis drugs is an issue of major concern. The inosine 5′-monophosphate dehydrogenase (IMPDH) of Babesia gibsoni has been identified and characterized as a molecular drug target in our previous studies. In the present study, inhibitory effects of IMPDH inhibitors (mycophenolate mofetil, mizoribine, ribavirin, 7-nitroindole, and mycophenolic acid) were evaluated in vitro or in vivo. In the inhibition assay of recombinant B. gibsoni IMPDH activity, mycophenolate mofetil was the most potent inhibitor (IC 50 = 2.58 ± 1.32 μM) while ribavirin was the least potent. The inhibitory effects of mycophenolate mofetil, mizoribine, ribavirin, and 7-nitroindole on the in vitro growths of B. gibsoni and Babesia bovis were also assessed. The results revealed that mycophenolate mofetil was the most potent inhibitor of the multiplications of both B. gibsoni (IC 50 = 0.13 ± 0.05 μM) and B. bovis (IC 50 = 0.97 ± 0.49 μM). Ribavirin was also the least potent for both B. gibsoni and B. bovis in vitro. Mycophenolic acid, a metabolite of mycophenolate mofetil, caused an inhibition of Babesia microti in mice with noticeable improvement in hematological parameters of the infected mice (ED 50 = 44.15 ± 12.53 mg/kg). Although the report provides a non-exhaustive view of potential treatment strategy without addressing the potential adverse effect of immune suppression on infections, these results indicated that the IMPDH might be a molecular target of MPA for B. microti . Altogether, we provide a basis for development of antibabesia prodrugs by targeting IMPDH of the parasites in the treatment of babesiosis.

A cDNA encoding the Babesia microti 32-kDa protein was identified by serological immunoscreening of a cDNA expression library and designated as BmP32. The full length of BmP32 contains an open reading frame of 918 base pairs consisting of 306 amino acids having a significant homology with B. microti secreted antigen 1. Antiserum raised against recombinant protein (rBmP32) specifically reacted with a 32-kDa native protein of the parasite lysate using western blot analysis. The indirect immunofluorescent antibody test showed a preferable localization of BmP32 in the cytoplasm of the intra- and extracellular parasites. Moreover, BmP32 was secreted in the cytosol of infected erythrocytes, especially during the peak parasitemia and the recovery phase of the infection. Next, the antigenicity of rBmP32 was examined by an enzyme-linked immunosorbent assay (ELISA) and sera from mice experimentally infected with either B. microti or closely related parasites. ELISA was highly specific and sensitive when used for the detection of B. microti antibody in a mouse model. Furthermore, mice immunized with rBmP32 emulsified with Freund's adjuvant were not significantly protected against challenge infection with B. microt i. However, high antibody titer was detected just before the challenge infection. Our data suggest that rBmP32 may be a specific diagnostic antigen but not a subunit vaccine.

Here we report on an epidemiological study regarding the molecular prevalence of different genotypes of Theileria orientalis present among domestic cattle and water buffalo populations bred in Thailand. A phylogenetic analysis based on the parasitic gene encoding a major piroplasm surface protein revealed the presence of 5 genotypes (Types 1, 3, 5, 7, and N-3) in cattle and 7 genotypes (Types 1, 3, 4, 5, 7, N-2, and N-3) in water buffaloes. Types 4, 7, and N-3 of T. orientalis were reported for the first time in water buffaloes. The previously reported C and Thai types from Thailand clustered as types 7 and 6, respectively, in the present analysis. Great similarities were observed among nucleotide sequences of isolates of the same genotype from cattle and water buffaloes, and, therefore, water buffaloes were considered to serve as a reservoir for these genotypes of T. orientalis in Thailand. In conclusion, T. orientalis parasites circulating in Thailand are more diverse in their genetic characters than previously anticipated.

Actin is a ubiquitous and highly conserved microfilament protein that is hypothesized to play a mechanical force-generating role in the unusual gliding motility of sporozoan zoites and their active penetration of host cells. We have identified and isolated an actin gene from a Babesia gibsoni cDNA library by random sequencing. The complete nucleotide sequence of the actin gene is 1,243 bp; a single open reading frame encodes a polypeptide of 377 amino acid residues. The deduced amino acid sequence showed a high homology with actins from other species, especially with reported apicomplexan protozoans. The antiserum against recombinant actin expressed in Escherichia coli recognizes a 42-kDa native protein, which is consistent with its expected size. Immunofluorescence and confocal microscopic observation revealed that the protein is diffusely distributed throughout the B. gibsoni parasites.

In this study, P23 of Cryptosporidium parvum sporozoites, an immunodominant surface protein, was stably expressed in Toxoplasma gondii (Tg/P23) and its protective effects were evaluated in a mouse model. The molecular weight and antigenic property of P23 expressed by Tg/P23 were similar to those of the native P23. Mice immunized with lysed Tg/P23 tachyzoites produced specific neutralizing antibodies against C. parvum . These findings indicate that the T. gondii vector may provide a new tool for the production of a recombinant vaccine against cryptosporidiosis in animals.

The surface antigen 1–related sequence 2 of Neospora caninum (NcSRS2) is considered as an immunodominant antigen. In this study, the gene encoding truncated NcSRS2 (NcSRS2t) lacking an N-terminal signal peptide and C-terminal hydrophobic regions was expressed in Escherichia coli , and its diagnostic potential in an enzyme-linked immunosorbent assay (ELISA) was evaluated. ELISA could discriminate clearly between known N. caninum –positive and –negative sera from cattle. Field serum samples collected from cattle in Brazil were examined for the diagnosis of N. caninum infection using ELISA. Of the 197 samples analyzed, 64 (32.5%) samples were positive for antibodies to N. caninum . Of the 64 ELISA-positive samples, 58 (90.6%) were confirmed as positive by Western blot analysis with whole-parasite antigens. These results suggest that ELISA with recombinant NcSRS2t is an effective method for diagnosis of N. caninum infection in cattle.

The entire P50 gene encoding a surface protein of Babesia gibsoni was cloned into the bacteria expression vector pGEX-4T-3 and subsequently expressed in Escherichia coli as a glutathione S-transferase fusion protein. The purified recombinant P50 was evaluated in an enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of B. gibsoni infection in dogs. ELISA was able to differentiate clearly among B. gibsoni –infected, Babesia canis –infected, and uninfected dog sera. The antibody response against the recombinant P50 was maintained at a high level until the chronic stage of infection in dogs experimentally infected with B. gibsoni . When serum samples collected from domestic dogs in Japan were examined for the diagnosis of B. gibsoni infection by the ELISA, 3 of 209 samples (1.4%) were positive for the antibody to B. gibsoni . This result was completely identical to those of Western blot analysis and the indirect fluorescent antibody test. These results indicate that the recombinant P50 expressed in E. coli is a useful diagnostic antigen for practical use in the diagnosis of B. gibsoni infection in dogs.

In order to develop a vaccine against cryptosporidiosis in cattle, we constructed a recombinant bovine herpesvirus-1 (BHV-1) expressing an immunodominant surface protein, p23, of Cryptosporidium parvum sporozoites. In the recombinant virus, the p23 gene under the control of a CAG promoter and a gene coding for an enhanced green fluorescent protein were integrated into the gG gene of BHV-1. Despite a low frequency of homologous recombination, cloning of the recombinants was easy because of the specific fluorescence of the plaques formed by recombinants. These plaques were among the plaques of the nonfluorescent parental virus. All clones selected for fluorescence also contained the p23 gene. In MDBK cells infected with the recombinant BHV-1, the antibody against the p23 protein recognized the p23 protein as an approximately 23-kDa specific band in Western blotting analysis. Rabbits immunized with the recombinant produced IgG against the p23 protein. It was also demonstrated that the sera of immunized rabbits reduced infection of C. parvum sporozoites in HCT-8 cells. The serum of an immunized rabbit reduced infection compared with the normal rabbit serum control. These results indicate that the recombinant BHV-1 induces neutralizing antibodies in rabbits.

Canine red blood cell-substituted severe combined immune deficiency (Ca-RBC-SCID) mice were prepared for canine Babesia gibsoni infection. The Ca-RBC-SCID mice infected with B. gibsoni developed a high level of parasitemia, and showed clinical symptoms such as anemia and hemoglobinuria, which are similar to those observed in dogs infected with B. gibsoni. The B. gibsoni parasites grown in Ca-RBC-SCID mice showed marked morphological changes, including a significantly larger size of parasites than those in dogs and abundant RBCs containing 4, 8, 16, and 32 parasites. The multiple infection may have resulted from 1 parasite because the posterior end of each parasite in a multiply infected cell was connected. The parasites grown in SCID mice retained their infectivity and virulence to dogs and their morphology was dramatically restored to the original state when they were returned to dogs.