Youn-Kyoung Goo

Molecular and serological prevalence of Babesia bovis and Babesia bigemina in water buffaloes in the north region of Brazil

Bovine babesiosis is a tick-borne disease caused mainly by Babesia bovis and Babesia bigemina, which are associated to considerable economic losses in cattle herds worldwide. Approximately 60% of buffalo herds in South America are located in Northern Brazil. Little is known about the impact of babesiosis on buffalo herds in Brazil. The present work aimed to verify the occurrence of B. bovis and B. bigemina in 542 water buffaloes in the state of Pará, Northern Brazil, using molecular and serological techniques. The percentage of seropositive animals for B. bovis and B. bigemina was 41.2% and 19.0%, respectively, by ELISA. B. bovis and B. bigemina DNA were detected in 15 and 16% of sampled buffaloes, respectively. A high correlation (Kappa index of 0.9) between serological and molecular tests suggests that the combination of the utilized techniques in the present study is suitable for babesiosis diagnosis in an endemic unstable area. Significantly difference of positivity for serological and molecular assays was verified to localities and reproductive status of sampled animals, but not between buffalo breeds. The immune status of sampled buffaloes associated to the circulation of babesiosis agents in sampled population suggests that the studied area is at risk to clinical babesiosis outbreaks. Furthermore, this study demonstrated that this region can be classified as endemically unstable.

Spherical Body Protein 4 Is a New Serological Antigen for Global Detection of Babesia bovis Infection in Cattle

ABSTRACT Five Babesia bovis recombinant proteins, including merozoite surface antigen 2c (BbMSA-2c), C-terminal rhoptry-associated protein 1 (BbRAP-1/CT), truncated thrombospondin-related anonymous protein (BbTRAP-T), spherical body protein 1 (BbSBP-1), and spherical body protein 4 (BbSBP-4), were evaluated as diagnostic antigens to detect the infection in cattle. The recombinant proteins were highly antigenic when tested with experimentally B. bovis-infected bovine serum in Western blot analysis. Furthermore, five antisera that had been raised against each of the recombinant proteins reacted specifically with the corresponding authentic protein, as determined in Western blot analysis. Next, enzyme-linked immunosorbent assays (ELISAs) using these recombinant proteins were evaluated for diagnostic use, and the sensitivity and specificity of each protein were demonstrated with a series of serum samples from experimentally B. bovis-infected cattle. Furthermore, a total of 669 field serum samples collected from cattle in regions of B. bovis endemicity in seven countries were tested with the ELISAs, and the results were compared to those of an indirect fluorescent antibody test (IFAT), as a reference. Among five recombinant antigens, recombinant BbSBP-4 (rBbSBP-4) had the highest concordance rate (85.3%) and kappa value (0.705), indicating its reliability in the detection of specific antibodies to B. bovis in cattle, even in different geographical regions. Overall, we have successfully developed an ELISA based on rBbSBP-4 as a new serological antigen for a practical and sensitive test which will be applicable for epidemiologic survey and control programs in the future.

Artesunate, a potential drug for treatment of Babesia infection.

The effects of artesunate, a water-soluble artemisinin derivative, against Babesia species, including Babesia bovis, Babesia gibsoni and Babesia microti were studied. Cultures of B. bovis and B. gibsoni were treated with 0.26, 2.6, 26 and 260microM artesunate, showing inhibition of parasite growth at concentrations equal to and greater than 2.6microM artesunate by days 3 post-treatment for B. gibsoni and B. bovis in a dose-dependent manner. Consistent with in vitro experiments, artesunate was effective in the treatment of mice infected with B. microti at doses equal to and greater than 10mg/kg of body weight on days 8-10 post-infection. Taken together, these results suggest that artesunate could be a potential drug against Babesia infection.

Identification and characterization of a novel secreted antigen 1 of Babesia microti and evaluation of its potential use in enzyme-linked immunosorbent assay and immunochromatographic test

Here, we identified a novel secreted antigen designated as Babesia microti secreted antigen 1 (BmSA1) by immunoscreening a B. microti cDNA expression library using the sera from hamsters immunized with plasma, putatively containing secreted antigens, from B. microti-infected hamsters. Antibodies raised in mice immunized with recombinant BmSA1 (rBmSA1) recognized a native 33-kDa parasite protein. An enzyme-linked immunosorbent assay (ELISA) of rBmSA1 detected specific antibodies as early as 6 and 4 days post-infection in sera from a hamster experimentally infected with B. microti Gray strain (US type) and a mouse experimentally infected with B. microti Munich strain (rodent isolate), respectively. Moreover, a rapid immunochromatographic test (ICT) using rBmSA1 detected specific antibodies in a hamster experimentally infected with B. microti from day 6 to at least day 270 post-infection, which was quite consistent with the results of the ELISA. In addition, analysis of the sera involved in the first case of human babesiosis in Japan (Kobe type) showed that specific antibodies were detectable in the patient and the positive donor by ELISA using rBmSA1, and the ICT result was identical to the ELISA data. Taken together, these results indicated that BmSA1 could be a promising and universal target for developing both ELISA and ICT for the serodiagnosis of human babesiosis and for an epidemiological survey of its rodent reservoir.

Epidemiological survey of Babesia gibsoni infection in dogs in Japan by enzyme-linked immunosorbent assay using B. gibsoni thrombospondin-related adhesive protein antigen.

A nationwide epidemiological survey of Babesia gibsoni infection in non-fighting dogs was conducted using an improved ELISA with recombinant B. gibsoni thrombospondin-related adhesive protein (BgTRAP). A total of 1206 dogs from 27 prefectures were examined and 128 (10.6%) tested positive. In the eastern part of Japan, 39 dogs out of the 559 (7.0%) examined were positive, while 89 dogs out of 647 (13.8%) tested positive in the western part of Japan. Although the percentage of dogs that tested positive was significantly (p=0.0001) lower in the eastern part compared to the western part of Japan, overall these results indicate that B. gibsoni infection of dogs has a widespread geographic distribution throughout the country. A history of tick infestation was identified as a significant risk factor for B. gibsoni infection (p=0.0091), while sex (p=0.9411), age (p=0.0920) and breed (p=0.0549) of dogs were not statistically significant risk factors. These results indicate that tick infestation is the most dominant risk factor for B. gibsoni infection of non-fighting dogs in Japan and suggest that other B. gibsoni transmission routes, such as fighting and transplacental transmission, may be less important.

Molecular and serological prevalence of Babesia bovis and Babesia bigemina in cattle from central region of Syria

A total of 207 bovine blood samples were collected from clinically healthy cattle bred in central region of Syria and examined by Giemsa-stained blood smears, nested PCR, ELISA, and IFAT to determine the molecular and serological prevalence of Babesia bovis and B. bigemina. All samples were negative to Babesia spp. by microscopic examination of blood smears. On the other hand, the overall prevalence of B. bovis and B. bigemina was 9.18% and 15.46% by nPCR, 15.46% and 18.84% by ELISA, and 18.36% and 21.74% by IFAT, respectively. Mixed infections were detected in a total of 5 samples (2.4%) by nPCR, 16 (7.73%) by ELISA and 27 (13.04%) by IFAT. Statistically significant differences in the prevalence of the two infections were observed on the basis of age and location. These data provide valuable information regarding the occurrence and epidemiology of B. bovis and B. bigemina infections in Syrian cattle, which can be employed in developing rational strategies for disease control and management.

Molecular and immunological characterization of Babesia gibsoni and Babesia microti heat shock protein‐70

Serological immunoscreening was used to identify a gene encoding heat shock protein‐70 from Babesia gibsoni (BgHSP‐70) that showed high homology with HSP‐70s from other apicomplexan parasites. This gene corresponded to a full‐length cDNA containing an open reading frame of 1968 bp predicted to result in a 70‐kDa mature protein consisting of 656 amino acids. Analysis of the expression levels of BgHSP‐70 indicated elevated transcription from cultured parasites incubated at 40°C for 1 h, but not at 30°C. Interestingly, antiserum raised against recombinant BgHSP‐70 protein reacted specifically not only with a 70‐kDa protein of B. gibsoni but also with a corresponding native protein of B. microti (BmHSP‐70), indicating the high degree of conservation of this protein. The BmHSP‐70 gene was then isolated and characterized and the immunoprotective properties of recombinant BgHSP‐70 (rBgHSP‐70) and rBmHSP‐70 were compared in vitro and in vivo. Both proteins had potent mitogenic effects on murine and canine mononuclear cells as evidenced by high proliferative responses and IFN‐γ production after stimulation. Immunization regimes in BALB/c and C57BL/6 mice using rBgHSP‐70 and rBmHSP‐70 elicited high antibody levels, with concurrent significant reductions in peripheral parasitaemias. Taken together, these results emphasize the potential of HSP‐70s as a molecular adjuvant vaccine.

A Novel Korean Red Ginseng Compound Gintonin Inhibited Inflammation by MAPK and NF-κB Pathways and Recovered the Levels of mir-34a and mir-93 in RAW 264.7 Cells

The beneficial health promoting effects of ginseng from vitalizing the body to enhancing long life have been well explored very rapidly in the past few years. Up till now many ginsenosides have been discovered for their marvelous therapeutic effects. However during past three years, a novel ginseng compound has been discovered, called gintonin, that differs from other ginsenosides on the basis of its signal transduction and chemical nature. Gintonin has been widely studied for its anti-Alzheimers disease activities and other neuropathies. However, its anti-inflammatory activity remained unexplored. In our study we have reported for the first time the anti-inflammatory activity of gintonin on RAW 264.7 cells. We found that gintonin potently suppresses the nitric oxide production without any cytotoxicity at given doses and also efficiently suppressed the levels of proinflammatory cytokines. Moreover, it mediaes its signal transduction via MAPK and NF-κB pathways and revives the levels of mir-34a and mir-93. These findings are valuable for the anti-inflammatory effects of this new compound with particular reference to microRNA involvement in the ginseng family.

Further Evidence of Increasing Diversity of Plasmodium vivax in the Republic of Korea in Recent Years.

Background Vivax malaria was successfully eliminated from the Republic of Korea (ROK) in the late 1970s but re-emerged in 1993. Two decades later as the ROK enters the final stages of malaria elimination, dedicated surveillance of the local P. vivax population is critical. We apply a population genetic approach to gauge P. vivax transmission dynamics in the ROK between 2010 and 2012. Methodology/Principal Findings P. vivax positive blood samples from 98 autochthonous cases were collected from patients attending health centers in the ROK in 2010 (n = 27), 2011 (n = 48) and 2012 (n = 23). Parasite genotyping was undertaken at 9 tandem repeat markers. Although not reaching significance, a trend of increasing population diversity was observed from 2010 (HE = 0.50 ± 0.11) to 2011 (HE = 0.56 ± 0.08) and 2012 (HE = 0.60 ± 0.06). Conversely, linkage disequilibrium declined during the same period: IAS = 0.15 in 2010 (P = 0.010), 0.09 in 2011 (P = 0.010) and 0.05 in 2012 (P = 0.010). In combination with data from other ROK studies undertaken between 1994 and 2007, our results are consistent with increasing parasite divergence since re-emergence. Polyclonal infections were rare (3% infections) suggesting that local out-crossing alone was unlikely to explain the increased divergence. Cases introduced from an external reservoir may therefore have contributed to the increased diversity. Aside from one isolate, all infections carried a short MS20 allele (142 or 149 bp), not observed in other studies in tropical endemic countries despite high diversity, inferring that these regions are unlikely reservoirs. Conclusions Whilst a number of factors may explain the observed population genetic trends, the available evidence suggests that an external geographic reservoir with moderate diversity sustains the majority of P. vivax infection in the ROK, with important implications for malaria elimination.

Autophagy Inhibitors as a Potential Antiamoebic Treatment for Acanthamoeba Keratitis

ABSTRACT Acanthamoeba cysts are resistant to extreme physical and chemical conditions. Autophagy is an essential pathway for encystation of Acanthamoeba cells. To evaluate the possibility of an autophagic Acanthamoeba encystation mechanism, we evaluated autophagy inhibitors, such as 3-methyladenine (3MA), LY294002, wortmannin, bafilomycin A, and chloroquine. Among these autophagy inhibitors, the use of 3MA and chloroquine showed a significant reduction in the encystation ratio in Acanthamoeba cells. Wortmannin also inhibited the formation of mature cysts, while LY294002 and bafilomycin A did not affect the encystation of Acanthamoeba cells. Transmission electron microscopy revealed that 3MA and wortmannin inhibited autophagy formation and that chloroquine interfered with the formation of autolysosomes. Inhibition of autophagy or autolysosome formation resulted in a significant block in the encystation in Acanthamoeba cells. Clinical treatment with 0.02% polyhexamethylene biguanide (PHMB) showed high cytopathic effects on Acanthamoeba trophozoites and cysts; however, it also revealed high cytopathic effects on human corneal epithelial cells. In this study, we investigated effects of the combination of a low (0.00125%) concentration of PHMB with each of the autophagy inhibitors 3MA, wortmannin, and chloroquine on Acanthamoeba and human corneal epithelial cells. These new combination treatments showed low cytopathic effects on human corneal cells and high cytopathic effects on Acanthamoeba cells. Taken together, these results provide fundamental information for optimizing the treatment of Acanthamoeba keratitis.