Serodiagnosis of Toxoplasma gondii infection in dairy cows in Thailand.

Toxoplasma gondii is a zoonotic protozoan parasite of both medical and veterinary importance worldwide. The parasite can cause severe complications in immunocompromised individuals such as AIDS patients and transplant recipients, where up to 25% of patients will develop toxoplasmic encephalitis. Toxoplasmosis is a zoonosis that causes a public health concern in both developed and developing countries such as Thailand. Livestock development particularly in dairy cows of Thailand have been hampered by low production of milk and slow growth rate because of many pathogens including T. gondii. The objective of this study was to evaluate the serodiagnostic tool to be used for detection of T. gondii infection in dairy cows of Thailand. During 2006-2007, the sera of 700 cows from 55 small farm holders of the highest number of dairy cow population in the northern provinces were collected and analysed. Antibodies to T. gondii were assayed by latex agglutination test (LAT), enzyme-linked immunosorbent assay (ELISA) and indirect fluorescent antibody test (IFAT). The overall prevalence of T. gondii infection in dairy cows was 9.4% (66/700) by LAT and 17% (119/700) by ELISA. Sixty-three seropositive samples by LAT (95.5%) and 107 by ELISA (89.9%) were confirmed by IFAT. These results demonstrated that LAT had the highest specificity for detection of T. gondii infection.

Inhibitory effects of (-)-epigallocatechin-3-gallate from green tea on the growth of Babesia parasites.

(-)-Epigallocatechin-3-gallate (EGCG) is the major tea catechin and accounts for 50-80% of the total catechin in green tea. (-)-Epigallocatechin-3-gallate has antioxidant, anti-inflammatory, anti-microbial, anti-cancer, and anti-trypanocidal activities. This report describes the inhibitory effect of (-)-Epigallocatechin-3-gallate on the in vitro growth of bovine Babesia parasites and the in vivo growth of the mouse-adapted rodent babesia B. microti. The in vitro growth of the Babesia species was significantly (P<0.05) inhibited in the presence of micromolar concentrations of EGCG (IC50 values=18 and 25 microM for B. bovis, and B. bigemina, respectively). The parasites showed no re-growth at 25 microM for B. bovis and B. bigemina in the subsequent viability test. The drug significantly (P<0.05) inhibited the growth of B. microti at doses of 5 and 10 mg/kg body weight, and the parasites completely cleared on day 14 and 16 post-inoculation in the 5 and 10 mg/kg treated groups, respectively. These findings highlight the potentiality of (-)-Epigallocatechin-3-gallate as a chemotherapeutic drug for the treatment of babesiosis.

Molecular characterizations of three distinct Babesia gibsoni rhoptry-associated protein-1s (RAP-1s).

Three cDNAs encoding rhoptry-associated protein 1 (RAP-1) homologues were found in the Babesia gibsoni EST database. Based on similarities to BgRAP-1a, which was identified previously by serological screening of a cDNA merozoite library, the two new genes were designated BgRAP-1b (33.7%) and BgRAP-1c (57%). Mice antiserum raised against each recombinant protein reacted specifically with B. gibsoni parasites as determined by Western blotting, which showed native molecular sizes of the BgRAP-1a (51 kDa), BgRAP-1b (53 kDa) and BgRAP-1c (47 kDa) consistent with predictable molecular weights. Immunofluoresence using these antibodies revealed localization of all BgRAP-1s within the matrix of merozoites; however, BgRAP-1a appeared to diverge from the other two when it was found secreted into the cytoplasm of infected erythrocytes. Apical localization of all 3 BgRAP-1s during the extracellular stage of the parasite combined with their ability to bind a canine erythrocyte membrane fraction was suggestive of a role for these proteins in erythrocyte attachment. Lastly, the ability of these recombinant proteins to be used as diagnostic reagents was tested by ELISA and the sensitivities of BgRAP-1a and BgRAP-1c were found increased through N-terminal truncation. Taken together, our data suggest divergent roles for the 3 BgRAP-1s in the merozoite stage of B. gibsoni.

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 degrees C for 1 h, but not at 30 degrees 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-gamma 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.

Characterization of a leucine aminopeptidase of Babesia gibsoni.

Peptidases of parasitic protozoa are currently under intense investigation in order to identify novel virulence factors, drug targets, and vaccine candidates, except in Babesia. Leucine aminopeptidases in protozoa, such as Plasmodium and Leishmania, have been identified to be involved in free amino acid regulation. We report here the molecular and enzymatic characterization, as well as the localization of a leucine aminopeptidase, a member of the M17 cytosolic aminopeptidase family, from B. gibsoni (BgLAP). A functional recombinant BgLAP (rBgLAP) expressed in Escherichia coli efficiently hydrolysed synthetic substrates for aminopeptidase, a leucine substrate. Enzyme activity of the rBgLAP was found to be optimum at pH 8.0 and at 37 degrees C. The substrate profile was slightly different from its homologue in P. falciprum. The activity was also strongly dependent on metal divalent cations, and was inhibited by bestatin, which is a specific inhibitor for metalloprotease. These results indicated that BgLAP played an important role in free amino acid regulation.

Seroprevalence of Cryptosporidium parvum infection of dairy cows in three northern provinces of Thailand determined by enzyme-linked immunosorbent assay using recombinant antigen CpP23.

Cryptosporidium parvum is the most frequent parasitic agent that causes diarrhoea in AIDS patients in Thailand. Cryptosporidiosis outbreaks in humans may be attributed to contamination of their drinking water from infected dairy pastures. A 23-kDa glycoprotein of C. parvum (CpP23) is a sporozoite surface protein that is geographically conserved among C. parvum isolates. This glycoprotein is a potentially useful candidate antigen for the diagnosis of cryptosporidiosis by enzyme-linked immunosorbent assay. Therefore, we investigated the seroprevalence of C. parvum infection in dairy cows in northern Thailand using an ELISA based on recombinant CpP23 antigen. Sera were randomly collected from 642 dairy cows of 42 small-holder farmers, which had the top three highest number of the dairy cows' population in Northern Thailand, that included Chiang Mai, Chiang Rai and Lumpang provinces. The overall seroprevalence of the infection was 4.4%, and the seropositive rates for the three provinces were 3.3% in Chiang Mai, 5.1% in Chiang Rai and 3% in Lumpang. These results suggest that cattle could play a role in zoonotic cryptosporidiosis in Thailand.

Prevalence of Theileria equi and Babesia caballi infections in horses belonging to resource-poor farmers in the north-eastern Free State Province, South Africa.

The prevalence of Theileria equi and Babesia caballi infections in the north-eastern Free State Province of South Africa was determined by examination of thin and thick Giemsa-stained blood smears, IFAT and PCR. No parasites were detected by microscopy from any blood samples collected at five study sites, Qwaqwa, Kestell, Harrismith, Vrede and Warden. Of the tested serum samples, 28/29 (96.5%), 20/21 (95.2%) and 42/42 (100%) were positive by IFAT for T. equi infections in Harrismith, Kestell and Qwaqwa, respectively, and 5/29 (17.2%), 13/21 (61.9%) and 30/42 (71.4%) were sero-positive for B. caballi infections in Harrismith, Kestell and Qwaqwa, respectively. All DNA samples from the study sites were negative for B. caballi infections by PCR, but five samples, two from each of Kestell and Warden and one from Vrede, were PCR positive for T. equi infections. The high prevalence of antibodies against T. equi and B. caballi in the sampled horses indicates that the animals had been exposed to T. equi and B. caballi infections but the absence of parasitaemia and very low number of positive PCR samples, however, imply that T. equi and B. caballi are endemically stable in the north-eastern Free State Province.

Diagnosis of Babesia caballi and Theileria equi infections in horses in Sudan using ELISA and PCR.

The purpose of this study was to estimate the prevalence of equine piroplasmosis in Sudan. The presence of antibodies against Babesia caballi and Theileria equi was determined in serum samples obtained from 158 horses raised in different locations in Sudan by enzyme-linked immunosorbent assay (ELISA). The B. caballi 48-kDa and the T. equi EMA-2 purified recombinant proteins were used as antigens in the ELISA test. Results showed that seven (4.4%) were positive for B. caballi and 80 (63.5%) were positive for T. equi. Polymerase chain reaction (PCR) assays have been applied using primers targeting the B. caballi 48-kDa merozoite antigen, the T. equi SSUrRNA and the T. equi EMA-1 genes. PCR performed on 131 blood spots in filter paper revealed that 33 (25.2%) samples were positive for T. equi but no positives were found for B. caballi. It is concluded that equine piroplasmosis is endemic in the country. This is the first study on serological and molecular epidemiological diagnosis on equine piroplasmosis in Sudan.

C3 contributes to the cross-protective immunity induced by Babesia gibsoni phosphoriboprotein P0 against a lethal B. rodhaini infection.

We have studied the impact of complement component 3 (C3) deficiency on the progression of lethal Babesia rodhaini infection in immune mice. A B. gibsoni ribosomal phosphoprotein P0 (BgP0) previously reported to be a cross-protective antigen against Babesia infection was used to immunize C57BL/6 wild-type (WT) and C3-deficient (C3-/-) mice. Test mice were immunized intraperitoneally (i.p.) with recombinant BgP0 (rBgP0), while controls either were immunized with PBS or did not receive any immunization. Following the immunization regime, test WT mice induced a specifically strong humoral response consisting of mixed immunoglobulins IgG1 and IgG2 associated with high production of IFN-gamma in the supernatant of splenocytes. While test C3-/- mice had significantly decreased total IgG, IgG1 and IgG2b responses, the secretions of IL-12 and IFN-gamma tended to be lower than those in WT mice. Furthermore, partial protection was only observed in rBgP0-immunized WT mice but not in C3-/- mice or controls. Indeed, rBgP0-immunized WT mice showed significant reductions in the initiation of parasitaemia correlated with delayed mortalities and considerable survival rates. Taken together, our results indicate that cross-protection was impaired in C3-/- mice in view of the decrease in the antibody responses and cytokine production and the high susceptibility to infection.

Comparison of polymerase chain reaction methods for the detection of Theileria equi infection using whole blood compared with pre-extracted DNA samples as PCR templates.

Rapid, efficient, and reproducible procedures for isolating DNA before PCR gene amplification are essential for the diagnosis of piroplasms. In this study, we evaluated the ease and reliability of detecting Theileria equi by PCR using pre-extracted DNA samples (by QIAamp DNA Mini Kit and phenol-chloroform methods) compared with blood spotted on FTA cards as PCR templates. Although minimal variations in limit of detection were observed among the methods compared, overall, the use of pre-extracted DNA samples and blood spotted on FTA cards had comparable detection limits. These results indicate that T. equi infection can be efficiently detected directly from FTA cards by PCR without the need for pre-extraction of DNA from blood samples.

Cyclin-dependent kinase inhibitors block erythrocyte invasion and intraerythrocytic development of Babesia bovis in vitro.

SUMMARYCyclin-dependent kinases (CDKs) are essential for the regulation of the eukaryotic cell cycle. A number of chemicals, which selectively inhibit the CDK activities, have been synthesized for the development of anti-cancer drugs. This report describes the inhibitory effect of purine derivatives known to be CDK inhibitors on the asexual growth of Babesia bovis. The 4 compounds, roscovitine, purvalanol A, CGP74514A, and CDK2 Inhibitor II, showed significantly suppressive effects on the in vitro growth of B. bovis. Three (roscovitine, purvalanol A, and CDK2 Inhibitor II) showed an inhibitory effect on the early stages of intraerythrocytic development of B. bovis. CGP74514A (CDK1-specific inhibitor) blocked the erythrocyte invasion by merozoites. Our data suggest the chemotherapeutic potential of the CDK inhibitors for babesiosis, and the target molecules of the compounds would participate in the process of successful erythrocyte invasion or intraerythrocytic development of B. bovis.

Molecular characterization of a novel 32-kDa merozoite antigen of Babesia gibsoni with a better diagnostic performance by enzyme-linked immunosorbent assay.

We cloned and expressed a novel gene encoding a 32-kDa merozoite protein of Babesia gibsoni (BgP32). The length of nucleotide sequence of the cDNA was 1464 bp with an open reading frame of 969 bp. The truncated recombinant BgP32 (rBgP32) without a signal peptide and C-terminal hydrophobic sequence was expressed in Escherichia coli as a soluble glutathione-S-transferase (GST) fusion protein. Western blotting demonstrated that the native protein was 32-kDa, consistent with molecular weight of the predicted mature polypeptide. Enzyme-linked immunosorbent assay (ELISA) using rBgP32 detected specific antibodies from 8 days to 541 days post-infection in the sequential sera from a dog experimentally infected with B. gibsoni. Moreover, the antigen did not cross-react with B. canis subspecies and closely related protozoan parasites, indicating that rBgP32 is a specific diagnostic antigen. Analysis of 47 sera taken from dogs with anaemic signs revealed that rBgP32 detected a higher proportion of B. gibsoni seropositive samples (77%) than its previously identified rBgP50 (68%) homologue. These results indicate that the BgP32 is a novel immunodominant antigen of B. gibsoni, and rBgP32 might be useful for diagnosis of B. gibsoni infection.

Amount of cholesterol in host membrane affects erythrocyte invasion and replication by Babesia bovis.

Cholesterol is a major component of the erythrocyte membrane. In the present study, we investigated the effects of cholesterol reduction in host bovine erythrocytes (RBC) on the growth of Babesia bovis, a major bovine haemoprotozoon. An in vitro growth assay with bovine RBC that had been prepared by pre-treatment with a cholesterol depletion agent (methyl-beta-cyclodextrin, MCD) showed that the culture with 5 mM MCD-treated RBC inhibited the growth of B. bovis significantly as compared with that with the control RBC. In further experiments, the treatment with 5 mM MCD was proved to suppress both activities of the parasite, erythrocyte invasion and replication within the infected RBC. In contrast, a slight reduction in the membrane cholesterol by 1 mM MCD treatment promoted both their growth and erythrocyte invasion activity. These results indicate that erythrocyte invasion and replication by B. bovis are affected by the amount of cholesterol in the host erythrocyte membrane.

Calcium-ions are involved in erythrocyte invasion by equine Babesia parasites.

Ethylene glycol bis (beta-aminoethylether)-N,N,N,N-tetraacetic acid (EGTA) is a chelating agent capable of binding to positively-charged metal ions, including a calcium-ion (Ca2+). Here, we demonstrated the inhibitory effect of the chemical on the in vitro asexual growth of the equine protozoan parasites, Babesia caballi and Babesia equi. The growth of both B. caballi and B. equi was significantly inhibited in the presence of EGTA (IC50=1.27 and 2.25 mM, respectively). Under microscopical observation, increased percentages of extracellular merozoites in the total parasites were detected in both of the cultures treated with high concentrations of EGTA. In contrast, further addition of Ca2+ to the EGTA-treated cultures prevented the parasites from clearing and the percentages of extracellular merozoites from increasing. As for B. caballi, an invasion test using high-voltage pulsing proved that EGTA has an inhibitory effect to their erythrocyte invasion. These results suggest that Ca2+ is involved in erythrocyte invasion by equine Babesia parasites.

Effects of protein kinase inhibitors on the in vitro growth of Babesia bovis.

Staurosporine, Ro-31-7549, and KN-93, which are inhibitors of serine/threonine protein kinase, protein kinase C, and calcium-modulin kinase, respectively, were tested for their effects on the in vitro growth of Babesia bovis. Staurosporine was the most effective inhibitor, completely clearing the parasitaemia as early as the first day of exposure at a concentration of 100 microM. Moreover, staurosporine caused a significant increase in the percentage of extracellular merozoites, most likely due to the inhibition of erythrocyte invasion by the parasite. Although 5 mM Ro-31-7549 and KN-93 had a suppressive action, this was not enough to destroy the parasite. Interestingly, concentrations of 0.5 to 5 mM KN-93 influenced the parasitic development within the infected erythrocytes. The present study suggests that B. bovis requires, to a certain extent, the phosphorylations mediated by parasite- or host erythrocyte-protein kinases, in particular, for the processes of successful invasion of erythrocytes and intraerythrocytic development.

Molecular characterization of a putative protein disulfide isomerase from Babesia caballi.

We produced a mAb against the Babesia caballi extracellular merozoite termed mAb 2H2 and used it to screen a cDNA expression library prepared from B. caballi merozoite mRNA for highly expressed proteins. The complete nucleotide sequence of the cloned gene had 1547 nucleotides and contained a 36-nucleotide intron. The 1398 nucleotide open reading frame predicts a 51 kDa protein showing similarity to protein disulfide isomerase (PDI) from other species. The PDI gene had a predicted N-terminal signal sequence of 19 amino acids and a C-terminal tetrapeptide sequence (His-Thr-Glu-Leu; HTEL) for retention in lumen of the endoplasmic reticulum (ER). The recombinant protein expressed in baculovirus showed an apparent mass of 51 kDa, identical to that the native B. caballi protein. Moreover, the ER retention signal site (HTEL) of the recombinant protein retained its function in ER of insect cells. This 51 kDa protein was strongly expressed by extracelluar B. caballi merozoites in indirect immunofluorescence antibody tests, and was not expressed in the early phase of trophozoite development. Interestingly, detailed observation showed that the reaction of anti-P51 antibody and mAb 2H2 against pear-shaped forms was very erratic, some displaying one or two brightly fluorescent patterns.

Development of a practical immunochromatographic test with recombinant P50 for the diagnosis of Babesia gibsoni infection in dogs.

An immunochromatographic test (ICT), using recombinant truncated P50 (P50t), for the detection of antibodies to Babesia gibsoni was developed and evaluated. Whereas all sera from specific pathogen-free dogs were clearly negative, all sera from dogs experimentally infected with B. gibsoni were clearly positive in the ICT. In addition, the ICT detected no cross-reactivity with sera from dogs experimentally infected with closely related parasites, B. canis canis, B. canis vogeli, and B. canis rossi, or with Neospora caninum, and Leishmania infantum. Sequential sera from a dog experimentally infected with B. gibsoni were tested with the ICT; it was shown that the specific antibodies are detectable as early as 6 days post-infection (p.i.) and that strong antibody responses remained until the end of the experiment (144 days p.i.). To evaluate the clinical application of the ICT, a total of 54 serum samples collected from domestic dogs that had been identified as having signs of anaemia at veterinary hospitals in Japan, were tested with the ICT, the previously established enzyme-linked immunosorbent assay (ELISA) and with the indirect fluorescent antibody test (IFAT). Twenty-four of the tested samples (44.4%) were positive in both ICT and ELISA, and (51.8%) in IFAT. The concordance between ELISA and ICT was found to be 100%, and 85.7% with IFAT. Taken together, the results above suggest that the ICT using P50t is rapid, simple, accurate, and suitable for use at clinical sites for the diagnosis of B. gibsoni infection in dogs.

Host serum modifies the drug susceptibility of Babesia bovis in vitro.

Babesia parasites generally require a defined percentage of serum in the culture medium for their in vitro growth. In this study, we attempted to culture Babesia bovis in a serum-free condition. The growth pattern and morphology of B. bovis in serum-free (plain) GIT medium were unaltered as compared to those of the standard growth condition containing 40% bovine serum in M199. When exposed to the test drugs, the parasite in plain GIT medium showed clearly lower IC50 values than those in 40% serum-containing G IT medium, indicating that several serum components may interfere with the drug bio-availability. Therefore, the serum-free culture system is useful for standardizing drug test protocols and understanding the roles of serum factors in the drug test.

Natural IgM antibodies in sera from various animals but not the cat kill Toxoplasma gondii by activating the classical complement pathway.

Sera from swine, rabbit, and dog, that had never been exposed to Toxoplasma gondii, demonstrated significant killing of T. gondii tachyzoites in vitro, while cat serum did not. Swine and rabbit sera contained natural IgM antibody against the tachyzoites, and the classical complement pathway was activated by the binding of natural IgM antibody to the tachyzoites, leading to lysis. Anti-T. gondii antibodies, induced in swine or cat infected with T. gondii, had no killing effect by themselves but killed the tachyzoites in the presence of swine complement. However, the anti-T. gondii antibodies of swine or cat demonstrated a very low killing effect in the presence of cat complement. This suggests that T. gondii tachyzoites have an evasion mechanism to prevent lysis which is specific for cat complement.

Clotrimazole, ketoconazole, and clodinafop-propargyl inhibit the in vitro growth of Babesia bigemina and Babesia bovis (Phylum Apicomplexa).

We evaluated the growth inhibitory efficacy of the imidazole derivatives, clotrimazole (CLT) and ketoconazole (KC), and the herbicide clodinafop-propargyl (CP), in in vitro cultures of Babesia bovis and B. bigemina. Clotrimazole was effective in a dose range of 15 to 60 microM (IC50: 11 and 23.5 microM), followed by KC (50 to 100 microM; IC50: 50 and 32 microM) and CP (500 microM; IC50: 265 and 390 microM). In transmission electron microscopy, extensive damage was observed in the cytoplasm of drug-treated parasites. Combinations of CLT/KC, CLT/CP and CLT/KC/CP acted synergistically in both parasites. In contrast, the combination of KC/CP was exclusively effective in B. bovis, but not in B. bigemina.