Seroprevalence and risk factors associated with Theileria parva infection among calves in Narok County, Kenya.

Background and Aim: East Coast fever (ECF), caused by Theileria parva, is a devastating disease that causes significant economic losses to cattle production in sub-Saharan Africa. Prevention and control of ECF are challenging in pastoral settings due to inadequate epidemiological information. This study aimed to estimate the seroprevalence and risk factors associated with T. parva infection among calves in different production systems to help design appropriate control interventions. Materials and Methods: Blood samples were collected from 318 calves and tested using an indirect enzyme-linked immunosorbent assay targeting antibodies against polymorphic immunodominant molecules found on the surface of T. parva. Information on calf characteristics and management practices was also collected during sampling. Descriptive statistics and logistic regression were used to analyze potential risk factors, such as age and acaricide application, where p < 0.05 was considered significant. Results: Of the 318 calves sampled, 41 (12.89%) were positive for T. parva, with a higher proportion in pastoral systems (36.58%) than in mixed farming systems (34.10%) and agropastoral systems (29.27%). From univariate analysis, calf age (p = 0.002), body weight (p = 0.001), suckling status (p = 0.026), rectal temperature (p = 0.06), calves on pasture (p = 0.022), other feeds (p = 0.004), feed grown within the farm (p = 0.004), acaricide application (p = 0.001), and acaricide application frequency (p = 0.001) were significantly associated with seropositivity. However, calf age (odds ratio [OR], 0.96; 95% confidence interval [CI], 0.91-0.99; p = 0.04), other feeds (OR, 8.82; 95% CI, 1.74-44.63; p = 0.009), and suckling status (OR, 0.38; 95% CI, 0.15-0.99; p = 0.05) were significantly associated with T. parva infection in the multivariable mixed logistic model. Conclusion: T. parva is circulating in young calves in the study area (and possibly in cattle populations due to maternal transfer of antibodies to the calves). There is a need for molecular surveillance to determine the presence and burden of T. parva infection.

Prevalence of Ticks Infesting Dairy Cattle and the Pathogens They Harbour in Smallholder Farms in Peri-Urban Areas of Nairobi, Kenya.

This study aimed at determining the tick species infesting dairy cattle in Nairobi, Kenya, and the pathogens they harbour. While ticks are well-known vectors of major bacterial pathogens of both veterinary importance and public health importance, few studies have investigated the range of the tick species and the associated pathogens, especially present in unique dairy production systems, which compromise animal welfare, such as those in peri-urban areas. A cross-sectional study was undertaken involving 314 randomly selected dairy cattle in 109 smallholder farms. Each animal was examined for attached ticks followed by morphological tick identification at the species level. Genomic DNA was extracted from each of the ticks, and 16S rDNA gene was amplified for pathogen identification. Sequencing of the amplicons and subsequent BLASTn analysis, multiple sequence alignment, and phylogenetic reconstruction were performed to confirm the species of the pathogens. Sixty-six (21.0%) of the cattle examined had ticks. A total of 94 adult ticks were found on the cattle, and of these, 63 (67.0%), 18 (19.1%), and 13 (13.8%) were in the genera Rhipicephalus, Amblyomma, and Hyalomma, respectively. Twelve tick species in Rhipicephalus genus and two in Amblyomma and Hyalomma genera were identified. Although Rh. decoloratus was the most prevalent tick (24.5% (23/94)), the emerging Rh. microplus (6.4% (6/94)) was also identified. The DNA of Rickettsia was detected in the ticks, with Rickettsia conorii in H. rufipes and A. variegatum, and Rickettsia aeschlimannii in Rh. microplus and H. rufipes, while Ehrlichia ruminantium and E. canis were in A. variegatum. In conclusion, the study reported a wide range of tick species present in the study area including Rhipicephalus microplus, which is an emerging tick species in parts of Kenya. The ticks harboured DNA of Rickettsia and Ehrlichia, highlighting possible animal and human health concerns. Hence, effective tick control strategies remain paramount to prevent potential diseases associated with the harboured pathogens.

Developing and validating a rapid assessment tool for small ruminant reproduction and production in pastoralist flocks in Kajiado, Kenya.

The collection of production performance data in small ruminants pastoralists flocks is essential to evaluate their efficiency and assess how different challenges, such as diseases, droughts or changes in land use, affect their sustainability. Although different methodologies exist, capturing these data is difficult, particularly in nomadic pastoral flocks. In this study, a method for rapid assessment of flock production performance based on farmer recall was designed, implemented and tested. Through literature review and consultation with pastoralists and key informants, a questionnaire was developed to capture small ruminant flock data disaggregated by age (≤2 years old and >2 years old), species and sex. Data on flock dynamics and on reproduction parameters were collected for a period of 12 months. A survey of 130 pastoralists with medium to large flocks was then conducted in Kajiado County (Kenya). Values were calculated for 12 reproduction performance indicators and 7 additional production performance indicators. In addition, a flock efficiency indicator (FEI) is proposed that classifies mixed flocks (i.e., with sheep and goats) into low, medium or high efficiency. Results showed that in flocks with low efficiency, the median value for net fecundity rate was 0.43 lambs/ewe (range 0.08-1.00) and 0.41 kids/doe (range 0.07-0.73), and the median production rate was 6% (range [-47%] to 20%) for sheep and 11% (range [-38%] to 0.21%) for goats. In flocks with high efficiency the median net fecundity rate was 0.77 lambs/ewe (range 0.48-2.73) and 0.88 kids/doe (range 0.49-1.80), and the median production rate was 25% (range 11-47) for sheep and 28% (range 15-46) for goats. Sixty-two (47.7%) of the pastoralists surveyed reported usually buying animals into their flocks, and, consequently were considered as 'pastoralists and traders'. Their flocks had significantly lower FEI scores (median = 7.5, interquartile range [IQR] 6-9), compared with "pastoralists only" flocks (median = 8, IQR 6-10, Kruskal-Wallis rank sum test, p-value <0.001). Since this method is based on pastoralist recall, values obtained should be considered as approximations. Nonetheless, the proposed assessment tool can be used by individuals with low resources or recording capacity, and in large scale programmes to monitor pastoralist flock dynamics, set-up benchmarking programmes, estimate the impact of diseases and shocks, identify those flocks which are most vulnerable to these shocks and evaluate the effectiveness of policies and interventions on herd performance.

Risk factors for the incursion, spread and persistence of the foot and mouth disease virus in Eastern Rwanda.

BACKGROUND: Identification of risk factors is crucial in Foot-and-mouth disease (FMD) control especially in endemic countries. In Rwanda, almost all outbreaks of Foot-and-Mouth Disease Virus (FMDV) have started in Eastern Rwanda. Identifying the risk factors in this area will support government control efforts. This study was carried out to identify and map different risk factors for the incursion, spread and persistence of FMDV in Eastern Rwanda. Questionnaires were administered during farm visits to establish risk factors for FMD outbreaks. Descriptive statistical measures were determined and odds ratios were calculated to determine the effects of risk factors on the occurrence of FMD. Quantum Geographic Information System (QGIS) was used to produce thematic maps on the proportion of putative risk factors for FMD per village. RESULTS: Based on farmers' perceptions, 85.31% (with p < 0.01) experienced more outbreaks during the major dry season, a finding consistent with other reports in other parts of the world. Univariate analysis revealed that mixed farming (OR = 1.501, p = 0.163, CI = 95%), and natural breeding method (OR = 1.626; p = 0.21, CI = 95%) were associated with the occurrence of FMD indicating that the two risk factors could be responsible for FMD outbreaks in the farms. The occurrence of FMD in the farms was found to be significantly associated with lack of vaccination of calves younger than 12 months in herds (OR = 0.707; p = 0.046, CI = 95%). CONCLUSIONS: This is the first study to describe risk factors for persistence of FMDV in livestock systems in Rwanda. However, further studies are required to understand the role of transboundary animal movements and genotypic profiles of circulating FMDV in farming systems in Rwanda.

Molecular prevalence of emerging Anaplasma and Ehrlichia pathogens in apparently healthy dairy cattle in peri-urban Nairobi, Kenya.

BACKGROUND: Anaplasma and Ehrlichia species are tick-borne pathogens of both veterinary and public health importance. The current status of these pathogens, including emerging species such as Ehrlichia minasensis and Anaplasma platys, infecting cattle in Kenya, remain unclear, mainly because of limitation in the diagnostic techniques. Therefore, we investigated the Anaplasma and Ehrlichia species infecting dairy cattle in Nairobi, Kenya using molecular methods. RESULTS: A total of 306 whole blood samples were collected from apparently healthy dairy cattle. Whole blood DNA was extracted and tested for presence of Anaplasma and Ehrlichia DNA through amplification and sequencing of the 16S rDNA gene. Sequence identity was confirmed using BLASTn analysis while phylogenetic reconstruction was performed to determine the genetic relationship between the Kenyan isolates and other annotated genotypes available in GenBank. Anaplasma and Ehrlichia species were detected in 19.9 and 3.3% of all the samples analyzed, respectively. BLASTn analysis of the sequences against non-redundant GenBank nucleotide database revealed infections with A. platys (44.8%), A. marginale (31%) and A. bovis (13.8%). All four sequenced Ehrlichia spp. were similar to Ehrlichia minasensis. Nucleotide polymorphism was observed for A. platys, A. bovis and E. minasensis. The Anaplasma species clustered in four distinct phylogenetic clades including A. marginale, A. platys, A. bovis and some unidentified Anaplasma spp. The Kenyan Ehrlichia minasensis clustered in the same clade with isolates from America and Australia but distant from E. ruminantium. CONCLUSION: This study provides the first report of infection of dairy cattle in Kenya with A. platys and E. minasensis, which are emerging pathogens. We conclude that cattle in peri-urban Nairobi are infected with various species of Anaplasma and E. minasensis. To understand the extent of these infections in other parts of the country, large-scale screening studies as well as vector identification is necessary to inform strategic control.

Identification and characterization of profilin antigen among Babesia species as a common vaccine candidate against babesiosis.

We have characterized a member of the profilin (PROF) family protein as a common antigen in three pathogens-Babesia bovis (B. bovis), Babesia bigemina (B. bigemina), and Babesia microti (B. microti)-and evaluated its immunogenic and cross-protective properties against a challenge infection with B. microti in BALB/c mice. The recombinant PROF proteins of B. bovis, B. bigemina, and B. microti were successfully expressed in Escherichia coli (E. coli) as soluble GST fusion proteins (rBboPROF, rBbigPROF, and rBmPROF, respectively), and they were found to be antigenic. On probing with mouse anti-rPROF serum, green fluorescence was observed on the parasites' cytosols by confocal laser microscopy. Immunization regimes in BALB/c mice using rPROFs induced cross-protective immunity against B. microti infection based on high levels of cytokines and immunoglobulin (IgG) titers, a reduction in peak parasitemia levels, and earlier clearance of the parasite as compared with control mice. The findings of the present study indicate that PROF is a common antigen among bovine and murine Babesia parasites, and it might be used as a common vaccine candidate against babesiosis.

Characterization of Toxoplasma gondii glyoxalase 1 and evaluation of inhibitory effects of curcumin on the enzyme and parasite cultures.

BACKGROUND: The glyoxalase pathway, which includes two enzymes, glyoxalase 1 and 2 (Glo1 and Glo2), is a ubiquitous cellular system responsible for the removal of cytotoxic methylglyoxal produced during glycolysis. Protozoan parasites, including Toxoplasma gondii (T. gondii) tachyzoites, produce methylglyoxal because of increased glycolytic fluxes. A Glo1 inhibitor such as curcumin could be considered a drug candidate for anti-protozoan, anti-inflammatory, and anti-cancer therapy. METHODS: The T. gondii Glo1 gene (TgGlo1) was cloned and the recombinant protein was produced. Enzyme kinetics of TgGlo1 and five mutants were evaluated by adding methylglyoxal and glutathione to a reaction mixture. Finally, the inhibitory effects of various concentrations of curcumin on recombinant TgGlo1 were evaluated using in vitro cultures of T. gondii. RESULTS: Active recombinant TgGlo1 was successfully produced and the active sites (E166 and E251) of TgGlo1 were verified by point mutagenesis. Curcumin at the tested doses inhibited the enzymatic activity of recombinant TgGlo1 as well as the parasitic propagation of in vitro-cultured T. gondii. The Ki and IC50 were 12.9 ± 0.5 µM and 38.3 ± 0.9 µM, respectively. CONCLUSION: The inhibitory effect of curcumin on the enzymatic activity of TgGlo1 and parasitic propagation of T. gondii could be explored in the potential development of a potent drug for the treatment of toxoplasmosis. However, considering the fact that curcumin is known to have many effects on other molecules in the micromolar range, further elucidation of curcumin's direct inhibition of the glyoxalase system of T. gondii will be needed.

Molecular detection and characterization of Babesia bovis, Babesia bigemina, Theileria species and Anaplasma marginale isolated from cattle in Kenya.

BACKGROUND: Infections with Babesia bovis, Babesia bigemina, Theileria species and Anaplasma marginale are endemic in Kenya yet there is a lack of adequate information on their genotypes. This study established the genetic diversities of the above tick-borne hemoparasites infecting cattle in Kenya. METHODS: Nested PCR and sequencing were used to determine the prevalence and genetic diversity of the above parasites in 192 cattle blood samples collected from Ngong and Machakos farms. B. bovis spherical body protein 4, B. bigemina rhoptry-associated protein 1a, A. marginale major surface protein 5, Theileria spp. 18S rRNA, T. parva p104 and T. orientalis major piroplasm surface protein were used as the marker genes. RESULTS: B. bovis, B. bigemina, T. parva, T. velifera, T. taurotragi, T. mutans and A. marginale were prevalent in both farms, whereas T. ovis, Theileria sp. (buffalo) and T. orientalis were found only in Ngong farm. Co-infections were observed in more than 50 % of positive samples in both farms. Babesia parasites and A. marginale sequences were highly conserved while T. parva and T. orientalis were polymorphic. Cattle-derived T. parva was detected in Machakos farm. However, cattle and buffalo-derived Theileria were detected in Ngong farm suggesting interactions between cattle and wild buffaloes. Generally, the pathogens detected in Kenya were genetically related to the other African isolates but different from the isolates in other continents. CONCLUSIONS: The current findings reaffirm the endemicity and co-infection of cattle with tick-borne hemoparasites, and the role of wildlife in pathogens transmission and population genetics in Kenya.

Molecular Characterization of Babesia bovis M17 Leucine Aminopeptidase and Inhibition of Babesia Growth by Bestatin.

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 Km and Vmax values of the recombinant enzyme were 139.3 ± 30.25 and 64.83 ± 4.6 µM, respectively, while the Ki was 2210 ± 358 µM after the inhibition. Bestatin was a more potent inhibitor of the growth of B. bovis [IC50 (50% inhibition concentration) = 131.7 ± 51.43 µM] than B. gibsoni [IC50 = 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.

Mycophenolic acid, mycophenolate mofetil, mizoribine, ribavirin, and 7-nitroindole inhibit propagation of Babesia parasites by targeting inosine 5'-monophosphate dehydrogenase.

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.

Prime-boost vaccination with plasmid DNA followed by recombinant vaccinia virus expressing BgGARP induced a partial protective immunity to inhibit Babesia gibsoni proliferation in dogs.

A heterologous prime-boost vaccination regime with DNA and recombinant vaccinia virus (rvv) vectors expressing relevant antigens has been shown to induce effective immune responses against several infectious pathogens. In this study, we describe the effectiveness of the prime-boost strategy by immunizing dogs with a recombinant plasmid followed by vaccinia virus, both of which expressed the glutamic acid-rich protein (BgGARP) of Babesia gibsoni. The dogs immunized with the prime-boost regime developed a significantly high level of specific antibodies against BgGARP when compared with the control groups. The antibody level was strongly increased after a booster immunization with a recombinant vaccinia virus. Two weeks after the booster immunization with a recombinant vaccinia virus expressing BgGARP, the dogs were challenged with B. gibsoni parasite. The dogs immunized with the prime-boost regime showed partial protection, manifested as a significantly low level of parasitemia. These results indicated that this type of DNA/rvv prime-boost immunization approach may have use against B. gibsoni infection in dogs.

Molecular characterization and antigenic properties of a novel Babesia gibsoni glutamic acid-rich protein (BgGARP).

Identification and molecular characterization of Babesia gibsoni proteins with potential antigenic properties are crucial for the development and validation of the serodiagnostic method. In this study, we isolated a cDNA clone encoding a novel B. gibsoni 76-kDa protein by immunoscreening of the parasite cDNA library. Computer analysis revealed that the protein presents a glutamic acid-rich region in the C-terminal. Therefore, the protein was designated as B. gibsoni glutamic acid-rich protein (BgGARP). A BLASTp analysis of a translated BgGARP polypeptide demonstrated that the peptide shared a significant homology with a 200-kDa protein of Babesia bigemina and Babesia bovis. A truncated BgGARP cDNA (BgGARPt) encoding a predicted 13-kDa peptide was expressed in Escherichia coli (E. coli), and mouse antisera against the recombinant protein were used to characterize a corresponding native protein. The antiserum against recombinant BgGARPt (rBgGARPt) recognized a 140-kDa protein in the lysate of infected erythrocytes, which was detectable in the cytoplasm of the parasites by confocal microscopic observation. In addition, the specificity and sensitivity of enzyme-linked immunosorbent assay (ELISA) with rBgGARPt were evaluated using B. gibsoni-infected dog sera and specific pathogen-free (SPF) dog sera. Moreover, 107 serum samples from dogs clinically diagnosed with babesiosis were examined using ELISA with rBgGARPt. The results showed that 86 (80.4%) samples were positive by rBgGARPt-ELISA, which was comparable to IFAT and PCR as reference test. Taken together, these results demonstrate that BgGARP is a suitable serodiagnostic antigen for detecting antibodies against B. gibsoni in dogs.

TgGRA23, a novel Toxoplasma gondii dense granule protein associated with the parasitophorous vacuole membrane and intravacuolar network.

Toxoplasma gondii is an intracellular protozoan parasite, which relies on a specialized compartment, the parasitophorous vacuole (PV), to survive within host cells. Dense granules within the parasite release a large variety of proteins to maintain the integrity of the vacuole structure. Here, we identified a novel dense granule protein in T. gondii, TgGRA23, which is a homolog of the Sarcocystis muris dense granule protein, SmDG32. Recombinant TgGRA23 (rTgGRA23) expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein was used to raise antisera in mice and rabbits. Immunoblotting showed that antisera from the immunized mice and rabbits reacted with parasite lysates to yield a 21-kDa native protein. In addition, immuno-electron microscopic examination showed that TgGRA23 resides in the dense granules, PV membrane and intravacuolar network of the parasite. To confirm the precise subcellular localization of TgGRA23 in T. gondii, an immunofluorescent antibody test was performed using dense granule markers. Notably, TgGRA23 co-localized with other dense granule proteins including TgGRA4 and TgGRA7, in the extracellular-stage parasites. Biochemical experiments indicated that TgGRA23 is insoluble and may form an electrostatic complex that is resistant to non-ionic detergents. Furthermore, specific antibodies to TgGRA23 were detected during the chronic stage of Toxoplasma infection in mice. Our results suggest that TgGRA23 is an as yet unknown member of the T. gondii dense granule proteins, and that it may be involved in remodeling or maintenance of the PV.

Epidemiological survey of Babesia bovis and Babesia bigemina infections of cattle in Philippines.

A total of 250 blood samples were collected from clinically healthy cattle in five provinces of Philippines. DNA was extracted from the samples and analyzed by nested PCR assays for an epidemiological survey of Babesia bovis and Babesia bigemina infections. Out of the 250 samples, 27 (10.8%) and 16 (6.4%) were positive for B. bovis infection and B. bigemina infection, respectively. Mixed infections were detected in a total of 4 samples (1.6%). Our data provide baseline information regarding the epidemiology of B. bovis and B. bigemina infections in cattle in Philippines, which can be utilized in developing proper strategies for disease control and management.

Identification and characterization of an interspersed repeat antigen of Babesia microti (BmIRA).

In this report, a novel gene encoding an interspersed repeat antigen from Babesia microti (BmIRA) was identified and described. The full-length cDNA containing an open reading frame of 1,947 bp was obtained by immunoscreening a B. microti cDNA expression library. The full-length of BmIRA gene was expressed as a GST fusion recombinant BmIRA (rBmIRA) in Escherichia coli. Sera of mice immunized with the rBmIRA detected a native parasite protein with a molecular mass of 76 kDa on Western blot analysis. The same protein was detected in the parasites by immunofluorescent antibody test (IFAT). An enzyme-linked immunosorbent assay (ELISA) using rBmIRA detected specific antibodies as early as 11 days post-infection in sera from a hamster experimentally infected with B. microti Gray stain (US type). Furthermore, a rapid immunochromatographic test (ICT) using rBmIRA detected specific antibodies in a hamster experimentally infected with B. microti from day 11 to at least day 180 post-infection. The results indicate the antibody response against the rBmIRA was maintained during the chronic stage of infection. On the other hand, an immunoprotective property of rBmIRA as a subunit vaccine was evaluated in hamsters against B. microti challenge, but no significant protection was observed. Our data suggest that the immunodominant antigen BmIRA could be a useful serodiagnostic antigen for screening of B. microti infection.

Cloning, characterization and validation of inosine 5'-monophosphate dehydrogenase of Babesia gibsoni as molecular drug target.

The inosine monophosphate dehydrogenase (IMPDH) enzyme has been characterized and validated as a molecular drug target in other apicomplexans but not in the genus Babesia. Subsequently, we cloned and expressed a Babesia gibsoni IMPDH (BgIMPDH) cDNA in Escherichia coli. We also determined the inhibitory effect of mycophenolic acid (MPA) on recombinant BgIMPDH (rBgIMPDH) activity and the Babesia-growths in vitro. The translated BgIMPDH peptide contained thirteen amino acid residues responsible for substrate and cofactor binding in its catalytic domain with Gly374 in BgIMPDH being replaced by Ser388 in mammalian IMPDH. The native BgIMPDH enzyme in the parasite was approximately 54-kDa a mass similar to His-tag rBgIMPDH protein. The Km values of the rBgIMPDH were 8.18±0.878 (mean±standard error of the mean) µM and 360.80±43.41µM for IMP and NAD(+), respectively. MPA inhibited the rBgIMPDH activity yielding a Ki value of 20.93±1.83µM with respect to NAD(+). For Babesia growths, the IC50s were 0.95±0.21 and 2.88±0.49µM for B. gibsoni and B. bovis, respectively. Therefore, our results suggest that MPA may inhibit the replication of Babesia parasites by targeting IMPDH enzyme of the purine pathway.

Development of a rapid immunochromatographic test using a recombinant thrombospondin-related adhesive protein of Babesia gibsoni.

We developed an immunochromatographic test (ICT) with the full-length of thrombospondin-related adhesive protein of Babesia gibsoni expressed by the modified expression method. The developed ICT showed high sensitivity, specificity, and kappa value with a reference test (100%, 93.78%, and 0.8976, respectively), indicating that the ICT could be a new practical diagnostic test for B. gibsoni infection.

Molecular detection and identification of Babesia bovis and Babesia bigemina in cattle in northern Thailand.

Although Babesia bovis and Babesia bigemina infections cause economic losses in the cattle industry in northern Thailand, there is inadequate information on Babesia isolates present in the area. Therefore, to determine the prevalence and genetic relationship between Babesia isolates, we screened 200 blood samples of cattle from Chiang Rai, Chiang Mai, and Lumpang provinces of northern Thailand. A nested polymerase chain reaction using primers targeting B. bovis spherical body protein 2 (BboSBP2) and B. bigemina rhoptry-associated protein 1a (BbiRAP-1a) genes revealed a prevalence of 12 and 21 % for B. bovis and B. bigemina, respectively, while that of mixed infections was 6.5 % samples. The prevalences of B. bovis in Chiang Rai, Chiang Mai, and Lumpang were 9.5, 3.7, and 25.5 %, respectively. For B. bigemina, the prevalences were 15.8, 12.9, and 39.2 % in Chiang Rai, Chiang Mai, and Lumpang, respectively. Mixed infections with B. bovis and B. bigemina were 6.3 % in Chiang Rai, 1.9 % in Chiang Mai, and 13.7 % in Lumpang. The identical sequences of either BboSBP2 gene or BbiRAP-1a gene were shared among the Babesia isolates in the three provinces of northern Thailand. Further analysis using the internal transcribed spacer gene revealed at least four genotypes for B. bovis and five genotypes for B. bigemina in northern Thailand, while the sequences present great genetic diversities in the different isolates. Overall, we have demonstrated a high prevalence and polymorphism of Babesia parasites in northern Thailand calling for the need to design effective control programs for bovine babesiosis.

Protective effect of a prime-boost strategy with plasmid DNA followed by recombinant adenovirus expressing TgAMA1 as vaccines against Toxoplasma gondii infection in mice.

A heterologous prime-boost strategy with priming plasmid DNA followed by recombinant virus expressing relevant antigens is known to stimulate protective immunity against intracellular parasites. In this study, we have evaluated a heterologous prime-boost strategy for immunizing mice against Toxoplasma gondii infection. Our results revealed that the prime-boost strategy using both plasmid DNA and adenoviral vector encoding TgAMA1 may stimulate both humoral and Th1/Th2 cellular immune responses specific for TgAMA1. Moreover, C57BL/6 mice immunized with the pAMA1/Ad5Null, pNull/Ad5AMA1, and pAMA1/Ad5AMA1 constructs showed survival rates of 12.5%, 37.5%, and 50%, respectively. In contrast, all the pNull/Ad5Null immunized mice died after infection with the PLK-GFP strain of T. gondii. Brain cyst burden was reduced by 23% in mice immunized with pAMA1/Ad5AMA1 compared with the pNull/Ad5AMA1 immunized mice. These results demonstrate that the heterologous DNA priming and recombinant adenovirus boost strategy may provide protective immunity against T. gondii infection.

Four promising antigens, BgP32, BgP45, BgP47, and BgP50, for serodiagnosis of Babesia gibsoni infection were classified as B. gibsoni merozoite surface protein family.

We determined the molecular characteristics of four proteins, BgP32, BgP45, BgP47, and BgP50, of Babesia gibsoni. Localization by subcellular fractionations followed by Western blotting revealed that the corresponding native proteins belong to merozoite surface protein family of B. gibsoni (BgMSP). Moreover, antisera against either rBgP45 or rBgP47 cross-reacted with all the proteins of the BgMSP family on ELISA and IFAT analyses. Of the four candidate antigens, ELISA with rBgP45 yielded high sensitivity, and ELISA with rBgP32 resulted in high specificity and in concordance with IFAT results.