Patrick Vudriko

Molecular detection and genetic characterization of Babesia, Theileria and Anaplasma amongst apparently healthy sheep and goats in the central region of Turkey

Babesia spp., Theileria spp. and Anaplasma spp. are significant tick-borne pathogens of livestock globally. In this study, we investigated the presence and distribution of Babesia ovis, Theileria ovis and Anaplasma ovis in 343 small ruminants (249 sheep and 94 goats) from 13 towns in the Central Anatolia region of Turkey using species-specific PCR assays. The PCR were conducted using the primers based on the B. ovis ssu rRNA (BoSSUrRNA), T. ovis ssu rRNA (ToSSUrRNA) and A. ovis major surface protein 4 (AoMSP4) genes, respectively. Fragments of these genes were sequenced for phylogenetic analysis. PCR results revealed that the overall infections of A. ovis, T. ovis and B. ovis were 60.0%, 35.9% and 5.2%, respectively. Co-infection of the animals with two or three pathogens was detected in 105/343 (30.6%) of the ovine samples. The results of sequence analysis indicated that AoMSP4 were conserved among the Turkish samples, with 100% sequence identity values. In contrast, the BoSSUrRNA and ToSSUrRNA gene sequences were relatively diverse with identity values of 98.54%-99.82% and 99.23%-99.81%, respectively. Phylograms were inferred based on the BoSSUrRNA, ToSSUrRNA and AoMSP4 sequences obtained in this study and those from previous studies. B. ovis isolates from Turkey were found in the same clade as the isolates from other countries in phylogenetic analysis. On the other hand, the Turkish T. ovis isolates in the present study formed a monophyletic grouping with the isolates from other countries in a phylogeny based on ToSSUrRNA sequences. Furthermore, phylogenetic analysis using AoMSP4 sequences showed the presence of three genotypes of A. ovis. This study provides important data for understanding the epidemiology of tick-borne diseases in small ruminants and the degree of genetic heterogeneities among these pathogens in Turkey. To our knowledge, this is the first study on the co-infection of Babesia, Theileria and Anaplasma in sheep and goats in Turkey.

Molecular detection and genetic identification of Babesia bigemina, Theileria annulata, Theileria orientalis and Anaplasma marginale in Turkey.

Babesia spp., Theileria spp. and Anaplasma spp. are significant tick-borne pathogens of livestock globally. In this study, we investigated the presence and distribution of Babesia bigemina, Theileria annulata, Theileria orientalis and Anaplasma marginale in cattle from 6 provinces of Turkey using species-specific PCR assays. The PCR were conducted using the primers based on the B. bigemina rhoptry-associated protein 1a (BbiRAP-1a), T. annulata merozoite surface antigen-1 (Tams-1), T. orientalis major piroplasm surface protein (ToMPSP) and A. marginale major surface protein 4 (AmMSP4) genes, respectively. Fragments of B. bigemina internal transcribed spacer (BbiITS), T. annulata internal transcribed spacer (TaITS), ToMPSP and AmMSP4 genes were sequenced for phylogenetic analysis. PCR results revealed that the overall infections of A. marginale, T. annulata, B. bigemina and T. orientalis were 29.1%, 18.9%, 11.2% and 5.6%, respectively. The co-infection of two or three pathogens was detected in 29/196 (15.1%) of the cattle samples. The results of sequence analysis indicated that BbiRAP-1a, BbiITS, Tams-1, ToMPSP and AmMSP4 were conserved among the Turkish samples, with 99.76%, 99-99.8%, 99.34-99.78%, 96.9-99.61% and 99.42-99.71% sequence identity values, respectively. In contrast, the Turkish TaITS gene sequences were relatively diverse with 92.3-96.63% identity values. B. bigemina isolates from Turkey were found in the same clade as the isolates from other countries in phylogenetic analysis. On the other hand, phylogenetic analysis based on T. annulata ITS sequences revealed significant differences in the genotypes of T. annulata isolates from Turkey. Additionally, the T. orientalis isolates from Turkish samples were classified as MPSP type 3 genotype. This is the first report of type 3 MPSP in Turkey. Moreover, AmMSP4 isolates from Turkey were found in the same clade as the isolates from other countries. This study provides important data for understanding the epidemiology of tick-borne diseases and it is expected to improve approach for diagnosis and control of tick-borne diseases in Turkey.

Molecular epidemiology of bovine Babesia spp. and Theileria orientalis parasites in beef cattle from northern and northeastern Thailand.

Beef cattle production represents the largest cattle population in Thailand. Their productivity is constrained by tick-borne diseases such as babesiosis and theileriosis. In this study, we determined the prevalence of Babesia bigemina, Babesia bovis and Theileria orientalis using polymerase chain reaction (PCR). The genetic markers that were used for detection of the above parasites were sequenced to determine identities and similarity for Babesia spp. and genetic diversity of T. orientalis. Furthermore the risk factors for the occurrence of the above protozoan parasites in beef cattle from northern and northeastern parts of Thailand were assessed. A total of 329 blood samples were collected from beef cattle in 6 provinces. The study revealed that T. orientalis was the most prevalent (30.1%) parasite in beef cattle followed by B. bigemina (13.1%) and B. bovis (5.5%). Overall, 78.7% of the cattle screened were infected with at least one of the above parasites. Co-infection with Babesia spp. and T. orientalis was 30.1%. B. bigemina and T. orientalis were the most prevalent (15.1%) co-infection although triple infection with the three parasites was observed in 3.0% of the samples. Sequencing analysis revealed that B. bigemina RAP1 gene and B. bovis SBP2 gene were conserved among the parasites from different cattle samples. Phylogenetic analysis showed that the T. orientalis MPSP gene from parasites isolated from cattle in north and northeast Thailand was classified into types 5 and 7 as reported previously. Lack of tick control program was the universal risk factor of the occurrence of Babesia spp. and T. orientalis infection in beef cattle in northern and northeastern Thailand. We therefore recommend training of farmers on appropriate tick control strategies and further research on potential vectors for T. orientalis and elucidate the effect of co-infection with Babesia spp. on the pathogenicity of T. orientalis infection on beef in northern and northeastern Thailand.

Genetic mutations in sodium channel domain II and carboxylesterase genes associated with phenotypic resistance against synthetic pyrethroids by Rhipicephalus (Boophilus) decoloratus ticks in Uganda

We previously reported emergence of super synthetic pyrethroid (SP) resistant Rhipicephalus (Boophilus) decoloratus ticks in Uganda. This study investigated the genetic basis of phenotypic resistance against SP in R. (B.) decoloratus and sought to identify novel diagnostic mutations for rapid detection of SP resistance in the above tick species. Genomic DNA was extracted from pooled larvae of 20 tick populations (19 of known SP susceptibility and 1 unknown susceptibility). The voltage sensitive sodium channel (VSSC) domain II S4-5 linker (SP target) and partial carboxylesterase (SP metabolizing enzyme) genes were amplified by PCR, cloned and sequenced. The resultant sequences were analyzed to determine single nucleotide polymorphisms (SNPs) associated with phenotypic resistance in the various tick populations investigated. Novel SNPs that introduced Eco RI and Eco RII restriction sites in carboxylesterase gene were identified in silco and validated with restriction fragment length polymorphism (RFLP) against 18 tick populations of known SP susceptibility. The study identified a super knock down resistance (kdr) mutation T58C in R. (B.) decoloratus VSSC associated with stable SP resistance. We further identified multiple nonsynonymous mutations in carboxylesterase of SP resistant ticks; one of which conferred novel EcoRII (G195C) restriction site for PCR-RFLP detection of SP resistance. In conclusion, this study is the first to report super kdr mutation in sodium channel domain II and multiple mutations in carboxylesterase genes that may concurrently mediate stable resistance against synthetic pyrethroids in R. (B.) decoloratus ticks from Uganda. The Eco RII based PCR-RFLP is a useful tool for rapid detection of stable SP resistant R. (B.) decoloratus ticks.

Babesia bovis Dihydroorotate Dehydrogenase(BboDHODH) is a Novel Molecular Target of Drug for Bovine Babesiosis

ABSTRACT The emergence of drug resistance and adverse side effects of current bovine babesiosis treatment suggest that the search of new drug targets and development of safer and effective compounds are required. This study focuses on dihydroorotate dehydrogenase (DHODH), the fourth enzyme of pyrimidine biosynthesis pathway as a potential drug target for bovine babesiosis. Recombinant Babesia bovis DHODH protein (rBboDHODH) was produced in Escherichia coli and used for characterization and measurement of enzymatic activity. Furthermore, the effects of DHODH inhibitors were evaluated in vitro. The recombinant B. bovis DHODH histidine fusion protein (rBboDHODH) had 42.4-kDa molecular weight and exhibited a specific activity of 475.7 ± 245 Unit/mg, a Km = 276.2 µM for L-dihydroorotate and a Km= 94.41 µM for decylubiquinone. A 44-kDa band of native BboDHODH was detected by Western blot analysis and found in parasites mitochondria using a confocal microscope. Among DHODH inhibitors, atovaquone (ATV) and leflunomide (LFN) significantly inhibited the activity of rBboDHODH as well as the growth of B. bovis in vitro. The half maximal inhibitory concentration (IC50) of ATV and LFN was 2.38 ± 0.53 nM and 52.41 ± 11.47 µM, respectively. These results suggest that BboDHODH might be a novel target for development of new drug for treatment of B. bovis infection.

Transient transfection of intraerythrocytic Babesia gibsoni using elongation factor-1 alpha promoter

The development of gene manipulation techniques has been reported in many protozoan parasites over the past few years. However, these techniques have not yet been established for Babesia gibsoni. Here, we report for the first time, the successful transient transfection of B. gibsoni. The plasmid containing the firefly luciferase reporter gene (pBS-ELA) was transfected into B. gibsoni by an AMAXA 4D Nucleofector™ device. Transfection using program FA113 and Lonza buffer SF showed the highest luciferase expression. Twenty micrograms of plasmid produced the highest relative transfection efficiency. The fluorescent protein-expressing parasites were determined by GFP-containing plasmid (pBS-EGA) at 48 and 72h post transfection. This finding is the first step towards a stable transfection method for B. gibsoni, which may contribute to a better understanding of the biology of the parasite.

Retrospective study on cattle and poultry diseases in Uganda

Abstract Cattle and poultry enterprises are among the major contributors to food security and socioeconomic empowerment of households in Uganda. However, various diseases constrain their productivity. A two-year retrospective study between April 2012 and March 2014 was conducted using records for cattle and poultry diseases diagnosed at the Central Diagnostic Laboratory (CDL) to determine prevalent diseases in Uganda. The laboratory received 836 samples from poultry (36.3%) and cattle (63.7%). Of the 836 samples, 47.5% had a definitive diagnosis of disease causation. Most of the cattle and poultry diseases diagnosed were protozoan diseases (39.3%) followed by bacterial (21.4%), viral (17.1%), helminthiasis (11.1%), nutritional diseases (4%) and others (7.1%). For poultry, viral diseases (29.5%) and protozoan diseases (27.1%) especially newcastle disease (44.3%) and coccidiosis (100%) respectively, were the most diagnosed. While for cattle, hemo-protozoan parasites (52.1%) were the most prevalent, of which 92.9% were east coast fever infection. Bacterial infection (20.5%) in cattle were the second most diagnosed diseases and mastitis was the most diagnosed (46.2%). In summary, coccidioisis, collibacillosis, newcastle disease, gumboro disease, and avian helminthiasis were the most prevalent poultry diseases while in cattle, east coast fever, helminthiasis, mastitis, brucellosis and rabies were the most frequently diagnosed diseases. This study has identified the major diseases that hinder poultry and cattle production in Uganda. The data generated by CDL could be used for surveillance, monitoring and designing strategic interventions for control of poultry and cattle diseases in Uganda.

Milk Hygiene in Rural Southwestern Uganda: Prevalence of Mastitis and Antimicrobial Resistance Profiles of Bacterial Contaminants of Milk and Milk Products

Mastitis and antimicrobial resistance are a big challenge to the dairy industry in sub-Saharan Africa. A study was conducted in Kashongi and Keshunga subcounties of Kiruhura District (in Uganda) where the government and private sector have deliberate programs to improve production efficiency, quality, and safety of milk and its products. The study aimed to determine the prevalence of mastitis, its common causative agents, antimicrobial sensitivity of mastitis causing organisms, and contaminants of processed milk products: yoghurt and ghee. Seventy-one milk, fourteen yoghurt, and three ghee samples were collected from nine farms. Of the 71 cows tested, 54 (76.1%) had mastitis. The mastitis cases from Keshunga were 32 (59.3%) and Kashongi contributed 22 (40.7%) of the cases. The common mastitis causative agents were Staphylococcus spp. (30.8%), Streptococcus spp. (12.3%), Corynebacterium spp.(15.4%), and E. coli (7.7%). Some of the isolates were resistant to tetracycline and penicillin. Prevalent contaminants of yoghurt were Staphylococcus spp. (8.3%), Streptococcus spp. (8.3%), Corynebacterium spp. (8.3%), and E. coli (8.3%), whereas all ghee contained Streptococcus spp. (100%). Prevalence of mastitis, antimicrobial resistance, and contamination of milk products are high in the study area. Targeted programs to prevent and control mastitis as well as antibiotic resistance are recommended.

Genetic variations of four immunodominant antigens of Babesia gibsoni isolated from dogs in southwest Japan

Babesia gibsoni is a tick-borne apicomplexan parasite of dogs that often causes fever and hemolytic anemia. Previous reports have shown that the apical membrane antigen 1 (BgAMA1), the 50 kDa surface antigen (BgP50), the secreted antigen 1 (BgSA1), and the thrombospondin-related adhesive protein (BgTRAP) are promising diagnostic antigens and vaccine candidates against B. gibsoni. In the present study, we investigated the genetic polymorphisms and natural selection of these four genes of B. gibsoni isolated from dogs in southwest Japan. The prediction B-cell epitopes showed high antigenic score in the insert and indel regions of BgSA1 and BgTRAP. Sequence analyses have revealed that BgAMA1 had the highest nucleotide diversity, followed by BgP50, BgSA1 and BgTRAP. Meanwhile, the Tajimas D value test suggested balancing selection for BgAMA1 and BgP50. However, BgSA1 and BgTRAP have purifying selection making them potential vaccine candidate and diagnostic antigens since they are highly conserved. Our findings provide the genetic basis for designing and testing the efficacy of diagnostic antigens as well as vaccine candidates against B. gibsoni.

Molecular and Kinetic Characterization of Babesia microti Gray Strain Lactate Dehydrogenase as a Potential Drug Target

Babesia microti is an emerging zoonotic protozoan organism that causes “malaria-like” symptoms that can be fatal in immunocompromised people. Owing to lack of specific therapeutic regiment against the disease, we cloned and characterized B. microti lactate dehydrogenase (BmLDH) as a potential molecular drug receptor. The in vitro kinetic properties of BmLDH enzyme was evaluated using nicotinamide adenine dinucleotide (NAD+) as a co-factor and lactate as a substrate. Inhibitory assay was also done using gossypol as BmLDH inhibitor to determine the inhibitory concentration 50 (IC50). The result showed that the 0.99 kbp BmLDH gene codes for a barely soluble 36 kDa protein (332 amino acids) localized in both the cytoplasm and nucleus of the parasite. In vitro enzyme kinetic studies further revealed that BmLDH is an active enzyme with a high catalytic efficiency at optimal pH of 10.2. The Km values of NAD+ and lactate were 8.7 ± 0.57 mM and 99.9 ± 22.33 mM, respectively. The IC50 value for gossypol was 0.345 &mUM, while at 2.5 &mUM, gossypol caused 100% inhibition of BmLDH catalytic activity. These findings, therefore, provide initial evidence that BmLDH could be a potential drug target, although further in vivo studies are needed to validate the practical application of lactate dehydrogenase inhibitors against B. microti infection.