Novel Diagnostic Methods Development And Genome Sequencing Of Babesia Orientalis

Babesia orientalis as a tick-borne hemoprotozoan parasite, is the causative agent of babesiosis in water buffalo, it transmitted by Rhipicephalus haemaphysaloides transovarially with the infected stage being the adult tick. This disease is viewed as one of the most important diseases of buffalo in central and southern parts of China, and recently was also reported to the north side of Yangtse river, and causes enormous economis losses. The clinic manifestations are fever, anemia, icterus, hemoglobinuria and even mortality. In this study, three detection methods, reverse line blot (RLB), loop-mediated isothermal amplification (LAMP) and a TaqMan real-time PCR assay, were developed based on the v4 variable region of 18S rRNA for the detection of B. orientalis. These methods were used to investigating Theileria and Babesia prevalence in small ruminants both in China and South Africa. Three positive clones were obtained by immunoscreening the cDNA library of B. orientalis. Prokaryotic expression and western blot indicated that those three positive clones, B04-nuclear dynamic protein, B05-hypothetical protein and B41-heat shock protein 70 (HSP70) can be recognized by the serum from B. orientalis infected water buffalo and the native B. orientalis HSP70 in the merozoite stage was also identified. The full length of B. orientalis mitochondrion DNA was clone and the polymorphism was analysed, and the genome sequencing of B. orientalis was performed.(1) Development of reverse line bot (RLB) for B. orientalisSpecific primers were designed based on the v4 variable region of the 18S rRNA gene of B. orientalis, and the RLB detection method was developed subsequently.304 water buffalo blood samples,24 sika deer blood samples,82 sheep and 244 goats blood samples were collected from nine distinct geographical locations of Hubei province of south China, Hubei province Deer Center and Pretoria, South Africa, respectively. The hypervariable v4 region of the 18S rRNA gene was amplified using Theileria and Babesia genus-specific primers. The amplified PCR products were screened using the RLB hybridization assay.For the results of water buffalo in China, four species were found, including Theileria buffeli (19.1%), the most frequently found species in all of the locations, followed by Babesia orientalis (8.9%), B. bovis (1.0%) and B. bigemina (0.7%). Only 12 (3.9%) of the samples had mixed infections. The 18S rRNA of 11 positive samples were cloned, including 8 T. buffeli,2 B. bigemina and 1 B. orientalis. Phylogenetic analysis showed that the eight T. buffeli 18S rRNA clones grouped into four clusters, of which three grouped with the known T. buffeli type B and D. The remaining five grouped separately from the previously describe T. buffeli types. The two B. bigemina 18S rRNA gene clones grouped with B. bigemina Kunming. This is the first report of B. bigemina in the Hubei province. The B. orientalis Daye 18S rRNA gene clone grouped closely with the previously reported B. orientalis Wuhan strain and with Babesia sp. Kashi1 and Kashi2. For sika deer detection,17 samples were negative, the rest 7 samples'PCR products hybridized with Babesia/Theileria genus-specific probes, but failed to hybridize with any of the Babesia or Theileria species-specific probes. This suggested the presence of a novel species or variant species.18S rRNA and internal transcribed spacer (ITS) genes amplified from seven isolates were cloned and sequenced. Alignment and BlastN of the cloned sequences revealed high identities to the homologous 18S rRNA genes and ITS genes of Theileria cervi (AY735122), T. sp. CNY1A (AB012194) and T. sp. ex Yamaguchi (AF529272). Phylogenetic analysis based on the 18S rRNA gene and ITS sequences showed that all cloned sequences were grouped within the Theileria clade. Phylogeny based on the 18S rRNA gene divided the organisms into two groups. Group 1 was closest to Theileria sp. ex Yamaguchi (AF529272), while group 2 was distinct from all other identified Theileria and Babesia species. These results suggest the existence of Theileria infection in sika deer in China. To our knowledge, this is the first report of cervine Theileria in China.For the sheep and goats samples,23/82 sheep and 2/244 goats were tested positive for one or more Theileria hemoparasites, no Babesia spp. was found. And 20 of positive samples were mixed infections. Six mix-infections sheep samples were subjected to cloning and sequencing. Alignment of T. sp sable and T. bicornis probe sequences and the cloned 18S rRNA sequences suggested that there may be cross reactions of those two probes with T. separata, which means new probes of T. sp sable and T. bicornis should be designed. Phylogenetic analysis indicated that the cloned sequences falls into two groups. Group A was most closely to T. separata, whereas group B was in the clade of T. sp. Maleane sable (AY748462) and Theileria sp. VT12 which was reported in dog recently. These results showed that at least 2 groups of Theileria spp., which close to T. separata do exist in sheep and goats in Pretoria, South Africa.(2) Development of loop-mediated isothermal amplification (LAMP) for B. orientalisFour specific primers which recognized 6 position in the target gene, were designed based on the v4 hypervariable region of the 18S rRNA B. orientalis. Blood samples were collected from 165 water buffalo from eight different regions of Hubei Province, and from 66 african buffalo which were used for negative control. Genomic DNA was extracted, subjected to the LAMP assay and compared with results obtained using a previously described semi-nested PCR. The LAMP assay proofed to be B. orientalis specific and more sensitive than the semi-nested PCR. While previously B. orientalis had not been reported north of the Yangtse river, our results show that B. orientalis has spread to the north of the river. This could pose a serious threat to the water buffalo industry.(3) Development of TaqMan real-time PCR assay for detectng B. orientalisIn this study, a TaqMan real-time PCR assay was developed for quantitative detection of Babesia orientalis in water buffalo. Hybridization probe and oligonucleotide primers were designed based on the v4 region of 18S rRNA gene of B. orientalis. Blood samples were collected from B. orientalis experimentally infected water buffalo, as well as from 180 water buffalo from four different geographical locations of Hubei province, south China. The detection limit of the real-time PCR assay was determined to be 2 parasites/μl. The real-time PCR assay was compared with reverse line blot (RLB). B. orientalis was detected by real-time PCR on day 2 post-infection, while RLB was first detected on day 6 in experimentally infected water buffalo. For the results of 180 field samples, statistical analysis showed no significant difference in relative effectiveness of real-time PCR and RLB (x2=1.6312, P=0.2218) for identifying B. orientalis. The analysis also indicated that there was no difference on the prevalence of B. orientalis between the regions of south and north of Yangtse river by both the real-time PCR assay (x2=0.5238, P= 0.4595) and RLB detecting (x2=0.6310, P=0.5311). There were 16 samples'parasitemia was estimated to be from 2.0×103 to 2.0×102 parasites/μl This is the first report for developing a TaqMan real-time PCR assay for quantitative detection of B. orientalis in water buffalo blood samples.(4) Cloning and expression immuno-related genes of B. orientalisSixteen positive clones were obtained by immunoscreening the cDNA expression library with B. orientalis infected water buffalo sera. Three sets of specific primers were designed for cloning and expression the open reading frame (ORF) of three positive clones, B04-nuclear dynamic protein, B05-hypothetical protein, B41-heat shock protein 70 (HSP70). The amplicons were cloned into expression vectors, recombinant plasmids B04-pET-32a, B05-pET-28a and HSP70-pET-32a were constructed. Subsequently, the plasmids were introduced into E. coli RosettaTM strain and expressed as a Trx-fusion (B04-pET-32a and HSP70-pET-32a) and His-fusion (B05-pET-28a) protein. In western blot, the serum from B. orientalis infected water buffalo recognized each of those three Trx-fusion or His-fusion protein. The native B. orientalis HSP70 in the merozoite stage was also identified.(5) Genome sequencing of B. orientalisWhole-genome sequencing was performed by a hybrid sequencing method using Solexa technologies. A total of 23,898,916 reads were generated with an Illumina Solexa genome analyzer Iix and were mapped to 1284 scaffolds with the total length of 7,796,224 bp. Open reading frames containing more than 30 amino acids were predicted by Glimmer 3.0. The B. orientalis genome contains 4 chromosomes with 42.4% GC content which was similar to B. bovis 41.8% GC content.(6) Cloning the mitochondiral DNA (mtDNA) of B. orientalis and polymophysium analysisFor cloning mitochondrial genome of B. orientalis, specific primers were designed from the conserved region of mitochondrion sequences of hemoparasites including B. bovis, B. bigemina, B. caballi, T. parva etc.. Amplicons were cloned into pMD18-T, positive clones were subjected to sequencing. Assembling results indicated that B. orientalis mtDNA was similar with the mtDNAs of B. bovis. It contains three CDS which was called cytochrome C oxidaseⅠ(COXⅠ), cytochrome b (Cytb) and cytochrome C oxidaseⅢ(COXⅢ), the size of the CDS was 1428 bp,687 bp and 1092 bp, respectively. Phylogeny analysis based on COXⅠ, Cytb and the combined sequences of COXⅠand Cytb, indicated that B. orientalis fell into Babesia clade and most closed to B. bovis. These trees were similar to the trees based on 18S rRNA and heat shock protein (HSP70).