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Zhancheng Tian,
Guangyuan Liu,
Hong Yin,
Jianxun Luo,
Guiquan Guan,
Junren Xie, Jin Luo,
Jinfeng Zheng,
Meiyuan Tian,
Xiaosong Yuan,
Fangfang Wang,
Ronggui Chen,
Haijun Wang
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ABSTRACT: In this study a 552-bp region of the Cytochrome c oxidase subunit III (COX3) was amplified by polymerase chain reaction (PCR) and sequenced from individual Babesia species. Sequence variation between Babesia species from China ranged between 0-32.4%. We analyzed the phylogenetic performance of the partial sequence of the COX3 gene to resolve Babesia relationships as compared to the nuclear 18S rRNA and the mitochondrial cytochrome b (COB) gene, These data indicate that the COX3 gene seems to be superior to the COB gene and the 18S rRNA in recognizing close lineages among some Babesia species. Our work indicates that the COX3 gene does complement and corroborate the phylogenetic inferences observed with the nuclear 18S rRNA and the COB gene previously reported. The combined phylogenetic analysis based on the nuclear 18S rRNA and the COX3 gene significantly improved (bootstrap) intraspecies support of the phylogenetic relationship. The presence of additional variable sites in the COX3 gene allowed an improved interspecies differentiation of Babesia species in this study. The data could be applicable for the survey of parasite dynamics, epidemiological studies as well as prevention and control of the disease.
Infection, genetics and evolution: journal of molecular epidemiology and evolutionary genetics in infectious diseases 04/2013; · 3.22 Impact Factor
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Zhancheng Tian, Jin Luo,
Jinfeng Zheng,
Junren Xie,
Hui Shen,
Hong Yin,
Jianxun Luo,
Meiyuan Tian,
Xiaosong Yuan,
Fangfang Wang,
Guangyuan Liu
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ABSTRACT: In this study, a mitochondrial marker consisting of an approximately 550-bp region of the Cytochrome b genes (COB) was amplified by polymerase chain reaction (PCR) and sequenced from individual Babesia species. Sequence variation between Babesia species from China was 1.6-30.8%. The constructed phylogenetic tree based on the three unlinked gene sequences (partial COB gene, 18S rDNA and ITS) that evolve at different rates by the method of Neighbor-joining revealed the phylogenetic relationship of Babesia species in China compared with other published corresponding sequences from Babesia species. These data indicate that the 18S rDNA more reliably distinguish the deeper branches among some Babesia species than the partial COB gene and ITS, however, the partial COB gene sequence is better for recognizing close lineages among some Babesia species than the 18S rDNA and ITS sequences. So the combined phylogenetic analysis based on the multiple unlinked loci with different evolving rates can facilitate to establish the more reliable phylogenetic relationship of the Babesia genus. The data could be applicable for the survey of parasite dynamics, epidemiological studies as well as prevention and control of the disease.
Infection, genetics and evolution: journal of molecular epidemiology and evolutionary genetics in infectious diseases 10/2012; · 3.22 Impact Factor
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ABSTRACT: Seven Trypanosoma evansi isolates from China and a Trypanosoma congolense sp. gifted from Kenya were characterized genetically by the internal transcribed spacer 1 (ITS-1) of nuclear ribosomal DNA (rDNA). The ITS-1 rDNA with the length of 338-342 bp was amplified by polymerase chain reaction (PCR) and sequenced from individual isolates of T. evansi. Although sequence variation between T. evansi isolates from China only was 0.3-3.8%, the constructed phylogenetic tree based on the ITS-1 rDNA sequence by the method of neighbor-joining and maximum parsimony revealed the genetic diversity among T. evansi isolates from China. For T. congolense sp., the most phylogenetically related species was T. congolense IL1180. Although the sequence variation ranged 0.8-14.5% between T. congolense isolates, the phylogenetic tree can not reflected the genetic diversity among T. congolense isolates perhaps because of the fewer number of isolates and sequences. The data could be applicable for the survey of parasite dynamics, epidemiological studies as well as prevention and control of the disease.
Experimental Parasitology 08/2011; 129(3):303-6. · 2.12 Impact Factor
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ABSTRACT: The full-length cDNA encoding acid phosphatase (HL-3) from Haemaphysalis longicornis was obtained by 5' rapid amplification of cDNA ends (RACE). The cDNA contained a 1137 bp open reading frame (ORF) coding for 356 amino acids with a predicted theoretical isoelectric point (pI) of 6.35 and molecular weight of 41.0 kDa. The recombinant protein was expressed in Escherichia coli. The enzyme could hydrolyze para-nitrophenyl phosphate (pNPP) substrate at an optimum pH of 5.0. Real-time RT-PCR analysis showed that the HL-3 transcripts were expressed in various stages of unfed ticks and were significantly induced by blood feeding. Furthermore, the expression of HL-3 in midguts was significantly higher than in other tested tissues of partially fed adult ticks. The transcripts of the HL-3 mRNA in lipopolysaccharide (LPS)-injected ticks were 1.75 times of the PBS-injected control; Theileria sergenti infected larvae expressed 3.86 more times than that of uninfected ones. Western blot analysis showed that rabbit antiserum against the recombinant rHL-3 could recognize a native protein of approximately 41.0 kDa in the lysates from different stages of ticks. Vaccination of rabbits with the rHL-3 conferred partial protective immunity against ticks, resulting in 28% mortality and 10.6% reduction in engorgement weight of adult ticks, respectively. These results suggested that the HL-3 was involved in tick innate immunity and could be used as a potential candidate antigen for the development of anti-tick vaccines.
Veterinary Parasitology 06/2011; 182(2-4):287-96. · 2.58 Impact Factor
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ABSTRACT: A Haemaphysalis longicornis heat shock protein 70 (HLHsp70) was identified from a cDNA library synthesized from tick eggs. The HLHsp70 cDNA is 2311 bp in length and encodes 661 amino acid residues with the predicted molecular weight of 72.5 kDa and an isoelectronic point (pI) of 5.2. It also contains the highly conserved functional motifs of the Hsp70 family and a specific endoplasmic reticulum (ER) retention signal "KDEL" that is common among ER-localized proteins. The HLHsp70 exhibits 90% amino acid identity to the putative Hsp70 of Ixodes scapularis, and 85% to Gallus gallus 78 kDa glucose-regulated protein precursor. Real time RT-PCR analysis showed that the expression levels of the Hsp70 in ovaries and salivary glands were significantly higher than in other tested tissues in partially fed females. Although the expression level of the HLHsp70 was constantly low in unfed ticks, it was significantly induced by blood-feeding. Further, the expression was positively correlated to the temperature (4-37°C, tested). Western blot analysis showed that the rabbit antiserum against the recombinant HLHsp70 protein (rHLHSP70) recognized bands of approximately 100, 72, and 28 kDa from egg lysates, as well as a 72kDa fragment in protein extracts from partially fed larvae. Immunization of rabbits with the rHLHSP70 did not result in a statistically significant reduction of female tick engorgement and oviposition. These results suggest that although HLHSP70 plays a role in the physiological activities of ticks, as a constitutive protein it was not suitable for selection as a candidate vaccine antigen against ticks.
Veterinary Parasitology 04/2011; 181(2-4):282-90. · 2.58 Impact Factor
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ABSTRACT: In the present study, two hard tick species, Haemaphysalis longicornis and H. qinghaiensis from North-western China were characterized genetically by the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA and partial 16S rDNA. Based on a fragment within the hypervariable region of 16S rDNA with the length of approximately 453 bp, the phylogenetic trees were constructed by Neighbor-Joining and Maximum-parsimony methods. The results indicated that the phylogenetic status of H. qinghaiensis was distant from that of H. longicornis and closer to H. flava. Furthermore, the ITS-2 rDNA was amplified by PCR and sequenced from individual ticks. The length of ITS-2 is 1,606 bp for H. longicornis and 1,162 bp for H. qinghaiensis. Although sequence variation between the immature stages of H. longicornis was 0.1-0.4%, nucleotide differences between the tested species ranged 2.1-23.2%, indicating that ITS-2 rDNA sequences are genetic markers for the differentiation of the two hard ticks in China. Hence, a PCR-linked restriction fragment length polymorphism (RFLP) approach was developed for their unequivocal differentiation based on ITS-2 rDNA, which provides the foundation for further studies on ticks in China and has implications for studying the population genetic structure of the ticks and for identification and differentiation of closely related ticks.
Experimental and Applied Acarology 01/2011; 54(2):165-72. · 1.39 Impact Factor
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ABSTRACT: A fragment of ribosomal protein L24 was obtained from the complementary deoxyribonucleic acid (cDNA) library of Haemaphysalis longicornis eggs. The complete sequence of the clone was subsequently obtained using rapid amplification of the cDNA ends (RACE). Ribosomal protein L24 from H. longicornis had a high percentage similarity to this protein from different species. Conserved domains were also identified in RpL24. Real-time polymerase chain reaction (PCR) analysis showed that this gene is expressed in various tissues and different developmental stages of H. longicornis. Furthermore, HLL24 is mostly expressed in ovaries and salivary glands compared with other tissues in partially fed adult female ticks, and the expression level of HLL24 is significantly lower in eggs and larvas than in other developmental stages. RpL24 was also cloned from Haemaphysalis qinghaiensis and Hyalomma anatolicum anatolicum ticks, respectively. Comparison of their amino acid sequences revealed difference only in several amino acids. A vaccine based on the HLL24 recombinant protein could not protect rabbits against H. longicornis.
Parasitology Research 10/2010; 107(5):1213-20. · 2.15 Impact Factor
