Comparative analysis of Ancylostoma ceylanicum mitochondrial genome with other Ancylostoma species.

Ancylostoma ceylanicum may inhabit the small intestine of canids, felids and humans, can pose a potential risk to public health. This study is the first time to amplify complete mitochondrial genome sequence of A. ceylanicum from dog and to compare it with Ancylostoma tubaeforme, Ancylostoma duodenale and Ancylostoma caninum. The results showed that the complete mitochondrial genome of A. ceylanicum was 13,660 bp in length, including 12 protein-coding genes, 2 rRNA genes and 22 tRNA genes and 3 non-coding regions (AT-rich region, SNCR and LNCR). Its mtDNA was the shortest, biased toward A and T at base composition, and higher than other three Ancylostoma species at total AT content. Its nad5 and nad6 genes used TTG and ATT as initiation codons, while other three Ancylostoma species used ATT and GTG or ATG. The 22 tRNA genes were different in length among four Ancylostoma species, but their anticodons were the same. Among 12 protein-coding genes, the cox1 gene was the lowest at AT content and minimum at Ka/Ks while the nad2 gene was the opposite. The phylogenetic tree showed that in the lineage of Ancylostoma, A. ceylanicum occurred on a branch external to other three Ancylostoma species, and A. caninum and A. tubaeforme had closer phylogenetic relationship than A. duodenale. This study not only enhances the mitochondrial genome database of Ancylostomatidae nematodes, but also provides new data for further phylogenetic studies among Ancylostomatidae nematodes.

Prevalence and genotypes of Giardia lamblia from stray dogs and cats in Guangdong, China.

Giardia lamblia is a worldwide zoonotic intestinal parasite that infects humans and a wide range of mammals including dogs and cats, causing giardiasis with diarrhea. To investigate the infection and distribution of G. lamblia genotypes from stray dogs and cats in Guangdong, China according to different districts, gender and ages, fecal samples were collected and examined by microscopy, and all isolates were genotyped by PCR amplification using beta-giardin (bg) and triose phosphate isomerase (tpi) genes as molecular markers. The results showed that the prevalence of dogs and cats was 10.8% (57/527) and 5.8% (6/104), respectively. Sixty-one samples were detected by microscopy and 63 were amplified and successfully sequenced by the PCR. Based on the phylogenetic analysis, 25 canine isolates (24 assemblages AI and 1 assemblage D) were genotyped by tpi gene and 57 canine isolates (26 assemblages AI, 18 assemblages C and 13 assemblages D) genotyped by bg gene; 6 feline isolates were identified as assemblage AI by tpi gene, and as 3 assemblages AI and 3 assemblages F by bg gene. The dominant genotypes were assemblage AI in younger dogs (assemblage C in adult dogs) and assemblage C in male dogs (assemblage AI in female dogs). Mixed genotype infections were found in different age and gender groups. The results indicated that G. lamblia from stray dogs and cats in Guangdong province had a zoonotic potential with assemblage AI as the prevalent genotype. The different risk factors (age and sex) may have an impact on the infection of different genotypes.

Prokaryotic Expression of α-13 Giardin Gene and Its Intracellular Localization in Giardia lamblia.

To study prokaryotic expression and subcellular localization of α-13 giardin in Giardia lamblia trophozoites, α-13 giardin gene was amplified and cloned into prokaryotic expression vector pET-28a(+). The positive recombinant plasmid was transformed into E. coli BL21(DE3) for expression by using IPTG and autoinduction expression system (ZYM-5052). The target protein was validated by SDS-PAGE and Western blotting and purified by Ni-NTA Resin. Rabbits were immunized with purified fusion proteins for preparation of polyclonal antibody; then the intracellular location of α-13 giardin was determined by fluorescence immunoassay. The results showed that the length of α-13 giardin gene was 1038 bp, encoding a polypeptide of 345 amino acids. The expressed product was a fusion protein with about 40 kDa largely present in soluble form. The target protein accounted for 21.0% of total proteins after being induced with IPTG, while it accounted for 28.8% with ZYM-5052. The anti-α13-giardin polyclonal antibody possessed good antigenic specificity as well as excellent binding activity with recombinant α-13 giardin. Immunofluorescence assays revealed that α-13 giardin was localized in the cytoplasm of G. lamblia trophozoite, suggesting that it is a cytoplasm-associated protein. The present study may lay a foundation for further functional research on α-13 giardin of G. lamblia.

Development of T m -shift genotyping method for detection of cat-derived Giardia lamblia.

To develop T m -shift genotyping method for detection of cat-derived Giardia lamblia, two sets of primers with two GC-rich tails of unequal length attached to their 5'-end were designed according to two SNPs (BG434 and BG170) of ß-giardin (bg) gene, and specific PCR products were identified by inspection of a melting curve on real-time PCR thermocycler. A series of experiments on the stability, sensitivity, and accuracy of T m -shift method was tested, and clinical samples were also detected. The results showed that two sets of primers based on SNP could distinguish accurately between assemblages A and F. Coefficient of variation of T m values of assemblage A and F was 0.14 and 0.07% in BG434 and 0.10 and 0.11% in BG170, respectively. The lowest detection concentration was 4.52 × 10-5 and 4.88 × 10-5 ng/µL samples of assemblage A and F standard plasmids. The T m -shift genotyping results of ten DNA samples from the cat-derived G. lamblia were consistent with their known genotypes. The detection rate of clinical samples by T m -shift was higher than that by microscopy, and their genotyping results were in complete accordance with sequencing results. It is concluded that the T m -shift genotyping method is rapid, specific, and sensitive and may provide a new technological mean for molecular detection and epidemiological investigation of the cat-derived G. lamblia.

Immunolocalization of α18- and α12-giardin in Giardia lamblia trophozoites.

To study subcellular localization of α18- and α12-giardin in Giardia lamblia trophozoites, the α18- and α12-giardin genes were amplified from G. lamblia assemblage A, respectively. The PCR products were cloned into the prokaryotic expression vector pET-28a(+), and the positive recombinant plasmids were transformed into E. coli Rosetta (DE3) strain for the expression, and expressed α18- and α12-giardin fusion protein were purified by Ni-Agarose resin, respectively. Mice were immunized with purified fusion proteins for preparation of polyclonal antibody, and then the subcellular localization of α18- and α12-giardin was determined by fluorescence immunoassay. Results showed that the concentrations of purified α18- and α12-giardin fusion proteins were 1.20 and 0.86 mg/ml, respectively. The titers of anti-α18- and anti-α12-giardin polyclonal antibody were both as high as 1:25600 dilutions. Immunofluorescent analysis showed that α18- and α12-giardin proteins were mainly localized at four pairs of flagella and the cytoplasm of G. lamblia trophozoites, suggesting that α18- and α12-giardin are the flagella and cytoplasm-associated proteins, respectively. The above information would lay the foundation for research about the crystal structure and biological function of α18- and α12-giardin.

Sequence Analysis of Mitochondrial Genome of Toxascaris leonina from a South China Tiger.

Toxascaris leonina is a common parasitic nematode of wild mammals and has significant impacts on the protection of rare wild animals. To analyze population genetic characteristics of T. leonina from South China tiger, its mitochondrial (mt) genome was sequenced. Its complete circular mt genome was 14,277 bp in length, including 12 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 2 non-coding regions. The nucleotide composition was biased toward A and T. The most common start codon and stop codon were TTG and TAG, and 4 genes ended with an incomplete stop codon. There were 13 intergenic regions ranging 1 to 10 bp in size. Phylogenetically, T. leonina from a South China tiger was close to canine T. leonina. This study reports for the first time a complete mt genome sequence of T. leonina from the South China tiger, and provides a scientific basis for studying the genetic diversity of nematodes between different hosts.

Sequence analysis and prokaryotic expression of Giardia lamblia α-18 giardin gene.

To study the genetic variation and prokaryotic expression of α18 giardin gene of Giardia lamblia zoonotic assemblage A and host-specific assemblage F, the α18 genes were amplified from G. lamblia assemblages A and F by PCR and sequenced. The PCR product was cloned into the prokaryotic expression vector pET-28a(+) and the positive recombinant plasmid was transformed into Escherichia coli Rosetta (DE3) strain for the expression. The expressed α18 giardin fusion protein was validated by SDS-PAGE and Western blot analysis, and purified by Ni-Agarose resin. The putative sequence of α18 giardin amino acid was analyzed by bioinformatics software. Results showed that the α18 giardin gene was 861 bp in length, encoding 286 amino acids; it was 100% homologous between human-derived and dog-derived G. lamblia assemblage A, but it was 86.8% homologous with G. lamblia assemblage F (cat-derived). Giardin α18 was about 36 kDa in molecular weight, with good reactivity. Prediction based on in silico analyses: it had hydrophobicity, without signal peptide and transmembrane domain, and contained 11 alpha regions, 13 beta sheets, 1 beta turn and 7 random coils in secondary structure. The above information would lay the foundation for research about the subcellular localization and biological function of α18 giardin in G. lamblia.