Molecular characterization and tissue localization of glutathione S-transferase from adult Ancylostoma ceylanicum.

Glutathione S-transferases (GSTs) are a detoxifying enzyme family that is essential for parasite blood-feeding and survival, and represent potential targets for hookworm vaccine development. Multiple GST-encoding complementary DNAs (cDNAs) have been cloned from Ancylostoma caninum and Necator americanus, but there are no reports about the cloning of this enzyme from Ancylostoma ceylanicum, the animal-derived zoonotic hookworm. To study the molecular nature and tissue localization of GST of A. ceylanicum (Ace-GST), we designed primers based on the GST gene sequence of A. ceylanicum in GenBank, amplified the Ace-GST cDNA by reverse transcription polymerase chain reaction, and analysed its homology and genetic evolution relationship. The amplified product was cloned into the pET-32a vector and transformed into Escherichia coli BL21 (DE3) for expression. To prepare anti-GST polyclonal antibodies, the recombinant protein was purified and used to immunize Kunming mice. The level of immunoglobulin G (IgG) antibody in the serum of immunized mice was detected by indirect enzyme-linked immunosorbent assay, and the Ace-GST localization in adult worm was determined using the immunofluorescence method. The results showed that the full-length cDNA encoding Ace-GST was 468 bp, which had the highest homology with Ac-GST-1 (60.1%) and clustered into one branch (v-class) with Ac-GST-1 and Na-GST-1 in a phylogenetic tree. Mice immunized with recombinant Ace-GST showed specific IgG antibody response. Immunolocalization revealed that natural Ace-GST is mainly located in the epidermis, muscle and intestine of the adult. These results may lay a foundation for further studies on the biological function of Ace-GST.

Molecular differentiation of three canine and feline hookworms in South China through HRM analysis.

To investigate the prevalence of canine and feline hookworms in South China, and to assess the risk of zoonotic hookworms to humans, one pair of primers (HRM-F/HRM-R) was designed to establish a high-resolution melting (HRM) method based on internal transcribed spacer 1 (ITS-1) rDNA for the detection of Ancylostoma ceylanicum, A. caninum and A. tubaeforme infection. The results showed that the HRM for the three hookworms produced different melting-curve profiles, where melting temperature (Tm) values were 84.50°C for A. ceylanicum, 82.25°C for A. caninum and 81.73°C for A. tubaeforme, respectively. The reproducibility of intra- and inter-assay melting curves was almost perfect. The lowest concentration detected was about 5.69 ×10-4 g/µl. The HRM detection results from 18 canine and feline hookworm samples were in complete accordance with their sequencing results. The HRM method was more sensitive than the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique in the detection of 98 clinical samples. It is concluded that the HRM method can differentiate between A. ceylanicum, A. caninum, A. tubaeforme and their mixed infections, which may provide important technical support for the zoonotic risk assessment and molecular epidemiological survey of canine and feline hookworms.

The mitochondrial genome of Ancylostoma tubaeforme from cats in China.

Ancylostoma tubaeforme may infect canids, felids and humans, and pose a potential risk to public health. Polymerase chain reaction (PCR) techniques were used to amplify the complete mitochondrial (mt) genome sequence of A. tubaeforme from cats and to analyse its sequence characteristics after molecular identification based on the internal transcribed spacer ITS1+ sequence. The results show that the complete mt genome sequence (GenBank accession number KY070315) of A. tubaeforme from cats was 13,730 bp in length, including 12 protein-coding genes, 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes, two non-coding regions and an AT-rich region. The nucleotide content of A and T was 77.93%, biased toward A and T. Twelve protein-coding genes used ATT, TTG and GTG as initiation codons, and TAA, TAG, TA and T as termination codons. The length of the 22 tRNA genes ranged from 52 to 62 bp, their predicted secondary structures were D loops and V loops. The lengths of the two rRNAs were 958 and 697 bp. Phylogenetic analyses showed that A. tubaeforme from cats was in the lineage of Ancylostoma, having a close phylogenetic relationship with A. caninum. This study reports for the first time the mt genome of A. tubaeforme from cats in China, which could enhance the mt genome database of Ancylostomatidae nematodes, and it offers the scientific basis for further studies in the genetic diversity of hookworms among different hosts.

The mitochondrial genome of Dipetalonema gracile from a squirrel monkey in China.

Dipetalonema gracile is a common parasite in squirrel monkeys (Saimiri sciureus), which can cause malnutrition and progressive wasting of the host, and lead to death in the case of massive infection. This study aimed to identify a suspected D. gracile worm from a dead squirrel monkey by means of molecular biology, and to amplify its complete mitochondrial genome by polymerase chain reaction (PCR) and sequence analysis. The results identified the worm as D. gracile, and the full length of its complete mitochondrial genome was 13,584 bp, which contained 22 tRNA genes, 12 protein-coding genes, two rRNA genes, one AT-rich region and one small non-coding region. The nucleotide composition included A (16.89%), G (20.19%), T (56.22%) and C (6.70%), among which A + T = 73.11%. The 12 protein-coding genes used TTG and ATT as start codons, and TAG and TAA as stop codons. Among the 22 tRNA genes, only trnS1AGN and trnS2UCN exhibited the TΨC-loop structure, while the other 20 tRNAs showed the TV-loop structure. The rrnL (986 bp) and rrnS (685 bp) genes were single-stranded and conserved in secondary structure. This study has enriched the mitochondrial gene database of Dipetalonema and laid a scientific basis for further study on classification, and genetic and evolutionary relationships of Dipetalonema nematodes.