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.
To develop a Tm-shift method for detection of dog-derived Ancylostoma ceylanicum and A. caninum, three sets of primers were designed based on three SNPs (ITS71, ITS197, and ITS296) of their internal transcribed spacer 1 (ITS1) sequences. The detection effect of the Tm-shift was assessed through the stability, sensitivity, accuracy test, and clinical detection. The results showed that these three sets of primers could distinguish accurately between A. ceylanicum and A. caninum. The coefficient of variation in their Tm values on the three SNPs was 0.09% and 0.15% (ITS71), 0.18% and 0.14% (ITS197), and 0.13% and 0.07% (ITS296), respectively. The lowest detectable concentration of standard plasmids for A. ceylanicum and A. caninum was 5.33 × 10−6 ng/μL and 5.03 × 10−6 ng/μL. The Tm-shift results of ten DNA samples from the dog-derived hookworms were consistent with their known species. In the clinical detection of 50 fecal samples from stray dogs, the positive rate of hookworm detected by Tm-shift (42%) was significantly higher than that by microscopic examination (34%), and the former can identify the Ancylostoma species. It is concluded that the Tm-shift method is rapid, specific, sensitive, and suitable for the clinical detection and zoonotic risk assessment of the dog-derived hookworm.
Echinostoma revolutum is a zoonotic food-borne intestinal trematode that can cause intestinal bleeding, enteritis, and diarrhea in human and birds. To identify a suspected E. revolutum trematode from a red-crowned crane (Grus japonensis) and to reveal the genetic characteristics of its mitochondrial (mt) genome, the internal transcribed spacer (ITS) and complete mt genome sequence of this trematode were amplified. The results identified the trematode as E. revolutum. Its entire mt genome sequence was 15,714 bp in length, including 12 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes and one non-coding region (NCR), with 61.73% A+T base content and a significant AT preference. The length of the 22 tRNA genes ranged from 59 bp to 70 bp, and their secondary structure showed the typical cloverleaf and D-loop structure. The length of the large subunit of rRNA (rrnL) and the small subunit of rRNA (rrnS) gene was 1,011 bp and 742 bp, respectively. Phylogenetic trees showed that E. revolutum and E. miyagawai clustered together, belonging to Echinostomatidae with Hypoderaeum conoideum. This study may enrich the mitochondrial gene database of Echinostoma trematodes and provide valuable data for studying the molecular identification and phylogeny of some digenean trematodes.
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