Development of multi-ARMS-qPCR method for detection of hookworms from cats and dogs.

Hookworms are blood-sucking nematodes that infect dogs, cats, and humans, causing iron-deficiency anemia, abdominal pain, diarrhea, and skin inflammation. Amplification refractory mutation system (ARMS) is a modified technology based on allele-specific PCR, which is widely used in mutation detection and genotyping. However, no data about ARMS application in hookworm detection. This study aims to establish a multi-ARMS-qPCR method for the detection of three hookworm species from dogs and cats. A universal forward primer and three specific primers (ARMS-Cey, ARMS-Can, and ARMS-Tub) were designed based on the three ITS SNPs (ITS250, ITS78 and ITS153) of Ancylostoma ceylanicum, A. caninum, and A. tubaeforme, respectively. The results showed that the three designed ARMS primers generated specific melting curves for the three hookworms' standard plasmids. The melting temperature (Tm) values were 88.40 °C (A. ceylanicum), 83.15 °C (A. caninum), and 85.65 °C (A. tubaeforme), with good reproducibility of intra- and inter-assay. No amplification was observed with other intestinal parasites. The limit of detection using the established technique was 1, 2, and 104 egg per gram feces (EPG) for A. caninum, A. tubaeforme and A. ceylanicum, respectively. Using multi-ARMS-qPCR assay, 17 out of 50 fecal samples were positive for hookworms, including ten single and seven mixed infections, and single infections were quantified. In conclusion, the used multi-ARMS-qPCR method has the advantages of high efficiency, sensitivity, specificity, and quantitative analysis and can be used for the clinical detection, epidemiological investigation, and zoonotic risk assessment of canine and feline hookworms.

Prevalence and potential zoonotic risk of hookworms from stray dogs and cats in Guangdong, China.

Hookworm infection is globally prevalent among dogs and cats representing a major public health risk. Although previous studies have surveyed canine and feline hookworms in Guangzhou city, the status of these infection needs to be further explored in other regions of South China. To investigate the prevalence and zoonotic risk of canine and feline hookworms in eight cities (Guangzhou, Foshan, Shenzhen, Huizhou, Zhongshan, Shaoguan, Shantou and Chaozhou) of Guangdong province, China, we developed specific PCR methods based on ITS sequence for identifying three common hookworm species. The results showed that the prevalence of hookworms from stray dogs and cats was 20.23% (142/702) and 15.26% (47/308), respectively. The established PCR methods could identify Ancylostoma ceylanicum, A. caninum and A. tubaeforme. The mixed infections of A. caninum and A. ceylanicum were detected in stray dogs of Guangzhou and Shaoguan, with the rate of 8.3% and 21.2%, respectively. Among the stray dogs in Foshan, the infection rate of A. ceylanicum was higher than that of A. caninum. The stray cats in four of five investigated cities were infected with A. ceylanicum. The different region, age and rearing environments had an impact on the hookworm infection rates of stray dogs and cats. In conclusion, the reported higher infection rate of A. ceylanicum than other hookworm species in stray dogs and cats poses a potential risk to public health.

Establishment of a Tm-shift Method for Detection of Cat-Derived Hookworms.

Melting temperature shift (Tm-shift) is a new detection method that analyze the melting curve on real-time PCR thermocycler using SYBR Green I fluorescent dye. To establish a Tm-shift method for the detection of Ancylostoma ceylanicum and A. tubaeforme in cats, specific primers, with GC tail of unequal length attached to their 5 ́ end, were designed based on 2 SNP loci (ITS101 and ITS296) of the internal transcribed spacer 1 (ITS1) sequences. The standard curve of Tm-shift was established using the standard plasmids of A. ceylanicum (AceP) and A. tubaeforme (AtuP). The Tm-shift method stability, sensitivity, and accuracy were tested with reference to the standard curve, and clinical fecal samples were also examined. The results demonstrated that the 2 sets of primers based on the 2 SNPs could accurately distinguish between A. ceylanicum and A. tubaeforme. The coefficient of variation (CV) of Tm-values of AceP and AtuP was 0.07% and 0.06% in ITS101 and was 0.06% and 0.08% in ITS296, respectively. The minimum detectable DNA concentration was 5.22×10-6 and 5.28×10-6 ng/µl samples of AceP and AtuP, respectively. The accuracy of Tm-shift method reached 100% based on examination of 10 hookworm DNA samples with known species. In the clinical detection of hookworm in 69 stray cat fecal sample, the Tm-shift detection results were consistent with the microscopic examination and successfully differentiated between the 2-hookworm species. In conclusion, the developed method is a rapid, sensitive and accurate technique and can provide a promising tool for clinical detection and epidemiological investigation of cat-derived hookworms.

Tm-Shift Detection of Dog-Derived Ancylostoma ceylanicum and A. caninum.

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.

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.

New record of Ascaridia nymphii (Secernentea: Ascaridiidae) from macaw parrot, Ara chloroptera, in China.

Present study was performed to identify the species of ascarids from macaw parrot, Ara chloroptera, in China. Total 6 ascarids (3 males and 3 females) were collected in the feces of 3 macaws at Guangzhou Zoo in Guangdong Province, China. Their morphological characteristics with dimensions were observed under a light microscope, and their genetic characters were analyzed with the partial 18S rDNA, ITS rDNA and nad4 gene sequences, respectively. Results showed that all worms have no interlabia but male worms have two alate spicules, well-developed precloacal sucker and a tail with ventrolateral caudal alae and 11 pairs of papillae. The partial 18S rDNA, ITS rDNA and nad4 sequences were 831bp, 1015bp and 394bp in length, respectively. They showed the highest similarity of 99.8% (18S rDNA) with Ascaridia nymphii, 93.8% identities (ITS rDNA) with A. columbae and 98.5% to 99.5% identities (nad4) with Ascaridia sp. from infected parrot. All Ascaridia nematodes from the macaws were clustered into one clade and formed monophyletic group of Ascaridia with A. columbae and A. galli in two phylogenetic trees. It is observed that the combining morphological and sequencing data from three loci, the present Ascaridia species was identified as Ascaridia nymphii, which is the first record of A. nymphii from macaw parrot in China.

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.

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.

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.