In vitro immunoregulatory role of recombinant Ancylostoma ceylanicum calreticulin.

Ancylostoma ceylanicum is a zoonotic soil-derived nematode that parasitizes the intestines of humans and animals (dogs and cats), leading to malnutrition and iron-deficiency anemia. Helminth parasites secrete calreticulin (CRT), which regulates or blocks the host's immune response. However, no data on A. ceylanicum calreticulin (Ace-CRT) are available. We investigated the biological function of recombinant Ace-CRT (rAce-CRT). rAce-CRT showed reliable antigenicity and stimulated the proliferation of mouse splenocytes and canine peripheral blood mononuclear cells. Quantitative reverse-transcription PCR assays revealed that rAce-CRT primarily promoted the expression of T helper 2 cytokines, particularly IL-13, in canine peripheral blood lymphocytes. rAce-CRT inhibited complement-mediated sheep erythrocyte hemolysis in vitro. Our findings indicate that Ace-CRT plays an immunomodulatory role and may be a promising candidate molecule for a hookworm vaccine.

Phylogenetic relationship of Prohemistomum vivax to other trematodes based on the internal transcribed spacer region and mitochondrial genes.

Prohemistomum vivax is a zoonotic small cyathocotylid trematode that inhabits the intestines of fish-eating birds and mammals. Here, we amplified the internal transcribed spacer (ITS) sequence and six mitochondrial protein-coding genes (PCGs) from P. vivax. The ITS region was 1389 base pairs long and had a partial 18S ribosomal RNA gene, a full ITS1, 5.8S rRNA, and ITS2 sequence, and a partial 28S rRNA gene. The ITS region of P. vivax showed a minimum pairwise distance (0.3-0.6%) from the ITS sequences of Cyathocotylidae sp. 1 and 2 metacercariae from Clarias gariepinus. This result suggests that these metacercariae belong to P. vivax metacercariae. We first amplified mitochondrial genes from P. vivax, including cytochrome c oxidase subunit III (cox3) partial sequence; tRNA-His, cytochrome b (cytb), and NADH dehydrogenase subunit 4L (nad4L) complete sequences; and NADH dehydrogenase subunit 4 (nad4), cytochrome c oxidase I (cox1), and NADH dehydrogenase subunit 5 (nad5) partial sequences. P. vivax was most closely related to Cyathocotyle prussica (NC_039780) and Holostephanus sp. (OP082179), with cox1, cox3, and cytb genes conserved among the three trematodes. The ML phylogenetic tree of ITS sequences supports the order Diplostomida, divided into two main clades (the superfamily Diplostomoidea and Schistosomatoidea). The phylogeny of concatenated amino acid sequences of P. vivax six PCGs revealed that diplostomoids and Clinostomum sp. evolved in a clade with Plagiorchiida members, away from Schistosoma species. These results may yield ribosomal and mitochondrial genetic markers for molecular epidemiological investigations of cyathocotylid intestinal flukes.

Immune pathogenesis in pigeons during experimental Prohemistomum vivax infection.

Prohemistomum vivax is a small trematode belonging to the family Cyathocotylidae, infecting fish-eating birds and mammals, including humans. However, no data on molecular identification and immune pathogenesis are available, challenging effective diagnostic and therapeutic interventions. Here, we identified P. vivax based on combined morphological and molecular data and examined histopathological lesions and the differential cytokines expression in experimentally infected pigeons. Pigeons were orally infected with 500 prohemistomid metacercariae. Intestinal and spleen tissues were harvested 2, 4, 7, 14, 21, and 28 days post-infection (dpi). Gene expression levels of eleven cytokines (IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-15, IL-18, IFN-γ, and TGF-ß3) were assessed using quantitative reverse-transcription PCR (RT-qPCR). We identified the recovered flukes as Prohemistomum vivax based on morphological features and the sequence and phylogenetic analysis of the internal transcribed spacer 1 (ITS1), 5.8 ribosomal RNA, and ITS2 region. Histopathological lesions were induced as early as 2 dpi, with the intensity of villi atrophy and inflammatory cell infiltration increasing as the infection progressed. An early immunosuppressive state (2 and 4 dpi), with TGF-ß3 overexpression, developed to allow parasite colonization. A mixed Th1/Th2 immune response (overexpressed IFN-γ, IL-12, IL-2, IL-4, and IL-5) was activated as the infection progressed from 7 to 28 dpi. Inflammatory cytokines (IL-1, IL-6, IL-18, and IL-15) were generally overexpressed at 7-28 dpi, peaking at 7 or 14 dpi. The upregulated Treg IL-10 expression peaking between 21 and 28 dpi might promote the Th1/Th2 balance and immune homeostasis to protect the host from excessive tissue pathology and inflammation. The intestine and spleen expressed a significantly different relative quantity of cytokines throughout the infection. To conclude, our results presented distinct cytokine alteration throughout P. vivax infection in pigeons, which may aid in understanding the immune pathogenesis and host defense mechanism against this infection.

Inhibitory effect of morin on aldolase 2 from Eimeria tenella.

Eimeria tenella (E. tenella) is a protozoal parasite that can cause severe cecal lesions and death in chickens, seriously harming the chicken industry. Conventional control strategies mainly rely on anticoccidial drugs. However, the emerging problems of anticoccidial resistance and drug residues necessitate exploring potential drug targets for developing new anticoccidial drugs. Fructose-1,6-bisphosphate aldolase (ALD) is an essential enzyme for parasite energy metabolism that has been considered a potential drug target. In this study, we analyzed the molecular and biochemical properties of E. tenella ALD2 (EtALD2). EtALD2 mRNA expression was highest in second-generation merozoites, whereas the protein level was highest in unsporulated oocysts. Indirect immunofluorescence showed that EtALD2 was mainly distributed in sporozoite' cytoplasm. The natural product inhibitor (morin) was screened by computer-aided drug screening. Enzyme kinetic and inhibition kinetic assays showed that morin had a good inhibitory effect on EtALD2 activity (IC50 = 10.37 µM, Ki = 48.97 µM). In vitro inhibition assay demonstrated that morin had an inhibitory effect on E. tenella development, with an IC50 value of 3.98 µM and drug selection index of 177.49. In vivo, morin significantly improved cecal lesions (p < 0.05) and reduced oocyst excretion (p < 0.05) in E. tenella-infected chickens compared with the untreated group. The anticoccidial index of the group receiving 450 mg morin per kg feed was 162, showing a good anticoccidial effect. These findings suggest that EtALD2 could be a novel drug target for E. tenella treatment, and morin should be further evaluated as a therapeutic candidate for chicken coccidiosis.

Eukaryotic expression and immunogenicity of Ancylostoma ceylanicum calreticulin.

Ancylostoma ceylanicum is a zoonotic soil-derived nematode that parasitizes human and animal intestines, causing malnutrition and iron-deficiency anemia. Calreticulin is a multifunctional protein involved in all stages of parasitic infection. Studies have found that parasites can secret calreticulin to regulate the host's immune response. To explore the immunogenicity of the eukaryotic expression plasmid of Ancylostoma ceylanicum calreticulin (Ace-CRT), we constructed a recombinant Ace-CRT eukaryotic expression plasmid (pEGFP-N3-Ace-CRT). Successful expression of the target protein in Human Embryonic Kidney (HEK) 293 T cells was confirmed by indirect immunofluorescence and Western blot analysis. BALB/c mice were immunized with pEGFP-N3-Ace-CRT plasmid. Measuring IgG antibody levels in immunized mice sera by ELISA showed that the recombinant plasmid stimulated IgG antibody production in mice. Spleen lymphocytes were collected from vaccinated mice to determine the proportion of T cell subsets and the expression levels of cytokines. Flow cytometry revealed that the percentage of CD3 + CD4+ and CD3 + CD8+ T cells in mice spleen in the immunization group was significantly higher than that in the control group. Recombinant plasmid immunization increased IL-4, IL-10, IL-12, and IL-13 expression while decreasing IL-5, IL-6, and INF-γ in mice spleens. These results indicate that the eukaryotic plasmid constructed in this study had good immunogenicity and mainly induced a T helper 2 response in the host, laying a foundation for screening candidate molecules for anti-hookworm vaccines.

Corrigendum: Anti-Tumoral Effect and Action Mechanism of Exosomes Derived From Toxoplasma gondii-Infected Dendritic Cells in Mice Colorectal Cancer.

[This corrects the article DOI: 10.3389/fonc.2022.870528.].

Anti-Tumoral Effect and Action Mechanism of Exosomes Derived From Toxoplasma gondii-Infected Dendritic Cells in Mice Colorectal Cancer.

Toxoplasma gondii is an obligate intracellular protozoan with anti-tumor activity against a variety of cancers. However, the therapeutic effect of T. gondii on colorectal cancer is unclear, and using direct Toxoplasma infection in immunotherapy involves safety concerns. This study investigated the anti-tumoral effect and mechanism of exosomes derived from dendritic cells (DCs) infected with T. gondii (Me49-DC-Exo). We used differential ultracentrifugation to isolate exosomes from uninfected DCs (DC-Exo) and T. gondii Me49-infected DCs (Me49-DC-Exo). The isolated exosomes were identified by transmission electron microscopy, nanoparticle tracking analysis, and western blotting. Me49-DC-Exo significantly inhibited the tumor growth and reduced the proportion of M2 macrophages in the blood of tumor-bearing mice. In vitro, Me49-DC-Exo suppressed macrophage (RAW264.7) polarization to M2 phenotype. miRNA sequencing revealed that multiple miRNAs in Me49-DC-Exo were differentially expressed compared with DC-Exo, among which miR-182-5p, miR-155-5p, miR-125b-2-3p, and miR-155-3p were up-regulated, while miR-9-5p was significantly down-regulated. Transfecting mimics or inhibitors of these differential miRNAs into RAW264.7 cells showed that miR-155-5p promoted M1 macrophage polarization while inhibiting M2 macrophage polarization. Bioinformatics prediction and dual-luciferase reporter assay confirmed the suppressor of cytokine signaling 1 (SOCS1) as a direct target of miR-155-5p. Silencing SOCS1 gene expression in RAW264.7 cells increased CD86 + CD206 - M1 macrophage proportion, and inducible nitric oxide synthase and tumor necrosis factor-α mRNA levels. However, arginase-1 and transglutaminase 2 expression levels decreased. These results suggest that the exosomes inhibit macrophage polarization to M2 phenotype and regulate SOCS1 expression by delivering functional miR-155-5p. These findings provide new ideas for colorectal cancer immunotherapy.

The Potential Role of MicroRNA-124-3p in Growth, Development, and Reproduction of Schistosoma japonicum.

The microRNA-124-3p plays an important role in regulating development and neurogenesis. Previous microRNA sequencing analyses of Schistosoma japonicum revealed sja-miR-124-3p differential expression patterns in schistosomes from different hosts and at different developmental stages. This study explores the regulatory role of sja-miR-124-3p in S. japonicum development and reproduction. Quantitative reverse-transcription PCR (qRT-PCR) showed that the expression level of sja-miR-124-3p in S. japonicum from resistant hosts, such as Microtus fortis, and unsuitable hosts, such as rats and water buffalo, was significantly higher than that in mice and yellow cattle at the same developmental stage. Overexpressing sja-miR-124-3p in infected mice led to a hepatic egg reduction rate of 36.97%, smaller egg granulomas in the livers, increased liver weight, subsided hepatocyte necrosis, and diminished inflammatory cell infiltration. The width of female worms increased but decreased in males. The vitelline cells were irregular, swollen, or fused. The teguments and ventral sucker of males and females were swollen and broken, but the morphological changes were particularly notable in males. qRT-PCR and dual-luciferase reporter assay system were used to confirm the in-silico-predicted target genes, S. japonicum DEAD-box ATP-dependent RNA helicase 1 (sjDDX1) and DNA polymerase II subunit 2 (sjPOLE2). Our results showed that RNA interference (RNAi)-mediated sjDDX1 silencing in mice provided a 24.55% worm reduction rate and an 18.36% egg reduction rate, but the difference was not significant (p > 0.05). Thus, our findings suggest that sja-miR-124-3p has an important role in growth, development, and reproduction in S. japonicum. All these results will greatly contribute toward providing important clues for searching vaccine candidates and new drug targets against schistosomiasis.

Expression and biological functions of Ancylostoma ceylanicum saposin-like protein.

Ancylostoma ceylanicum is a common zoonotic nematode that inhabits the small intestine of humans, dogs, and cats. Saposin-like proteins (SLPs) have hemolytic and antibacterial activities and could be used as diagnostic or vaccine candidates. To explore the biological functions of Ancylostoma ceylanicum SLP (Ace-SLP-1), cDNA-encoding Ace-SLP-1 mature peptide was cloned into prokaryotic expression vector pET-28a and transformed into Escherichia coli BL21 (DE3) to induce expression. After incubation of canine red blood cell suspension with different concentrations of recombinant Ace-SLP-1, the supernatant was separated to measure OD value and calculate the hemolysis rate. The different concentrations of recombinant protein were co-cultured with E. coli and Enterococcus faecalis, and colony-forming units (CFU) were determined by the plate counting method. Peripheral blood mononuclear cells (PBMCs) from healthy dogs were incubated with different concentrations of recombinant Ace-SLP-1, and the cytokine expression was evaluated by relative quantitative PCR. Our results showed that the hemolytic activity of Ace-SLP-1 increased with the increase in protein concentration from 25 to 100 µg/mL. The recombinant protein had no antibacterial activity against the two kinds of bacteria but could stimulate the secretion of cytokines (IL-4, IL-10, IL-12, and IL-13) in canine PBMCs. These data suggest that Ace-SLP-1 is involved in hookworm blood-feeding and survival and has good immunogenicity, supporting its potential as a diagnostic and vaccine target molecule.

The mitochondrial genome sequence analysis of Ophidascaris baylisi from the Burmese python (Python molurus bivittatus).

Ophidascaris species are parasitic roundworms that inhabit the python gut, resulting in severe granulomatous lesions or even death. However, the classification and nomenclature of these roundworms are still controversial. Our study aims to identify a snake roundworm from the Burmese python (Python molurus bivittatus) and analyze the mitochondrial genome. We identified this roundworm as Ophidascaris baylisi based on the morphology and cytochrome c oxidase subunit I (cox1) sequence. Ophidascaris baylisi complete mitochondrial genome was 14,784 bp in length, consisting of two non-coding regions and 36 mitochondrial genes (12 protein-coding genes, 22 tRNA genes, and two rRNA genes). The protein-coding genes used TTG, ATG, ATT, or TTA as start codons and TAG, TAA, or T as stop codons. All tRNA genes showed a TV-loop structure, except trnS1AGN and trnS2UCN revealed a D-loop structure. The mitochondrial large ribosomal subunit 16S (rrnL) and small ribosomal subunit 12S (rrnS) were 956 bp and 700 bp long, respectively. Phylogenetic analysis based on O. baylisi mitochondrial protein-coding genes demonstrated that O. baylisi clustered with the family Ascarididae members and was most closely related to Ophidascaris wangi. These results may enhance the nematode mitochondrial genome database and provide valuable molecular markers for further research on the taxonomy, phylogeny, and genetic relationships of Ophidascaris nematodes.

Molecular cloning, expression and characterization of aspartyl protease inhibitor from Ancylostoma ceylanicum.

Aspartyl protease inhibitors (APIs) from parasitic intestinal nematodes are highly immunogenic and have been suggested as potential vaccine antigens. Ac-API-1 from Ancylostoma caninum showed strong immunogenicity and its polyclonal antibodies could specifically recognize the excretory/secretory products of adult worms. However, little is known about molecular characteristics and biological function of API from Ancylostoma ceylanicum (Ace-API). In this study, the Ace-API mature peptide coding sequence was cloned and expressed, and molecular characteristics of its full length sequence were analyzed. Ace-API cDNA was 684 bp in length, which encoded 228 amino acids. The similarity of the Ace-API amino acid sequence to Ac-API-1 and Adu-API-1 was 96.93% and 96.49%, respectively, and they clustered together in the phylogenetic tree. Escheria coli-expressed recombinant protein was mainly soluble in the supernatant of bacterial cell lysate. Western blot showed that Ace-API protein had good reactivity to the serum of infected dogs. Pepsin inhibition assay revealed that the recombinant protein had inhibitory activity on pepsin. Immunofluorescence results demonstrated that Ace-API was mainly localized to the epidermis, excretory glands, and pseudocoelomic fluid of the adult. Using the quantitative real-time PCR, the expression of Ace-api mRNA in adults was significantly higher than that in the third stage (L3) larvae. Together, these data indicate that Ace-API is secreted extracellularly by the parasite, and might play a role in protecting the parasite against the proteolytic digestion by the host proteases, which stimulate further studies to explore this protein as a potential hookworm vaccine candidate.

Autophagy induced by monensin serves as a mechanism for programmed death in Eimeria tenella.

Monensin (Mon), the first ionophoric antibiotic has widely been used for the treatment and prevention of coccidiosis in poultry until recently, however, at present; its efficacy has been compromised with the emergence of many Mon-resistant strains. Knowledge of the mode of the action of anti-parasitic agents is as important as for other antimicrobials, especially for discovery and long term use of the existing drugs. However, little is known about anti-parasitic drug: monensin's, mechanism of action and physiological alteration in Eimeria tenella. In this study, we explored Mon effects on the viability of Mon-Sensitive GZ (MonS-GZ) and Mon-Resistant GZ (MonR-GZ) Eimeria tenella strains using trypan blue staining and investigated Mon-induced autophagy using Western blotting, indirect immunofluorescence assay, and transmission electron microscopy. The results showed that monensin leads to programmed death of E. tenella parasites by inducing autophagy as a mechanism of anticoccidial action. Mon-induced autophagy was indicated by the decreased sporozoites survival rate, ATG8 over expression and localization, and intracellular vacuolar structures and autophagosomes formation in MonS-GZ strain while in MonR-GZ strains autophagy pathway was not triggered. The autophagy inhibitor 3-methyladenine (3-MA) effectively blocked programmed cell death and saved the MonS-GZ sporozoites. These findings indicated that autophagy serves as a potentially important mechanism of E. tenella cell death in response to Mon and disruption of the autophagy pathway may lead to emergence of drug resistance against this anti-parasitic drug.

Effect of different floatation solutions on E. tenella oocyst purification and optimization of centrifugation conditions for improved recovery of oocysts and sporocysts.

Saturated salt floatation method is widely used for coccidian oocyst purification. However, the repeated procedures and inefficient oocysts recovery rate are a continuous challenge. This study aimed to investigate the best suitable floatation solution, along with optimal centrifugation speed and time for Eimeria tenella (E. tenella) oocyst and sporocyst purification. Different floatation solutions i-e, saturated salt, Sheather's sugar and sodium hypochlorite (NaClO) at 20-60% concentrations were used to purify oocyst. It was found that about 96.99% oocysts (8609×g for 10 min) were recovered under these conditions without any effect on the viability of sporocysts. The recovery rate of oocysts using 50% NaClO (V/V) was significantly higher than 35% saturated salt flotation solution (P < 0.05). The optimal method for purification of oocysts based our experimentation was centrifugation at 8609×g for 3 min using 50% NaClO floatation solution, and the optimized centrifugation conditions for improved recovery of sporocysts (about 99.3%) were at 2152×g for 5 min. The present study provided a better method for the coccidian oocyst purification, which could be successfully adopted as a better alternative to existing techniques commonly used for investigations/research pertaining to coccidia.

Protein phosphorylation networks in spargana of Spirometra erinaceieuropaei revealed by phosphoproteomic analysis.

BACKGROUND: Sparganosis caused by Spirometra erinaceieuropaei spargana is a zoonotic parasitic infection that has been reported in many countries, including China, Japan, Thailand and Korea, as well as European countries and the USA. The biological and clinical significance of the parasite have previously been reported. Although the genomic and transcriptomic analysis of S. erinaceieuropaei provided insightful views about the development and pathogenesis of this species, little knowledge has been acquired in terms of post-translational regulation that is essential for parasite growth, development and reproduction. Here, we performed site-specific phosphoproteomic profiling, with an aim to obtain primary information about the global phosphorylation status of spargana. RESULTS: A total of 3228 phosphopeptides and 3461 phosphorylation sites were identified in 1758 spargana proteins. The annotated phosphoproteins were involved in a variety of biological pathways, including cellular (28%), metabolic (20%) and single-organism (17%) processes. The functional enrichment of phosphopeptides by Gene Ontology analysis indicated that most spargana phosphoproteins were related to the cytoskeleton cellular compartment, signaling molecular function, and a variety of biological processes, including a molecular function regulator, guanyl-nucleotide exchange factor activity, protein kinase activities, and calcium ion binding. The highly enriched pathways of phosphorylation proteins include the phosphatidylinositol signaling system, phagosome, endocytosis, inositol phosphate metabolism, terpenoid backbone biosynthesis, and peroxisome. Domain analysis identified an EF-hand domain and pleckstrin homology domain among the key domains. CONCLUSIONS: To our knowledge, this study performed the first global phosphoproteomic analysis of S. erinaceieuropaei. The dataset reported herein provides a valuable resource for future studies on the signaling pathways of this important zoonotic parasite.

Effect of Ancylostoma ceylanicum hookworm platelet inhibitor on platelet adhesion and peripheral blood mononuclear cell proliferation.

Ancylostoma ceylanicum is a zoonotic parasitic nematode that can cause iron-deficiency anemia and malnutrition in humans. A. ceylanicum hookworm platelet inhibitor (Ace-HPI) can inhibit platelet aggregation in the host to facilitate blood sucking, but whether it possesses platelet adhesion inhibitory activity or immunomodulatory role is yet unknown. To explore the effect of Ace-HPI on platelet adhesion, we expressed the recombinant protein in two competent cells, BL21 (DE3) and Rosetta-gami2 (DE3), and incubated this protein with canine platelets in a 96-well microplate. Ace-HPI was used to stimulate peripheral blood mononuclear cells (PBMC) in vitro to investigate the effect on PBMC proliferation and cytokine expression. Results showed that Ace-HPI expressed in Rosetta-gami2 (DE3) strain was mostly soluble. The inhibitory effect of this protein on platelet adhesion was relatively weak (7-8%). This protein stimulated the proliferation of PBMC and promoted the expression of Treg and Th2 cytokines, such as IL-10 and IL-13. These results lay a foundation for exploring the role of Ace-HPI in hookworm disease pathogenesis and as a candidate molecule for hookworm vaccines.

Mitochondrial Genome Sequence of Echinostoma revolutum from Red-Crowned Crane (Grus japonensis).

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.

Twenty-five-year research progress in hookworm excretory/secretory products.

Hookworm infection is a major public health problem that threatens about 500 million people throughout tropical areas of the world. Adult hookworms survive for many years in the host intestine, where they suck blood, causing iron deficiency anemia and malnutrition. Numerous molecules, named excretory/secretory (ES) products, are secreted by hookworm adults and/or larvae to aid in parasite survival and pathobiology. Although the molecular cloning and characterization of hookworm ES products began 25 years ago, the biological role and molecular nature of many of them are still unclear. Hookworm ES products, with distinct structures and functions, have been linked to many essential events in the disease pathogenesis. These events include host invasion and tissue migration, parasite nourishment and reproduction, and immune modulation. Several of these products represent promising vaccine targets for controlling hookworm disease and therapeutic targets for many inflammatory diseases. This review aims to summarize our present knowledge about hookworm ES products, including their role in parasite biology, host-parasite interactions, and as vaccine and pharmaceutical targets and to identify research gaps and future research directions in this field.

Identification and localization of hookworm platelet inhibitor in Ancylostoma ceylanicum.

Ancylostoma ceylanicum is a zoonotic hookworm, which mainly causes iron deficiency anemia (IDA) in humans and animals. Hookworm platelet inhibitor (HPI) has been isolated from adult Ancylostoma caninum and linked to the pathogenesis of hookworm associated intestinal hemorrhage and IDA. However, there is no available data about HPI from A. ceylanicum. To study the molecular characteristics of A. ceylanicum HPI (Ace-HPI), its corresponding cDNA was amplified from adult A. ceylanicum mRNA using the primers designed based on the Ac-HPI gene sequence, and its sequence homology and phylogenetic relationship were analyzed. The differential expression of Ace-hpi mRNA in the adult and third larval (L3) stages was compared using the quantitative real-time PCR. Ace-HPI reactivity and tissue localization were studied by Western blot and immunofluorescence, respectively. Platelet aggregation activity was monitored in a 96-well microplate reader. The results showed that the Ace-HPI encoding gene was 603 bp in length. Ace-HPI showed 91% homology to Ac-HPI, was closely related to Ac-ASP3, and belonged to the CAP superfamily. Ace-hpi transcripts were most abundant in the adult stage, followed by serum-stimulated infective larvae (ssL3), and finally in L3 stage, with a significant difference. Escherichia coli-expressed recombinant protein had good reactivity with the positive serum of A. ceylanicum-infected dogs. Immunolocalization indicated that Ace-HPI was located in the esophagus and cephalic glands of the adult. As well as, recombinant Ace-HPI inhibited the platelet aggregation in-vitro. HPI overexpression, anatomical location in adults, antigenicity and its in-vitro activity indicate its possible role in adult worm blood-feeding and as a valuable target for hookworm vaccine and drug development.

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.