Food-borne parasitic diseases caused by ingesting food and water containing infective parasites are still common parasitic diseases that are easily misdiagnosed and mistreated in clinical practice. With the participation of multi-disciplinary experts, and in the light of the latest research results at home and abroad, based on factors other than the quality of evidence (economics, patient preferences and values, trade-offs, accessibility, fairness, acceptability, etc.), the level of recommendation and the quality of evidence in evidence-based medicine were assessed using the World Health Organization-recommended evidence quality classification and strength of recommendation system, and a consensus of 24 items was reached to guide and improve the comprehensive diagnosis and treatment of food-borne parasitic diseases for clinical medical staff.
Objective To screen the differentially expressed genes (DEGs) by comparing the transcriptome of the brain tissues between the mice chronically infected with Toxoplasma gondii and normal mice, to analyze the relative transcription level of DEGs in the depression-related kynurenine (KYN) pathway and to provide a theoretical basis for exploring the mechanism of depression-like symptoms caused by Toxoplasma gondii chronic infecttion in mice. Methods SV129 male mice (n = 18) were randomly and equally divided into the infection group and the control group. Mice in the infection group were intraperitoneally injected with 120 tachyzoites of T. gondii ME49 strain (200 μl), and mice in the control group were injected with the same volume of PBS. After 3 months post-infection, mice brain tissues of the two groups were collected for extraction of total RNA to undertake transcriptome sequencing for screening DEG. With the DEGs obtained, cluster analysis, gene ontology (GO) functional annotation analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotation and enrichment analysis were performed. Eight DEGs [interferon-γ (IFN-γ), indoleamine 2,3-dioxygenase 1 (IDO1), IDO2, kynurenine-3-monooxygenase (KYNU), kynurenine-3-monooxygenase (KMO), 3-hydroxyanthranilate 3,4-dioxygenase (3-HAO), vimentin (Vim) and brain-derived neurotrophic factor (BDNF)] related to KYN pathway associated with depression were selected to examine each gene’s relative transcription level by quantitative real-time PCR (qRT-PCR), using glyceraldehyde-3-phosphate dehydrogenase gene as an internal reference. Results Transcriptome sequencing found 2 295 DEGs in the brain of the mice from the infection and control groups, of which 2 016 were up-regulated and 279 were down-regulated. GO analysis showed that localisation was the most significantly enriched biological process, with a total of 257 DEGs. The most significantly enriched in cellular components was the protein-containing complex, with a total of 425 DEGs. The most significantly enriched molecular function was molecular transducer activity, with 177 DEGs. The largest number of DEGs enriched in biological process, cell component and molecular function were cell process, cell part and binding, with 1 039, 1 240 and 1 088 DEGs, respectively. KEGG analysis showed that the top three up-regulated metabolic pathways were the immune system, signaling transduction, and viral infectious disease, and the top three down-regulated pathways were signal transduction, signaling molecules and interaction and immune system. Functional enrichment analysis showed that 77 pathways were significantly enriched. The signaling pathways related to depression include tumor necrosis factor signaling pathway, neuroactive ligand-receptor interaction, NF-kappa B signaling pathway, JAK-STAT signaling pathway, necroptosis, apoptosis, chemokine signaling pathway, KYN pathway. The qRT-PCR results showed that the relative transcription levels of IFN-γ, IDO1, IDO2, KYNU, KMO, 3-HAO and Vim genes in the infection group were 3 023.08%, 355.52%, 190.17%, 496.55%, 339.92%, 212.74% and 507.34%, if the relative transcript level of control mice was taken as 100%. Compared with the control group, the transcription was significantly up-regulated (t = 3.782, 3.749, 3.226, 2.908, 2.533, 5.656, 2.948; all P < 0.05 or 0.01). The relative transcription level of BDNF was 63.32%, which was significantly down-regulated (t = 2.398, P < 0.05). The fold change of IFN-γ, IDO1, IDO2, KYNU, KMO, 3-HAO, BDNF, Vim obtained by qRT-PCR was 4.96, 1.74, 0.89, 2.10, 1.60, 1.06, -0.94, 2.18, respectively. The fold change obtained by transcriptome sequencing was 7.30, 0.55, 0.80, 3.83, 2.75, 3.53, -0.86 and 1.93, respectively. The transcriptional trend obtained by qRT-PCR was consistent with that obtained by transcriptome sequencing. Conclusion DEGs from brain tissues of mice chronically infected with T. gondii were screened. Transcriptome analysis revealed that the immune response of central nervous system of the mice with chronic infection of T. gondii was continuously activated. Seven DEGs in KYN pathway related to depression showed up-regulated transcription level.
Trichinella spiralis infection can cause zoonotic trichinellosis that seriously endangers human health. During the entire T. spiralis life cycle stages, the parasite can manipulate the host's immune responses. The immune evasion mechanism, as a common natural selection mechanism, is an adaptation of pathogens to the immune system under natural selection. It enables the pathogens to relieve or alleviate the host's immune responses, avoid attacking from the immune system, and ensure that they can survive and reproduce. This paper reviewed the research advance of T. spiralis immune evasion mechanism in disturbing complement response, innate immune response, humoral immune response, and T cell immune response. Further research of the immune evasion mechanism of T. spiralis is of great significance for the prevention and control of trichinosis, the treatment of human autoimmune diseases, and allergic diseases.
Objective To explore the effect of drug-regulation on peroxisome proliferator-activated receptor-γ/retinoid X receptor-α (PPAR-γ/RXR-α) signalling pathway to improve lung injury caused by Paragonimus proliferus infection in rats. Methods The P. proliferus were isolated from crabs collected in Yunnan Province, and SD rats were infected with 8 metacercariae per rat via subcutaneous injection at abdominal wall. The infected rats were divided into the high-fat-diet group, rosiglitazone group and bexarotene group, with 10 rats in each group, and were given with high-fat-feeding (high-fat diet), rosiglitazone [3 mg/(kg·d)], bexarotene [10 mg/(kg·d)] by gavage for 7 days. The infected and healthy rats were used as the infection control group and healthy control group. On 28 d post-mediated, the rats were sacrificed to collect lung tissue and heart apical blood. The lung tissue sections were prepared and stained with hematoxylin-eosin to assess the tissue damage and score the alveolitis semi-quantitatively. Serum interleukin-1β (IL-1β) and tumor necrosis factor α (TNF-α) levels were detected by ELISA. The lung tissue total protein was extracted for examining relative expression levels of PPAR-γ/RXR-α signalling pathway-related key target proteins indluding PPAR-γ, RXR-α, fatty acid transporter 3 (FATP3), apolipoprotein A1 (ApoA1), nuclear factor kappa-B (NF-κB) signalling pathway proteins [p65, activator protein (AP1）], janus kinase (JAK)/signal transducer and activator of transcription (STAT) signalling pathway proteins （JAK2, STAT3） by Western blotting. The inter-group data were compared using single-factor ANOVA analysis. Results All rats were successfully infected except 2 rats in the bexarotin group. Parasitic cysts were found in the lungs or the chest cavity. HE staining showed that in the infection control group, a large number of inflammatory cells infiltrated the alveolar septum, the alveolar wall thickened, and the alveolar cavity collapsed or closed. In the high-fat diet group, obvious inflammation injury was shown. Compared with the infection control group, the alveolar cavity inflation was slightly improved. The inflammatory cells of alveolar septum in the rosiglitazone group and bexarotene group were significantly reduced. Compared to the infection control group, the degree of alveolar wall thickening was significantly reduced, and the alveolar cavity inflation was significantly improved. The semi-quantitative alveolitis scores were 0.300 ± 0.053, 2.200 ± 0.189, 1.900 ± 0.320, 1.300 ± 0.301 and 1.500 ± 0.112 (F = 12.033, P < 0.05) in the healthy control group, the infection control group, the high-fat diet group, the rosiglitazone group and the bexarotene group, respectively. ELISA tests show that serum IL-1β levels in the healthy control group, infected control group, high-fat diet group, rosiglitazone group and bexarotene group were (103.23 ± 3.37), (111.59 ± 20.49), (110.13 ± 12.95), (89.91 ± 14.84) and (96.34 ± 19.03) pg/ml, respectively. TNF-α levels were (144.81 ± 1.35), (180.21 ± 23.38), (171.76 ± 27.83), (155.37 ± 13.67) and (143.24 ± 23.66) pg/ml, respectively. There was a significant difference among the five groups (F = 17.236, 13.558; P < 0.05). Western blotting showed that in the lung tissue of rats in the healthy control group, the infection control group, high-fatdiet group, rosiglitazone group, and bexarotene group, PPAR-γ relative expression level were 0.51 ± 0.09, 0.67 ± 0.06, 0.75 ± 0.08, 0.34 ± 0.02 and 0.56 ± 0.04, respectively; RXR-α were 0.89 ± 0.05, 0.15 ± 0.03, 0.81 ± 0.09, 0.22 ± 0.02 and 0.61 ± 0.10, respectively; FATP3 were 0.59 ± 0.06, 0.64 ± 0.06, 0.68 ± 0.09, 0.59 ± 0.09 and 0.55 ± 0.03, respectively; ApoA1 were 0.58 ± 0.04, 0.83 ± 0.11, 0.92 ± 0.19, 0.71 ± 0.04 and 0.63 ± 0.08, respectively, and the differences among the five groups were statistically significant (F = 25.70, 67.12, 8.94, 11.58; All P < 0.05). P65 the relative expression level were 0.25 ± 0.19, 1.01 ± 0.21, 0.27 ± 0.15, 0.32 ± 0.01 and 0.22 ± 0.11, respectively; AP1 were 0.11 ±0.09, 1.12 ± 0.36, 0.08 ± 0.02, 0.03 ± 0.01 and 0.02 ± 0.01, respectively; JAK2 were 0.76 ± 0.18, 1.11 ± 0.24, 0.34 ± 0.06, 0.42 ± 0.01 and 0.35 ± 0.04, respectively; STAT3 were 0.80 ± 0.33, 1.11 ± 0.27, 0.68 ± 0.22, 0.77 ± 0.06 and 0.68 ± 0.19, respectively, the difference between the 5 groups were statistically significant (F = 43.77, 85.19, 37.22, 17.63; All P < 0.05). Among them, the relative expression levels of PPAR-γ, FATP3, ApoA1, p65, AP1, JAK2 and STAT3 in the infection control group were higher than those in the healthy control group, while those in rosiglitazone group and bexarotene group were lower than those in the infection control group (all P < 0.05). Conclusion A lung injury model of rat infected with P. proliferus was established. The rat model demonstrated that PPAR-γ/RXR-α signalling pathway plays an important role in the mechanism of lung injury caused by P. proliferus infection, and regulation of this signalling pathway by drugs may exert anti-inflammatory effects by inhibiting the NF-κB and JAK/STAT signalling pathways, thereby, reducing the extent of lung injury severity.
Objective This study investigates the effect of thioredoxin peroxidase (TPx) in the excretory-secretory antigen (ESA) of Cysticercus cellulosae on activation of dendritic cell (DC) in piglets. Methods Healthy piglet medulla-derived DC were cultured in vitroo, in which lipopolysaccharide (LPS) was added at a final concentration of 100 ng/ml on 7 d for stimulation for 2 days, and then continuously cultured for 2 more days to collect immature DC (imDC) and mature DC (mDC) separately. The morphological changes of DCs were observed by light microscopy and scanning electron microscopy on 1 to 9 d of culture. The expression of surface markers CD1 and major histocompatibility complex (MHC-Ⅱ) was detected by flow cytometry. The 7 d imDC was used in the assay with the assigned groups of negative control, TPx, ESA and LPS positive control, to which RPMI 1640 medium, TPx (50 μg/ml), ESA (50 μg/ml) and LPS (100 ng/ml) was added, respectively, to stimulate for 48 h for examining the expression of DC surface markers MHC-Ⅱ, CD80 and CD86 using flow cytometry and for detecting secretion levels of tumor necrosis factor-α (TNF-α), interleukin (IL-6), IL-10, IL-12 in DC culture supernatant by ELISA. One-way ANOVA was used for comparison between multiple groups, and LSD-t test was used for two-way comparison between groups. Results Under ligth microscope, imDC were ovoid in shape with single form at the first day of culture; with the extension of culture time, DC increased in size, appeared pseudopods and spines and other features, and changed from ovoid to irregular shape. Scanning electron microscopy showed that compared with imDC, mDC had irregular morphology, roughly long shuttle shape, and numerous protrusions of different lengths radiating from the cytosol, which were distributed in a dendritic pattern, a typical dendritic structure. Flow cytometry showed that the expression of CD1 and MHC-Ⅱ in imDC was (0.113 ± 0.005)% and (0.430 ± 0.016)%, respectively, which was lower than that of mDC (21.400 ± 0.327)% and (21.333 ± 0.450)% (t = 130.341, 92.906, both P < 0.05). The expression levels of MHC-Ⅱ, CD80, and CD86 in the TPx group were (15.300 ± 0.245)%, (22.900 ± 0.374)% and (13.033 ± 0.249)%, respectively, which were lower than those in the LPS positive control group (19.000 ± 0.374)%, (31.600 ± 0.082)%, and (21.300 ± 0.245)% (t = 11.53, 46.32, 43.84, all P < 0.05) and the ESA group (18.365 ± 0.618)%, (40.400 ± 0.356)% and (30.300 ± 0.283)%] (t = 9.55, 93.17, 91.57, all P < 0.05). The MHC-Ⅱ expression level in the TPx group was higher than that of the negative control group (12.133 ± 0.492)% (t = 9.87, P < 0.05). ELISA results showed that IL-6 level in DC of the TPx group was 15.682 ± 0.660, which was ower than that of 21.041 ± 0.901 in the control group (t = 6.51, P < 0.05); TNF-α (35.711 ± 4.196), IL-6, IL-10 (22.216 ± 1.357) and IL-12 (16.799 ± 0.523) were all lower than those of the LPS positive control group 169.037 ± 7.823, 42.118 ± 1.932, 34.730 ± 1.772, 52.504 ± 2.431 (t = 36.79, 32.09, 13.09, 35.05, all P < 0.05); IL-12 level were lower than that of the ESA group at 23.854 ± 1.020 (t = 6.93, P < 0.05). Conclusion TPx mediates immune tolerance by inducing high expression of DC surface molecules MHC-Ⅱ, low expression of CD80 and CD86, and reducing the secretion levels of IL-6.
Objective To understand the situation of soil-transmitted nematode infection in China in 2020 and provide support for evaluating the development of surveillance on soil-transmitted nematodiasis in various provinces/municipalities/autonomous regions, and improving and perfecting the control strategies. Methods Surveillance was carried out in 408 national surveillance sites (counties) in 31 provinces (municipalities, autonomous regions) in China in 2020. With the county as unit, each site was divided into 5 areas geographically: east, west, south, north, and central part, followed by selecting one township (town), and therein one administrative village was selected from wherein, 200 permanent residents over the age of 3 were sampled. A total of 1 000 people were surveyed at each surveillance site. Fecal samples were collected from the sampled villagers, and examined by using the modified Kato-Katz thick smear method (two slide-reading for each sample) for infection of hookworm, Ascaris lumbricoides, Trichuris trichiura and Enterobius vermicularis, to calculate the infection rate and intensity, respectively. In addition, soil samples were collected from fields or vegetable gardens of each village in the survey site, and examined for hookworm larvae using 5% saline at 45 ℃, and for Ascaris eggs by saturated sodium nitrate flotation method. Results In 2020, the overall infection rate of soil-transmitted nematode in residents was 0.84% (3 485/415 672) in 408 surveillance sites of 31 provinces (municipalities/autonomous regions), with the highest found in Hainan (6.34%, 199/3 141), followed by Yunnan (5.80%, 963/16 616) and Sichuan (3.66%, 592/16 168); infection rate in females was 0.91% (1 944/213 591), which was higher than that of 0.76% in males (1 541/202 081) (χ2 = 27.20, P < 0.01). The soil-transmitted nematode infection rate was the highest in the age group ≥ 60-years-old, which is 1.26% (1 376/109 251). The difference between each age group was statistically significant (χ2 = 382.28, P < 0.01). The infection rates of hookworm, A. lumbricoides, T. trichiura were 0.51% (2 016/415 672), 0.19% (805/415 672) and 0.16% (673/415 672), respectively. Among them, hookworm and T. trichiura had only mildly infected cases. The proportions of mild and moderate A. lumbricoides infections were 99.25% (799/805) and 0.75% (6/805), respectively. In 2020, 2 604 soil samples were examined and found that the positive rate of Ascaris eggs and hookworms was 3.07% (80/2 604) and 2.42% (63/2 604), respectively. Conclusion In 2020, the infection rate of soil-transmitted nematode in China remains at a low level in general, but the regional differences are still significant, and the areas with high infection rates still exist. At the same time, it is necessary to strengthen the control measures for the key groups of people over age of 60, women and children, and carry out health education.
In order to further understand the liver fibrosis and immune regulatory changes in mice infected with Clonorchis sinensis, BALB/c mice were randomly divided into 2 groups with 30 mice in each group. The infected group were administered 200 μl normal saline with 150 C. sinensis metacercariae through oral gavage and the control group received 200 μl normal saline. Six mice were randomly selected for dissection at 1, 2, 4, 8 and 16 weeks. The paraffin sections of the liver tissue were stained with hematoxylin-eosin (HE) and Masson to observe the progression of fibrosis. The relative transcription level and actin alpha 2 (ACTA2) expression in the liver were detected by qRT-PCR and western blotting separately. The spleen tissue were weighed for calculating the spleen index (spleen weight per 10 g weight, mg/10 g). The flow cytometry was performed to detect the dynamical changes of CD4+ T cells, helper T1 (Th1) cells, Th2 cells, Th17 cells and regulatory T (Treg) cells in spleen. The secretion levels of tumor necrosis factor (TNF), interleukin-6 (IL-6), interferon-γ (IFN-γ), IL-2, IL-4, IL-10, and IL-17A were quantified by cytometric bead array. The data was analyzed by GraphPad Prism 9.0. Two-way ANOVA was used for groups comparison and t test was used for pairwise comparison. The results showed that the mice weight decreased and spleen index increased after infected with C. sinensis and the inflammatory lesions were visible in liver tissue. HE staining showed that C. sinensis larvae and inflammatory cells could be found around the hepatobiliary duct at week 1 after infection. Masson staining revealed that the relative area of collagen fiber deposition was higher than the control group after infection (F = 20.190, P < 0.05). The qRT-PCR results showed that the hepatic ACTA2 relative transcription level of the infected group were higher than the control group at week 2, 4 and 16 after infection (t = 2.042, 2.475, 1.634; all P < 0.01) but lower than the control group at week 8 (t = 2.758, P < 0.05). Western blotting results showed that the hepatic ACTA2 protein relative expression level of the infected group were higher than the control group (F = 3.225, P < 0.01). The flow cytometry results showed that the percentage of splenic CD4+ T cells decreased to (13.4 ± 1.8)% at week 2 after infection and increased to (22.4 ± 1.5)% at week 16. The percentage of Th1 cells increased to (16.9 ± 5.3)% at week 2 and decreased to (3.9 ± 2.6)% at week 16. The percentage of Th2 cells decreased to (2.3 ± 0.6)% at week 4 and increased gradually after week 4. The percentage of Th17 cells increased to (5.3 ± 3.4)% at week 8 and decreased to (2.4 ± 1.4)% at week 16. The percentage of Treg cells decreased to (7.3 ± 1.5)% at week 4 and increased to (13.9 ± 1.2)% at week 16. The serum TNF content of infected group mice increased to (35.16 ± 11.28) pg/ml at week 1 after infection and decreased to (8.98 ± 1.66) pg/ml at week 8. The serum IL-6 content was higher than the control group after infection (t = 2.095, P < 0.05). The serum IL-2 content decreased to (0.09 ± 0.18) pg/ml at week 8 and increased to (3.81 ± 2.79) pg/ml at week 16 after infection. The content of serum IFN-γ, IL-4, IL-10 and IL-17A increased gradually after infection (F = 8.726, 8.068, 6.795, 14.840; all P < 0.05). As a conclusion, the dynamic changes of CD4+ T cells and serum cytokines were closely related to the hepatic fibrosis induced by C. sinensis.
Anisakis is a zoonotic parasite that is widely distributed in marine areas around the world. The adult nematodes are mainly parasitic in dolphins, seals, whales and other marine mammals, and the larvae are mainly parasitic in fish, cephalopods and other small marineorganisms. If the larvae of Anisakis are eaten by humans, they can cause anisakiasis and harm health. The traditional morphological analysis method is unable to accurately identify anisakis, which hinders the in-depth study of Anisakis population. With the development and application of molecular biotechnology, many important achievements have been made in the study of Anisakis. In this paper, research progress on the life history, host preference, morphology of adults and larvae, molecular biology classification, global distribution of Anisakis nematodes was reviewed.
Toxoplasma gondii is an obligate intracellular parasite that widely distributes in the world and causes zoonotic toxoplasmosis. In recent years, parasite infection in the brain has been paid more and more attention. T. gondii can cause central nervous system damage, often manifested as depression, schizophrenia, epilepsy and other symptoms. In this paper, the process and mechanism of T. gondii establishing chronic infection through blood-brain barrier, causing central nervous system injury and disease are reviewed.
After more than 70 years of effective control programme, China has made remarkable achievements in the control of key parasitic diseases, and is moving towards the goal of control and elimination. This paper analyzes the epidemic status and challenges of schistosomiasis, malaria, echinococcosis, leishmaniasis, clonorchiasis and soil-transmitted helminthiasis in recent years, and puts forward the future directions and key tasks of those diseases, in order to provide reference for accelerating the control and elimination programmes on key parasitic diseases in China.
Objective To analyze the effect of Toxoplasma gondii infection on the level of N6-methyladenosine （m6A） methylation of brain transcripts in mice. Methods C57BL/6J female mice (n = 20) were randomly divided into TgCtwh6 infection group (n = 7), LHG infection group (n = 7) and control group (n = 3 for the control of TgCtwh6 infection and LHG infection group, respectively). The TgCtwh6 infection group and the LHG infection group were inoculated by gavage with 0.2 ml brain tissue suspension (20 cysts/mouse) from the mice infected with T. gondii Chinese Ⅰ genotype wh6 strain or Chinese Ⅲ genotype LHG strain, respectively. The control group was given with the same amount of normal saline. At 15, 30 and 45 days post-infection, one mouse was randomly selected by drawing lots from each infected group, which was sacrificed under anesthesia to collect brain tissue for microscopic examination and counting of the cyst number in the cerebral cortex, hippocampus and olfactory bulb. At 45 days post-infection, the whole brain tissues of 3 mice in each group were collected, the total RNA was extracted, the genome library was prepared, the transcriptome was sequenced, differential methylation loci (DML) were screened, and differential m6A methylation sites and transcripts of mRNA in infected group and control group were recorded. The transcripts with methylation sites were analyzed by gene ontology functional annotation (GO), Kyoto Encyclopedia of Gene and Genome (KEGG), and methylated differential transcripts by gene set enrichment analysis (GSEA). Three m6A methylation modification protein genes, methyltransferase like 3 (METTL3), fat mass and obesity-associated protein (FTO) and YTH domain family 3 (YTHDF3), were selected, and their relative transcription levels were detected by real-time fluorescence quantitative reverse transcription PCR (qRT-PCR) using the glyceraldehyde-3-phosphate dehydrogenase gene as an internal reference. Results All the mice in the infection group were infected successfully. On day 45 after infection, the cysts in cerebral cortex, hippocampus and olfactory bulb were (5 676 ± 10), (4 773 ± 9) and (243 ± 10), respectively. A total of 760 650 methylation sites were detected in the infection group and control group. Among them, the methylation levels of four m6A motifs, GGACA, GGACC and ACGAT, accounted for 16.8% (127 923/760 650), 4.6% (35 164/760 650), 3.8% (28 983/760 650) and 0.4% (3 122/760 650), respectively. A total of 127 016 transcripts with high methylation sites were detected, including 71 727, 27 754, 24 556 and 2 979 transcripts with GGACA, GGACC, GGACT and ACGAT, respectively. The total number of DML sites for four m6A motifs, including ACGAT, GGACA, GGACC and GGACT, in the transcriptome of the infected and control group mice brain tissue was 9 233, including 4 832 high methylation sites and 4 851 low methylation sites. The GO analysis showed that the transcripts where DML was located were significantly enriched in cellular processes of biological processes, cell of cellular components, and binding in molecular functions. The KEGG enrichment analysis of the transcripts where DML was located showed that the intermolecular interaction network was mainly enriched in signaling pathway such as lysosome, spliceosomes, dopamine synapses. The GO enrichment analysis of biological processes showed that in the transcripts where DML was located, some transcripts related to biological processes were mainly enriched in processes such as protein glycosylation, negative regulation of neuronal apoptosis and proteion import into nucleus. The GSEA analysis showed the differential methylation between the control group and the two infection groups was highly enriched in transcriptome subsets related to the negative regulation pathway of fibroblast proliferation and Hippo signaling pathway. Among them, four key transcripts, ENSMUST00000105393, ENSMUST00000006523, ENSMUST00000055261 and ENSMUST00000038658, were screened, which encode costimulatory factor ligand (ICOSL), cysteine-rich intestinal protein 1 (CRIP1), MOB kinase activator 1A201 (MOB1A201) and MOB1A202, respectively. Compared with the control group, the expression of these four genes in TgCtwh6 infection group and LHG infection group were all up-regulated. The results of qRT-PCR showed that the relative expression level of mettl3 mRNA in brain tissue of TgCtwh6 infection group and LHG infection group was 5.47 ± 1.09, 1.63 ± 0.06, respectively, which was significantly different from that of the control group (1.01 ± 0.11) (t = 4.05, 5.03; both P < 0.05). The relative expression level of ythdf3 mRNA in the control group was 3.57 ± 0.08 and 1.80 ± 0.25, respectively, which was significantly different from that in the control group (1.01 ± 0.11) (t = 18.95, 2.85; both P < 0.05). The relative expression level of fto mRNA was 0.41 ± 0.04, 0.60 ± 0.12, respectively, which was significantly different from the control group (1.00 ± 0.06) (t = 7.67, 2.99; both P < 0.05). The qRT-PCR results were consistent with the transcriptional trend obtained by transcriptional sequencing. Conclusion Chronic T. gondii infection may enhance the m6A methylation in mouse brain transcripts, leading to changes in the biological processes and signal pathways related to host mental behavioural through modifying and regulating key differential transcripts including icosl, crip1 and mob1a.
Echinococcosis is a kind of parasitic zoonosis that seriously endangers human health and economic development, which is mainly caused by the parasitization and development of the Echinococcus larvae in the intermediate host. According to current research advances, the globally identified Echinococcus includes 9 valid natural species, which can cause 4 types of echinococcosis. This consensus has evolved alongside the development of research methods and scientific technology over the past two thousand years. Therefore, this article introduced the whole process of human exploration, recognition and cognition of Echinococcus and echinococcosis by summarizing and combing references. The landmark historical event is that in 1801, Rudolphi established an independent genus of Echinococcus for this flatworm. After that, several species of Echinococcus have been found in the world successively. In 1953, Rausch re-classified Echinococcus based on biological characteristics and gradually developed the concept and classification method of subspecies for Echinococcus; in 1967, Rausch further proposed the concept of Echinococcus strains, which was confirmed in the 1990s by Bowles through molecular biological methods. In 2013, Nakao introduced the phylogenetic species concept, revising Echinococcus into nine valid natural species that are still in use today. In addition, this paper summarized the discovery history of Echinococcus and echinococcosis in China. Since 1908, five species of Echinococcus have been reported in China, including E. shiquicus, which was unique to the Qingzang Gaoyuan region. This article provides a systematic review of the historical understanding of Echinococcus and echinococcosis, summarizing extensive historical data to further comprehend the taxonomic research advancements of Echinococcus.
Objective To reveal the effect of mild asymptomatic hookworm infection on intestinal microflora and metabolome in middle-aged and the elderly people, to provide reference information for study on interaction of helminth-intestinal microflora. Methods In October to November 2022, from the parasite infection survey in residents at the surveillance site for soil-transmitted nematodes infection in Qimen County and Huangshan District of Huangshan City, Anhui Province, the hookworm egg positives detected by modified Kato-Katz thick smear method were assigned in infection group, and the hookworm egg negatives in control group. Fresh fecal samples from the examinees were collected for extraction of intestinal microflora DNA, of which 16S rRNA gene sequence was amplified by PCR and sequenced. The abundance-based coverage estimator (ACE), PD-wholetree, Chao index and Shannon index were calculated. The principal coordinate analysis (PCoA) and β diversity analysis of the unweighted pair group method with arithmetic mean (UPGMA) hierarchical clustering were used to compare the difference in the distribution of intestinal microflora between the infection group and the control group, to screen the indicator flora by indicator value analysis. The metabolites in the fecal samples were extracted with methanol-water extraction method for metabolome analysis by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) for non-targeted sequencing. Orthogonal partial least squares discriminant analysis (OPLS-DA) was used to establish a model for screening differential metabolites between the infection group and the control group, and generate the volcano plot. The differential metabolites were screened based on P-values, variable weight value, and difference multiple. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used to search the metabolic pathways of related differential metabolites. Enrichment analysis of differential metabolites was performed, and the degree of linear correlation between two metabolites was analyzed by Pearson correlation analysis. Results A total of 22 subjects were studied, including 11 subjects of positive fecal eggs and 11 subjects of negative control. The average age of the infected group was 72.2 years old, and all of them presented mild asymptomatic hookworm infection. The average age of the control group was 53.1 years old. A total of 1 755 649 16S rRNA sequences were obtained and 1 245 amplicon sequence variants were clustered after quality control and assembly. A total of 389 species from 15 phyla, 22 classes, 58 orders, 94 families, and 191 genera were identified. The results of the diversity index analysis showed that the ACE, PD whole tree, Chao and Shannon indexes of intestinal flora diversity in the infection group were 188.768, 16.533, 192.667 and 5.195, respectively, whereas the control group were 167.829, 15.294, 157.371 and 4.898, respectively. No significant difference was seen between the two groups （t = 1.266, 0.952, 1.266, 0.962, P > 0.05）. The PCoA analysis demonstrated that the intestinal microbiota of the infected group and the control group overlapped to varying degrees but could still be divided into two distinct taxa. Additionally, the UPGMA hierarchical clustering analysis exhibited that both the infection group and the control were clustered in the same group first, and then clustered with the other group, and only one case overlapped. Multivariate statistical analysis showed seven flora dominating in the infection group, including Prevotella, Feacalibacterium, Klebsiella variicola, Parasutteralla, Coprococcus, Clostridium UCG-014 and Enterobacterium; while in the control group, only B. vulgatus was abundant. The results of indicator value analysis revealed the bacteria with the largest indicator value that affect the intestinal environment in the infection group, including Prevotella, Klebsiella, Bacteroides, Dialister, Enterobacterium, Coprococcus, UCG-002 and Faecalibacterium in the infection group; while in the control group, Clade_Ⅰa, Clade_Ⅲ, Blautia, Alistipes, Lachnoclostridium, Bilophila, Turicibacter and Muribaculaceae were dominant. The OPLS-DA model could effectively distinguish the metabolites in fecal samples of the infection group and the control. A total of 400 different metabolites were identified in the two groups, of which 156 were significantly increased and 244 decreased. The KEGG enrichment analysis indicated that the differential fecal metabolites were mainly enriched in protein digestion and absorption (P = 0.000) and central carbon metabolism (P = 0.000). Positive correlations were noted in 15 pairs metabolites i.e. between delta2-THA and (22E)-3 beta-hydroxy-5 alpha-chola-7,22-dien-oic acid (r = 0.935, P < 0.01), and negative correlations were seen in 9 pairs metabolites i.e. between lysoPE and aminovaleric acid betaine (r = -0.500, P < 0.05). Conclusion Asymptomatic hookworm infection in middle-aged and elderly people in rural area of Huangshan City may affect the composition and metabolism of intestinal microflora, which may create a symbiotic and colonization environment for probiotics
Antimalarial drug resistance presents the biggest challenge for treatment against malaria. Plasmodium parasites have developed different degrees of resistance to common traditional antimalarial drugs including artemisinin. Therefore, the improvement of traditional drugs and the research and development of new drugs are urgently needed. This paper discusses the malaria control strategies based on a systematic review of the resistance mechanisms against traditional antimalarial drugs, the improvement strategies and optimisation achievements based on traditional drugs, and the research advances of new antimalarial drugs.
Objective This study aimed to determine the complete mitochondrial genome sequence of Schistosoma japonicum featured with nocturnal cercarial emergence pattern and to explore its phylogenetic relationship with other S. japonicum geographical isolates from mainland China, providing new reference data for further study on population genetic structure and genetic diversity. Methods Field Oncomelania hupensis snails were collected from Shitai County of Anhui in 2020, and the infected snails were separated in the laboratory. ICR mice were infected with cercariae shed from the infected snails using abdominal patch method. The worms were obtained by perfusion from hepatic portal and mesenteric vein and randomly sampled for sequencing. The worm genomic DNA was extracted with blood/tissue DNA extraction Kit, and sequenced at paied-end using Illumina NovaSeq 6000 sequencing platform. The mitochondrial genome was assembled using GetOrganelle tool, and its structural features and base composition were analyzed using MEGA 11 software. The phylogenetic tree was constructed using the neighbour-joining (NJ) and maximum likelihood (ML) method based on 17 published mitochondrial genome sequences of S. japonicum from other parts of China. Results The complete circular mitochondrial genome of S. japonicum with nocturnal cercarial emergence pattern isolated from Shitai County of Anhui was 14 085 bp in length(Genbank accession no. ON637109), encoding 36 genes, consisting of 12 protein-coding genes (PCGs), two ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes, with the AT content of 71.35%. There were two gene-overlapping regions and 29 intergenic regions in the mitochondrial genome. Among the 22 tRNA genes, except for trnaC and trnaS1 genes, which missed the dihydrouridine (DHU) arm, the rest were able to form a typical cloverleaf secondary structure. Among the PCGs, the stop codon of cytb, nad4, nad2 and nad6 was TAA, which differed from other geographical strains in China with TAG as the stop codon. Phylogenetic trees constructed based on NJ and ML had similar topologies. The isolated strain of S. japonicum featured with “nocturnal cercarial emergence” in Shitai County, forms a single strain. S. japonicum strains from Taiwan, China and other regions of Chinese Mainland are separated to form one branch. Conclusion The stop codons of S. japonicum isolate from Shitai area of Anhui Province differed from those of other geographical isolates in China. Additionally, based on the phylogenetic analysis, the isolate featured with nocturnal cercarial emergence exhibited a more distant relationship with other regional isolates, showing greater
Objective To explore the N6 adenylate methylation (m6A) modification of circular RNA (circRNA) in male and female adult worms of Schistosoma japonicum, and predict its possible role in regulating miRNA. Methods S. japonicum cercariae (100 ± 2) were used to infect mice through abdominal skin for 20 min. On 28 d post-infection, the mice were anesthetized to collect adult worms using hepatic portal vein perfusion method, of which the female and male worms were separated for extracting total RNA, respectively. The RNA samples of 0.5 μg and 1.0 μg were applied by dotting on the nylon membrane, which was transferred to a UV-crosslinker for dot hybridization through successive incubation with m6A antibody (1∶1 000) and HRP labeled secondary antibody (1∶5 000). Immunoprecipitation separation was performed for 1 µg of total RNA from both female and male worms using RNA methylation co-precipitation kit. The RNA library kit was used for library construction. After quality testing with a biological analyzer, sequencing was performed using a 150 bp double-ended mode on a high-throughput sequencer. The Cutadapt software (v1.9.3) was applied to remove joints and low-quality sequences and obtain high-quality sequences. The sequence was aligned with the Schistosoma genome (Sja_WBPS14) using Bowtie2 software, and the Find_circ software was used to identify circRNA, while MACS software was used to recognize methylation sites. Cluster analysis of differentially expressed circRNAs was conducted using Heatmap2 software, and gene ontology (GO) enrichment analysis was performed for the source genes of statistically significant differentiated m6A modified circRNA. DiffReps software was used to analyze the differences in m6A modified circRNA between male and female worms. Using RNAhybrid software, the interaction between miRNA and circRNA was predicted. The reverse-transcribed miRNA was analyzed by qPCR, and all the relative expression level of miRNA was calculated with 2-ΔCt method. Results The dot blot hybridization results showed that there was m6A methylation modification in the total RNA of S. japonicum, and the m6A methylation signal tended to increase with the increase of RNA content. The high-throughput sequencing results showed that the proportion of high-quality sequences in S. japonicum was over 99.9%, and the RNA enrichment and library construction were effective. CircRNAs originating from overlapping regions account for 49%, followed by exons (29%) and intergenes (12%), with fewer circRNAs originating from introns (2%) and antisense chains (8%). Genes derived from highly enriched circRNAs in males may be related to cellular metabolic processes, cellular components, tissue biogenesis, and stress, while genes derived from highly enriched circRNAs in females may be involved in processes such as cell molecule binding. The number of m6A-modified circRNAs enriched in males was higher than that in females (P < 0.05, fold change ≥ 2) with 57 high abundance m6A-modified circRNAs in females and 81 in males. M6A-modified circRNAs in both male and female insects can potentially interact with multiple miRNAs, such as Sja-Bantam and Sja-miR-307, which are highly expressed in females, and Sja-miR-8-3p, Sja-miR-3504 and so on, which are highly expressed in males. Conclusion This study obtained the expression profile of m6A modified circRNA in adult worms of S. japonicum, and preliminarily revealed the miRNA that may be regulated by m6A modified circRNA.
Objective To investigate the role and mechanisms of CD73/adenosine/adenosine A2A receptor (A2AR) pathway in suppression of macrophage inflammatory response of Echinococcus granulosus. Methods Healthy C57BL/6 mice were intraperitoneally injected with aseptic starch broth (0.5 ml per mouse). Ascitic fluid was extracted from the mice on 3 d post-injection to separate macrophages, which were inoculated into a 6-well plate at a dose of 1 × 106/ml. After the cells were attached to the plate wall, E. granulosus cyst fluid was added at a final concentration of 0.8 mg/ml, and then cultured for 0 h, 6 h, 12 h, 18 h and 24 h to examine the relative transcriptional levels of CD73, A2AR, tumor necrosis factor-α (TNF-α) and arginase 1 (Arg-1) by qRT-PCR. The expression of CD73 was detected by flow cytometry, and the expression of extracellular signal-regulated kinase 1 (ERK1/2) and phosphorylated ERK1/2 (p-ERK1/2) was detected by Western blotting. The separated macrophages were inoculated onto a 6-well plate with 1 × 106 cells per well, and assigned to three groups: cyst fluid group, drug administration group and control group. Each group had 3 wells, to which E. granulosus cyst fluid (final concentration 0.8 mg/ml), E. granulosus cyst fluid (final concentration 0.8 mg/ml) and drugs (adenosine receptor inhibitor SCH58261, final concentration 50 μmol/L) and the same amount of medium was added, respectively. After 24 hours of culture, the relative transcription levels of TNF-α and Arg-1 were detected by qRT-PCR. The expression of ERK1/2 and p-ERK1/2 was detected by Western blotting. Thirty-six healthy C57BL/6 female mice aged 6-8 weeks were selected. 5 000 protoscolex of E. granulosus (20 μl per mouse) were injected under the liver capsule in the infection group, and the healthy group were not treated. One month later, the livers of 6 mice in each group were selected and embedded in paraffin to prepare slices. HE staining was used to observe liver tissue lesions, and the expression of A2AR was detected by immunohistochemical staining. The expression of CD73 in the proximal and distal vesicles was detected by flow cytometry. Infected mice were taken, and 1 mg/(kg•d) adenosine receptor inhibitor (SCH58261) and an equivalent amount of PBS were intraperitoneally injected into the treatment group (8 mice) and solvent group (8 mice), respectively. Healthy mice (8 mice) were not treated. After 22 days, the mice’s weight, liver, spleen, kidney and heart were weighed, and the ratio of each organ to body weight was calculated. After the mouse liver was embedded in paraffin, the sections were prepared, and the infiltration of inflammatory cells around the vesicles was observed by HE staining. Results The relative transcription level of CD73 mRNA in 18 h group (1.66 ± 0.17) and 24 h group (2.01 ± 0.15) was higher than that in 0 h group (1.00 ± 0.09) after 0 h, 6 h, 12 h, 18 h and 24 h treatment of macrophages with E. granulosus cyst solution (t = 3.35, P < 0.05; t = 5.83, P < 0.01). Flow cytometry showed that the percentage of CD73+ macrophages in 18 h group was (2.74 ± 0.43)%, which was higher than that in 0 h group (1.53 ± 0.10)% (t = 4.72, P < 0.01). The relative transcription levels of A2AR mRNA were 0.29 ± 0.03, 1.00 ± 0.14, 1.02 ± 0.02, 0.72 ± 0.08, 1.03 ± 0.03, respectively. All groups at 6 h, 12 h, 18 h and 24 h were higher than those in 0 h group (t = 4.84, 17.55, 5.21, 15.26; all P < 0.01). The western blotting results showed that the relative expression levels of A2AR protein in each group were 1.00 ± 0.00, 1.22 ± 0.05, 1.32 ± 0.02, 1.40 ± 0.05, and 1.46 ± 0.04, respectively. The relative expression levels of other groups were higher (t = 5.89, 18.35, 9.14, 15.06; all P < 0.01). Western blotting showed that the relative expression levels of A2AR protein in 6, 12, 18 and 24 h groups were 1.22 ± 0.05, 1.32 ± 0.02, 1.40 ± 0.05, and 1.46 ± 0.04，0.04, respectively. All of them were higher than those in the 0 h group (1.00 ± 0.00) (t = 5.89, 18.35, 9.14, 15.06; all P < 0.01). The relative transcription levels of TNF-α mRNA in the 6, 12 and 18 h groups were 1.00 ± 0.04, 0.31 ± 0.03, 0.12 ± 0.01, 0.05 ± 0.01, respectively, which were higher than those in the 0 h group (0.01 ± 0.00) (t = 22.37, 11.33, 11.48; all P < 0.01). The relative transcription level of Arg-1 mRNA in 18 h group (0.69 ± 0.09) and 24 h group (2.10 ± 0.07) was higher than that in 0 h group (0.004 ± 0.00) (t = 7.61, 28.64; both P < 0.01). Western blotting results showed that the relative expression of p-ERK1/2 protein in the 6 h group (3.07 ± 0.71), 12 h group (1.68 ± 0.18) and 18 h group (1.43 ± 0.14) was higher than that in the 0 h group (1.00 ± 0.00) (t = 4.15, 5.40, 4.50; all P < 0.05). The 6 h group was higher than the 18 h and 24 h groups (0.97 ± 0.34) (t = 3.23, 3.80; both P < 0.05). The relative transcription levels of TNF-α mRNA were 0.85 ± 0.05 and 1.56 ± 0.13 in the capsule fluid group and administration group, respectively, and the difference was statistically significant (t = 5.13, P < 0.01). The relative transcription level of Arg-1 mRNA in the capsule fluid group was 147.73 ± 10.06, which was higher than that in the drug administration group (13.94 ± 1.00) (t = 13.23, P < 0.01) and control group (59.59 ± 9.82) (t = 6.27, P < 0.01). The relative transcription level in the administration group was lower than that in the control group, and the difference was statistically significant (t = 4.62, P < 0.01). Western blotting results showed that the relative protein expression of P-ERK1/2 in the administration group (2.08 ± 0.38) was higher than that in the capsule fluid group (0.94 ± 0.29) and control group (1.00 ± 0.00) (t = 3.42, 4.04; both P < 0.05). The HE staining showed that inflammatory cell infiltration zones appeared in the liver tissue of infected mice. The immunohistochemical staining results showed that A2AR was positive in the infection group. The results of flow cytometry showed that the percentage of macrophages with CD73+ in the proximal vesicle was (12.31 ± 0.04)%, which was higher than that at the distal vesicle (5.95 ± 2.36)% (t = 3.81, P < 0.05). After administration, the ratio of weight of liver, spleen, kidney and heart to body mass in the healthy group, solvent group and administration group was 6.13 ± 0.66, 5.90 ± 0.48, 5.47 ± 0.87, 0.44 ± 0.18, 0.41 ± 0.29, 0.33 ± 0.10. There was no significant difference between 0.68 ± 0.03, 0.64 ± 0.05, 0.60 ± 0.09, 0.99 ± 0.15, 0.77 ± 0.13, 0.78 ± 0.19 (F = 0.95, 0.42, 1.46, 2.02; all P > 0.05). HE staining showed that perivascular inflammatory cells increased in the drug administration group compared with the solvent group. Conclusion E. granulosus evades host immunity by inducing macrophages to express CD73 and A2AR and thereby promoting the secretion of its inflammatory inhibitory factors.
Objective To observe the morphological characteristics of adult Histiostoma feroniarum under the electron microscope, and to study the genetic relationship between this species and other mite species of Acaridae. Methods The adult stage of H. feroniarum was isolated from the culture, and its external morphological features were observed using scanning electron microscopy (SEM), followed by extracting the mite DNA, for amplifying the cytochrome oxidase subunit 1 (cox1) and internal transcribed spacer (ITS) by PCR. The ITS and cox1 gene was sequenced and compared with the other 8 Acaridae species in GenBank. The phylogenetic tree was constructed by the maximum likelihood method. Results Under the electron microscope, the ventral epidermal protrusions of the mites were more developed. The epidermal protrusions of foot I healed into the chest plate, and the epidermal protrusions of foot Ⅱ extended to the centre and were not connected. There were 2 pairs of chitinous rings on the ventral surface, and the male adults were located between the Ⅱ-Ⅳ basal ganglia. The first pair of chitin rings of the female mite was located between the foot Ⅱ and Ⅲ, and the last pair of chitin rings were located at the level of the foot Ⅳ basal ganglia. Compared with the light microscope, the structures of male reproductive folds, leaflets and penis were clearer under the electron microscope. The cox1 and ITS sequences were obtained by PCR amplification, which was 539 bp and 1 633 bp. The ITS sequence similarity comparison results showed that the sequence similarity between H. feroniarum and Blomia tropicalis (GenBank accession no. KC215362) was the highest, which was at 90.41%, and the sequence similarity with other Acaridae species was 83.33%-89.73%. The cox1 sequence alignment showed that the highest sequence similarity was 83.55% between H. feroniarum and Lepidoglyphus destructor (GenBank accession no. MT075728), and the sequence similarity with other Acaridae species ranged from 79.78%-82.12%. The phylogenetic tree constructed based on ITS and cox1 sequences has differences in topological structure. showing that H. feroniarum is a single branch, but not at the same branch of other Acaridae species, which is likely consistent with morphological classification. The phylogenetic analysis based on ITS showed that H. feroniarum and Rhizoglyphus robini were clustered as a branch, which differs from morphological classification. Conclusion The morphological structure of male and female H. feroniarum could be clearly observed by scanning electron microscopy. Phylogenetic analysis of cox1 sequence reveals relatively distant genetic relationship seen between the H. feroniarum and other acaroid mites.
The individual malaria case investigation forms in 31 provinces/municipalities/autonomous regions (excluding Taiwan, Hong Kong and Macao) and Xinjiang Production and Construction Corps of China in 2023 were collected and sorted from “The Information System for Infectious Disease Surveillance”. The epidemiological characteristics were analyzed. In 2023, 2 488 malaria cases were reported, showing an increase of 194.4% compared to 2022 (845). Among them, there were 2 487 imported cases, 1 case of blood transfusion infection; 1 561 falciparum malaria cases (62.7%, 1 561/2 488), 615 vivax malaria (24.7%, 615/2 488), 234 ovale malaria (9.4%, 234/2 488), 66 malariae malaria (2.7%, 66/2 488) and 12 cases with mixed-infection (0.5%, 12/2 488). In the reported, 2 313 cases were of Chinese nationality (93.0%, 2 313/2 488) and 175 cases foreign nationality (7.0%, 175/2 488); the male-to-female ratio was 11.6∶1; the highest number of cases (29.1%, 725/2 488) was seen at the age group of 30-39 years showed. The malaria cases were reported from all the 31 provinces/municipalities/autonomous regions and Xinjiang Production and Construction Corps, with the top 5 provinces of Yunnan (398 cases), Henan (234 cases), Guangxi (195 cases), Shandong (178 cases) and Guangdong (174 cases) accumulately, a total of 1 179 cases (47.4%, 1 179/2 488) reported. Of the reported, 85 were severe cases (3.4%, 85/2 488) and 12 deaths (0.5%, 12/2 488). Although no local transmitted primary malaria cases have been reported in China for seven consecutive years, the risk of imported cases and re-transmission. It is imperative to continuousely strengthen the surveillance and response, find the cases timely and deliver standardized treatment, so as to reduce severe cases and death, preventing local re-transmission.
Objective To identify the microRNA (miRNA) of the Strongyloides stercoralis infective third-stage larvae (iL3) and parasitic female adult (pF), and analyze the differential expression of miRNA and the function of their target genes. Methods The iL3 was collected from the feces of dogs infected with S. stercoralis. Healthy dogs were subcutaneously injected with 4 000-5 000 iL3 at the back of the neck. The pF was collected from the dog intestine 21 days post-infection. The total RNA of iL3 and pF was extracted and sequenced. The sequencing data was aligned with the S. stercoralis genome and S. ratti miRNA to identify the conserved miRNA. The true miRNA sequences were screened out using miRDeep2 through loop structure analysis and mature sequence readings alignment. The miRNA expression profiles of iL3 and pF were analyzed to screen the differentially expressed miRNA, and predict their target genes. Functional analysis was performed to those of unannotated miRNAs target genes with the values of log2(Fold chang) > 1 and transcripts per million (TPM) > 1 000. GO enrichment analysis of differentially expressed miRNA target genes were analyzed using ClusterProfiler. Eleven unannotated or conserved differentially expressed miRNAs were selected for real-time fluorescent quantitative PCR (qRT-PCR) verification. The relative transcription level was calculated with the glyceraldehyde-3-phosphate dehydrogenase gene of S. stercoralis (GenBank accession number: BI773092.1) as reference gene. Results A total of 265 miRNA were identified, including 130 conserved miRNAs and 135 novel miRNAs, of them 134 were differentially expressed between iL3 and pF (77 conserved and 57 unannotated miRNAs), 251 miRNAs were shared by iL3 and pF, 10 were specific in iL3 and 4 were specific in pF. The prediction results of differentially expressed miRNA target genes showed that of the 57 unannotated miRNA, 12 miRNA with fold chang > 2 and TPM > 1 000 have 248 target genes homologous with Caenorhabditis elegans. 35.08% (87/248) of the target genes were related to development, while the rest were related to stress, exercise and molting. Most of the highly expressed unannotated miRNA in pF target SSTP_0000114700. GO analysis of differentially expressed miRNA target genes showed that a total of 5 595 target genes were enriched, among which the top 5 enriched components were membrane components (1 821), nucleic acid binding (561), nucleus (450), proteolysis (365) and signal transduction (284). The qRT-PCR analysis showed that the log2(fold change) of sst-miR-86-5p, sst-84-5p, sst-novel-104, sst-miR-92-3p, sst-miR-34a-3p, sst-miR-81a-5p, sst-miR-1-3p, sst-novel-108, sst-miR-124-5p, sst-miR-50-3p, sst-novel-51 were -2.12, 6.39, 4.46, -3.69, -3.69, 2.34, -2.48, -2.41, -2.30, 2.25, -3.32; and the log2(fold change) obtained from transcriptome analysis were -3.05, 4.98, 4.07, -4.9, -3.66, 0.98, -3.79, -2.61, -0.99, 0.63, -1.55, respectively. The upregulation and downregulation trends obtained from qRT-PCR and transcriptome analysis are consistent. Conclusion The miRNA expression profiles and differentially expressed miRNAs of S. stercoralis infective third-stage larvae and parasitic female adult are identified. The differentially expressed miRNAs are associated with the growth, development, and reproductive function of S. stercoralis.
