Third-stage Larvae Research Articles

EXPERIMENTAL TOXOCARIASIS IN RABBITS

Toxocariasis as a zoonosis represents a medical and veterinary problem. In the structure of human larval helminthiases, toxocariasis accounts for more than 60%. In non-specific hosts, including humans, toxocara larvae undergo visceral larva migrans, primarily damaging the liver, lungs, and other organs and tissues. Constant reinvasion accumulates second and third-stage larvae in the bodies of nonspecific (paratenic) hosts. In our research, we considered the problem of soil parasitological contamination on rabbit farms and the risks of rabbit infection, emphasising the need to study the involvement of rabbits as paratenic hosts in toxocariasis. We selected rabbits of the Soviet chinchilla breed as experimental animals. Toxocara eggs were administered orally to the animals. Hematological studies were conducted using commonly accepted methods. To detect specific IgG class antibodies, rabbit serum was analysed by enzyme-linked immunosorbent assay (ELISA). The presence of IgG class antibodies in the blood serum of rabbits at a positive titer (1:100) in ELISA allows us to conclude that rabbits, along with other animals, serve as paratenic hosts for Toxocara canis. Haematological research results indicated the absence of significant differences and remained within the physiological norm throughout the entire experiment.

Growth Performance and Ruminal Fermentation in Lambs with Endoparasites and In Vitro Effect of Medicinal Plants

We investigated growth performance and ruminal fermentation associated with gastrointestinal nematode (GIN) Haemonchus contortus in lambs and in vitro ruminal fermentation of mallow, chamomile, fumitory, wormwood (Herbmix), and chicory using inoculum from GIN-infected lambs. Twelve lambs were equally divided into two groups: uninfected animals (CON) and animals infected (INF) with approximately 5000 third-stage larvae derived from the MHCo1 strain of GIN H. contortus. Two lambs per group were killed on days 48, 49, and 50 after infection and ruminal content was collected separately from each lamb. Batch cultures of ruminal fluid from CON and INF were incubated for 24 h in vitro with 0.25 g meadow hay, Herbmix, and chicory using an in vitro gas production technique. Daily weight gain was relatively lower in the INF than the CON group, but not significantly (72.6 vs. 130.1 g/day). The ruminal populations of protozoa, bacteria, total Archaea, Methanobacteriales, and Methanomicrobiales were significantly higher in the INF than in the CON group. The substrates affected the concentrations of n-butyrate, iso-butyrate, n-valerate, iso-valerate, ammonia-N, total gas, and methane (p < 0.001) in vitro. GIN infection affected fermentation and microbial population in the rumens of the lambs, and chicory was a promising substrate to modulate ruminal fermentation in vitro.

Efficacy of a new fixed-dose combination injectable (0.2 mg/kg doramectin + 6.0 mg/kg levamisole hydrochloride) in Australian cattle against artificial infections of gastrointestinal nematodes

We describe a new fixed-dose combination injectable (FDCI) formulated with doramectin and levamisole hydrochloride (HCl) to target broad and overlapping spectra of gastrointestinal nematodes (GINs) through two distinct modes of action. Here, we demonstrate the superior efficacy of the FDCI against mixed populations of cattle GINs in two dose confirmation studies conducted in Australia using artificially induced adult (Study 1) and immature (Study 2) GIN infections. Artificial infections consisted of Cooperia spp., Haemonchus placei, Ostertagia ostertagi, and Trichostrongylus axei. In both studies, cattle were inoculated with third-stage larvae and infections were confirmed by fecal egg count (FEC). Treatment groups in both studies were as follows: (1) negative control (saline, 0.9% sodium chloride), (2) positive control injectable endectocide (Study 1–0.2 mg/kg ivermectin; Study 2–0.2 mg/kg doramectin), (3) positive control injectable anthelmintic (7.5 mg/kg levamisole HCl), and (4) FDCI (0.2 mg/kg doramectin + 6.0 mg/kg levamisole HCl). Cattle were treated either 28 days (Study 1) or 6 days (Study 2) post-infection. On Days 14–16 (Study 1) or Days 20–21 (Study 2) post-treatment, cattle were euthanized and necropsied for the recovery, identification, and enumeration of worms. Treatment efficacy was calculated as reduction in worm burdens of treated cattle compared to saline-treated cattle, and treatments were considered effective if the geometric mean worm burden in the treatment group was reduced by ≥ 95% compared to the negative control group. In both studies, saline-treated cattle remained positive for GIN infections for the study duration. Ivermectin was less than 95% effective against Cooperia spp. (80.2%) and H. placei (24.8%) in Study 1, and levamisole HCl was less than 95% effective against Ostertagia spp. (47.1%) in Study 2. In contrast, the novel FDCI was 100% effective in treating adult and immature life stages of all cattle GINs included in the artificial infections, with no worms recovered at necropsy from doramectin + levamisole HCl-treated cattle. These data show a single administration of the FDCI provides broad-spectrum treatment of economically important cattle GINs.

Therapeutic Management of Canine Dirofilariasis in Nagaland, India

Dirofilariasis is one of the most clinically important diseases in dogs. It is also called as heart-worm disease, which is a non-contagious parasitic disease caused by filarial or small thread-like worms, Dirofilaria immitis, and D. repens of the family Onchocercidae (Nelson et al., 2014; Adebayo et al., 2022). It is an arthropod-borne disease. The mosquitoes belonging to the genera Culex, Aedes, Anopheles, and Mansonia are principally responsible for the transmission of this disease (Borthakur et al., 2016). Transmission of the parasite occurs when a potential vector bites dogs during a blood meal. It takes about 6-7 months for the third-stage larvae (L3) to become adult parasites after entering the blood vessels of the hosts (Koutsu et al., 2022). After maturation, the adult worms reproduce and produce microfilariae (pre-L1), which are released in the host blood vessels and taken up by a feeding mosquito (Thilakarathne et al., 2023), and completed the lifecycle of the parasite.

Study on Morphological Changes and Interference in the Development of Aedes aegypti Caused by Some Essential Oil Constituents.

Dengue, Chikungunya and Zika are arboviruses, transmitted by the mosquito Aedes aegypti, that cause high mortality and serious health consequences in human populations. Efforts to control Ae. aegypti are important for preventing outbreaks of these diseases. Essential oil constituents are known to exhibit many activities, such as their use as larvicides. Given their potential, the present study aimed to characterize the larvicidal effect of dihydrojasmone, p-cymene, carvacrol, thymol, farnesol and nerolidol on the larvae of Ae. aegypti and their interference over the morphology of the mosquitos. The essential oil constituents were dissolved in dimethylsulfoxide at concentrations of 1-100 μg/mL and were applied in the breeding environment of third-stage larvae. The larvae from bioassays were fixed, dehydrated and embedded. Ultrathin sections were contrasted using 5% uranyl acetate and 1% lead citrate for observation through transmission electron microscopy. The oil with the highest larvicidal efficiency was found to be nerolidol, followed by farnesol, p-cymene, carvacrol, thymol and dihydrojasmone, with an LC50 of 11, 21, 23, 40, 45 and 66 µg/mL, respectively. The treated Ae. aegypti larvae caused alteration to the tegument or internal portions of larvae. The present study demonstrated which of these oils-dihydrojasmone, farnesol, thymol, p-cymene, carvacrol and nerolidol-have effective larvicidal activity.

Occurrence and prevalence of third-stage larvae of the Anisakidae family in Macrouridae species captured from bathyal depths of Sagami Bay, Japan

Anisakiasis is the prevailing form of foodborne illness in Japan, with Anisakidae larvae, specifically Anisakis simplex sensu stricto, serving as its causative agents. These larvae are commonly found in mackerel, sardines, and skipjacks retailed at fresh fish establishments. While the infection rates of Anisakidae larvae in these surface-dwelling fish have been extensively studied, their prevalence in deep-sea fish species remains inadequately understood. This investigation aims to determine the infection rates of Anisakidae larvae in three Macrouridae species—namely Coryphaenoides acrolepis, Coelorinchus japonicus, and Coelorinchus macrochir—which represent the most significant inhabitants of the deep-sea realm. Employing artificial digestion techniques, we successfully retrieved all internal contents of the fish, including the nematode larvae. Some Anisakidae larvae were morphologically identified as A. simplex sensu lato and further identified to species by PCR-RFLP analysis. Two larvae showed a band pattern that appeared to be a hybrid between A. simplex sensu stricto and A. berlandi, which after sequence analysis was considered to be an intraspecific variation of A. berlandi. On the other hand, species considered to be unknown were sequenced and phylogenetic analysis was carried out along with identified larvae. The findings indicate that A. simplex sensu stricto was notably more abundant in C. acrolepis, obtained at a depth of 1000 m. In C. japonicus and C. macrochir, captured at a depth of 400 m, a considerable number of Skrjabinisakis paggiae and Pseudoterranova ceticola, were detected in the deep-sea dwelling Kogia breviceps and K. sima as definitive hosts, were detected. Notably, a single C. macrochir harbored seven species of Anisakidae larvae, with an infection tally of nearly 200 individuals of S. paggiae. Thus, a substantial prevalence of Anisakidae larvae in the deep-sea-dwelling Macrouridae species potentially plays a significant role in the transmission pathway to cetaceans as their final hosts.

Supplementation with Rumen-Protected Methionine Reduced the Parasitic Effect of Haemonchus contortus in Goats.

The present study investigated the impact of rumen-protected (RP) methionine supplementation on the resistance and resilience to Haemonchus contortus experimental infection of goat kids. Twenty-seven 6-month-old goat kids (14.55 ± 2.7 kg body weight) were placed in individual pens during an experimental period of forty-two days. Each kid was placed under one of three distinct diets (n = 9 animals/diet) corresponding to the following experimental groups: Control (C, Hay + concentrate), Low Methionine (LM, Hay + concentrate + 3.5 g/Kg of Dry Matter (DM) of RP methionine, or High Methionine (HM, Hay + concentrate + 11.5 g/Kg of DM of RP methionine). After a 4-week period of adaptation to the diets, all the animals were experimentally infected with a single oral dose of 10,000 H. contortus third-stage infective larvae (L3). No significant effect of RP methionine supplementation was observed for feed intake, digestibility and growth performance. The faecal egg counts (FEC) and worm burdens were not impacted by RP methionine supplementation either. In contrast, Packed cell volume (PCV) and haemoglobin concentration were higher in kids supplemented with RP methionine. Similarly, the level of serum IgA directed against adult H. contortus excretion and secretion products (ESP) was higher in supplemented kids. These results suggested that RP methionine supplementation improved goat kids' resilience against H. contortus infection.

Chemical composition, larvicidal and molluscicidal activity of the essential oil Alpinia zerumbet

In this present study, we describe the chemical composition, larvicidal and molluscicidal activity of essential oil distilled from Alpinia zerumbet leaves against third-stage larvae of Ae.aegypti and the snail Biomphalaria glabrata, respectively. For this, we distilled the oil by hydrodistillation, identified its components by gas chromatography coupled to mass spectrometry and gas chromatography with flame ionization detection and calculated the lethal concentration of larvicidal and molluscicidal activity using the probit method. From these analyses, we showed that the oil is predominantly composed of monoterpenes, in which p-cymene (34.12%), 1,8-cineole (25.67%) and terpinen-4-ol (21.01%) stood out. In addition, we also proved that the oil has larvicidal and molluscicidal activity with lethal concentrations respectively of 43.74 and 36.52 μg/mL. Therefore, we conclude that the oil is active and has the potential to replace synthetic larvicides and molluscicides.

Rat lungworm survives winter: experimental overwintering of Angiostrongylus cantonensis larvae in European slugs.

The rat lungworm Angiostrongylus cantonensis is a metastrongyloid nematode that causes neurological disorders in its accidental hosts, including humans. This invasive pathogen is native to Southeast Asia and adjacent regions and is gradually expanding its distribution to tropical and subtropical areas with new foci discovered near temperate regions. The parasite has a complex life cycle with a range of gastropods serving as intermediate hosts. A broad spectrum of poikilotherm vertebrates and invertebrates can serve as paratenic hosts. Since it has already been demonstrated that other, non-zoonotic metastrongyloids can survive in their intermediate hosts during the winter, the aim of our study was to evaluate the survival of A. cantonensis third-stage larvae in experimentally infected slugs (Limax maximus) kept at 4.5–7°C for 60 days. Third-stage larvae of A. cantonensis survived the period of low temperature and remained capable of infecting definitive hosts (laboratory rats) afterwards, even though their numbers dropped significantly. These results suggest that further spread to higher latitudes or altitudes is possible in areas with sufficient abundance of definitive hosts, since low winter temperatures are not necessarily an obstacle to the spread of the parasite.

A study implementing real-time PCR to identify Strongyloides species of third-stage larvae in human stool samples from Southern Vietnam

BackgroundStrongyloidiasis, a neglected disease caused by intestinal nematodes of the genus, is endemic to tropical and subtropical areas such as Vietnam. Morphological methods only identify the genus, while DNA-molecular techniques are susceptible in Strongyloides spp. detection. The study aims to determine the prevalence of dominant Strongyloides species among the population in Duc Hoa district, Long An, Vietnam. MethodsA cross-sectional study used 1190 stool specimens collected from July 2017 to November 2018. All samples were transported within 2 h, stored at 2–8°C, and processed within 48 h for microscopy smear and culture at the Laboratory of Medical Parasitology, Pham Ngoc Thach University of Medicine (PNT). Then all positive samples with the above 2 methods were verified by real-time PCR technique. Real-time PCR amplification was conducted at the Laboratory of Molecular Biology, PNT. ResultsDirect microscopy and modified Harada-Mori culture detected Strongyloides spp. larvae in 79/1190 samples (6.6%). About 94.2% of the DNA samples were Strongyloides stercoralis, 2.9% were co-infections with Strongyloides ratti and S. stercoralis, and 2.9% were patients with S. ratti. The identity of 12/14 sequences was confirmed as S. stercoralis with a high level of similarity (91.3%–100%) and over 98% for S. ratti. ConclusionDNA-molecular techniques and sequence analysis are highly suitable for identifying Strongyloides species isolated from stool samples. It is remarkable evidence of the presence of zoonosis S. ratti disease in human, not just the known S. stercoralis. It is likely to result in a certain proportion of people being infected by this animal-borne infectious pathogen.

Regulation of immune response against third-stage Gnathostoma spinigerum larvae by human genes.

Gnathostomiasis is an important zoonosis in tropical areas that is mainly caused by third-stage Gnathostoma spinigerum larvae (G. spinigerum L3). This study aimed to prove whether G. spinigerum L3 produces extracellular vesicles (EVs) and investigate human gene profiles related to the immune response against the larvae. We created an immune cell model using normal human peripheral blood mononuclear cells (PBMCs) co-cultured with the larvae for 1 and 3 days, respectively. The PBMCs were harvested for transcriptome sequencing analysis. The EV ultrastructure was examined in the larvae and the cultured medium. Extracellular vesicle-like particles were observed under the larval teguments and in the pellets in the medium. RNA-seq analysis revealed that 2,847 and 3,118 genes were significantly expressed on days 1 and 3 after culture, respectively. The downregulated genes on day 1 after culture were involved in pro-inflammatory cytokines, the complement system and apoptosis, whereas those on day 3 were involved in T cell-dependent B cell activation and wound healing. Significantly upregulated genes related to cell proliferation, activation and development, as well as cytotoxicity, were observed on day 1, and genes regulating T cell maturation, granulocyte function, nuclear factor-κB and toll-like receptor pathways were predominantly observed on day 3 after culture. G. spinigerum L3 produces EV-like particles and releases them into the excretory-secretory products. Overall, genotypic findings during our 3-day observation revealed that most significant gene expressions were related to T and B cell signalling, driving T helper 2 cells related to chronic infection, immune evasion of the larvae, and the pathogenesis of gnathostomiasis. Further in-depth studies are necessary to clarify gene functions in the pathogenesis and immune evasion mechanisms of the infective larvae.

Quantifying metabolic activity of Ascaris suum L3 using resazurin reduction

BackgroundHelminth infections are an important public health problem in humans and have an even greater impact on domestic animal and livestock welfare. Current readouts for anthelmintic drug screening assays are stage development, migration, or motility that can be subjective, laborious, and low in throughput. The aim of this study was to apply and optimize a fluorometric technique using resazurin for evaluating changes in the metabolic activity of Ascaris suum third-stage larvae (L3), a parasite of high economic relevance in swine.MethodsAscaris suum L3 were mechanically hatched from 6- to 8-week embryonated and sucrose-gradient-enriched eggs. Resazurin dye and A. suum L3 were titrated in 96-well microtiter plates, and resazurin reduction activity was assessed by fluorometry after 24 h of incubation. Fluorescence microscopy was used to localize the resazurin reduction site within the larvae. Finally, we exposed A. suum L3 to various stress conditions including heat, methanol, and anthelmintics, and investigated their impact on larval metabolism through resazurin reduction activity.ResultsWe show that the non-fluorescent dye resazurin is reduced inside vital A. suum L3 to fluorescent resorufin and released into the culture media. Optimal assay parameters are 100–1000 L3 per well, a resazurin concentration of 7.5 µg/ml, and incubation at 37 °C/5% CO2 for 24 h. An intact L2 sheath around the L3 of A. suum completely prevents the uptake of resazurin, while in unsheathed L3, the most intense fluorescence signal is observed along the larval midgut. L3 exposed to methanol or heat show a gradually decreased resazurin reduction activity. In addition, 24 h exposure to ivermectin at 0.625 µM, mebendazole at 5 µM, and thiabendazole from 10 to 100 µM significantly decreased larval metabolic activity by 55%, 73%, and 70% to 89%, respectively.ConclusionsTogether, our results show that both metabolic stressors and anthelmintic drugs significantly and reproducibly reduce the resazurin reduction activity of A. suum L3, making the proposed assay a sensitive and easy-to-use method to evaluate metabolic activity of A. suum L3 in vitro.Graphical

An initial investigation of the predatory activity of the phoretic mites of dung beetles, Macrocheles sp. (Mesostigmata: Macrochelidae), on the gastrointestinal nematode of sheep Haemonchus contortus (Strongylida: Trichostrongylidae)

Worldwide increasing levels of anthelmintic resistance are pushing farmers and health advisors towards a more sustainable management of gastrointestinal nematodes (GIN), one of the greatest threats to the viability of grazing small ruminant production systems, and to these animals’ welfare. These Strongyloidea develop freely in livestock dung, where they are exposed to predatory organisms that can eventually break their cycle. The aim of this study was to investigate the effect of phoretic mites of dung beetles Macrocheles sp. (Macrochelidae) on the gastrointestinal nematode Haemonchus contortus (Rudolphi, 1803) that has a free-living larval stage that evolves in sheep feces. Direct observations under a stereomicroscope confirmed that the studied Macrocheles mites may have an active predatory behavior on the third-stage (L3) larvae of H. contortus. In order to quantify the effects of the mites’ predation on the survival of L3 nematode larvae, we designed experimental units, replicated across five trials, and containing free-living stages of the nematode either in the presence or absence of mites. The number of H. contortus larvae that reached the third-stage was significantly reduced in the presence of mites, despite a variable intensity of the predatory effects between trials. This is the first report of a predatory activity of phoretic mites of dung beetles’ on a livestock gastrointestinal nematode. This first step encourages further studies in order to have a better quantification of this interaction, and to investigate to which extent the phoretic mites of dung beetles can impact H. contortus populations in natural conditions.

Low prevalence of Contracaecum third-stage larvae parasitizing Sea of Galilee fisheries: 1-year survey after 57 years of no information

Freshwater and marine ecosystems are a suitable habitat for parasitic nematodes of the genus Contracaecum (family: Anisakidae) to complete their complex life cycle. Several fish species of the Sea of Galilee (Lake Kinneret) were reported in 1964 as second intermediate/paratenic hosts of Contracaecum spp. larvae. The lack of taxonomically relevant morphological features of these larvae hindered their proper identification. Here we report the results of a 1-year survey conducted in 2021, 57 years after the first (and only) such survey. We analyzed 352 specimens from 10 fish species (native and non-native) of the Sea of Galilee (Israel) ichthyofauna. We compared our results with those of the first parasitological survey conducted by Paperna in 1964; the overall prevalence of nematodes referable to Contracaecum larvae was 16.8% and 0.85% in 1964 and in 2021, respectively. Different from the first survey that identified Contracaecum larvae morphologically, we used both morphological and molecular tools. Two wild native cyprinids—Jordan himri (Carasobarbus canis) and Jordan barbel (Luciobarbus longiceps)—were infected (a single specimen each) with Contracaecum quadripapillatum larvae in their abdominal cavity. A single specimen of blue tilapia (Oreochromis aureus) was infected with two larvae of Contracaecum multipapillatum E, localized in the pericardial cavity. The findings of our study, which is part of a large project focused on Contracaecum spp. infecting both piscivorous birds and fish collected in Israel, advance our knowledge about the distribution and host range of this potentially zoonotic parasite in fishery products of the Sea of Galilee.

Fluralaner (Bravecto®) treatment kills Aedes aegypti after feeding on Dirofilaria immitis-infected dogs

BackgroundTransmission of canine heartworm (Dirofilaria immitis) from infected to naïve dogs is dependent on successful mosquito feeding and survival.MethodsTo determine whether treating heartworm-infected dogs with fluralaner (Bravecto®) limits the survival of infected mosquitoes, and potentially the transmission of D. immitis, we allowed female mosquitoes to feed on microfilaremic dogs and evaluated mosquito survival and infection with D. immitis. Eight dogs were experimentally infected with D. immitis. On day 0 (~ 11 months post-infection), four microfilaremic dogs were treated with fluralaner according to label directions while the other four were non-treated controls. Mosquitoes (Aedes aegypti Liverpool) were allowed to feed on each dog on days −7, 2, 30, 56, and 84. Fed mosquitoes were collected, and the number of live mosquitoes determined at 6 h, 24 h, 48 h, and 72 h post-feeding. Surviving mosquitoes held for 2 weeks were dissected to confirm third-stage D. immitis larvae; PCR (12S rRNA gene) was performed post-dissection to identify D. immitis in mosquitoes.ResultsPrior to treatment, 98.4%, 85.1%, 60.7%, and 40.3% of mosquitoes fed on microfilaremic dogs were alive at 6 h, 24 h, 48 h, and 72 h post-feeding, respectively. Similarly, mosquitoes fed on microfilaremic, non-treated dogs were alive 6 h post-feeding (98.5–100%) throughout the study. In contrast, mosquitoes fed on fluralaner-treated dogs 2 days after treatment were dead or severely moribund by 6 h post-feeding. At 30 and 56 days post-treatment, > 99% of mosquitoes fed on treated dogs were dead by 24 h. At 84 days post-treatment, 98.4% of mosquitoes fed on treated dogs were dead by 24 h. Before treatment, third-stage larvae of D. immitis were recovered from 15.5% of Ae. aegypti 2 weeks after feeding, and 72.4% were positive for D. immitis by PCR. Similarly, 17.7% of mosquitoes fed on non-treated dogs had D. immitis third-stage larvae 2 weeks after feeding, and 88.2% were positive by PCR. Five mosquitoes fed on fluralaner-treated dogs survived 2 weeks post-feeding, and 4/5 were from day 84. None had third-stage larvae at dissection, and all were PCR-negative.ConclusionThe data indicate that fluralaner treatment of dogs kills mosquitoes and thus would be expected to reduce transmission of heartworm in the surrounding community.Graphical

Optimizing the xWORM assay for monitoring hookworm larvae motility

Parasitic worms (helminths) infect almost all taxa across the animal kingdom, and pose significant challenges to public health and economies, particularly in developing countries. To address this problem, researchers have developed various tools to measure the motility and viability of helminths. However, the conditions used in anthelmintic screening assays are often not optimized, and can vary considerably between research teams. These unoptimized conditions may impact novel drug screens, as little is known about the effects of different conditions on the health of the target parasites. To improve future research, this study determined the effects of key assay parameters including, media type, media concentration, in-well parasite density, and assay duration on the infective third-stage larva (L3) of two types of hookworms, namely Nippostrongylus brasiliensis in rodents, and Necator americanus in humans. Conditions were screened over several days using the xCELLigence worm real-time motility assay (xWORM); a real-time impedance-based helminth motility assay using the xCELLigence system with 96-well microplates. While results varied depending on the species and media used, the study found that 500–1,000 L3/200-µL and a media concentration of 3.13–25% generally produced good to excellent assay conditions. The findings of this study can guide the future selection of xWORM assay parameters for novel drug trials involving these parasite species and serve as a suggested model for optimizing trial conditions for alternative parasite targets and assays.

Bioinformatics analysis and prokaryotic expression of Strongyloides stercoralis serine protease inhibitor 1

To predict the structure and antigenic epitope of the Strongyloides stercoralis serine protease inhibitor 1 (Ss-SRPN-1) protein using bioinformatics tools, and to construct prokaryotic expression plasmids for expression of recombinant Ss-SRPN-1 protein, so as to provide the basis for unraveling the function of the Ss-SRPN-1 protein. The amino acid sequence of the Ss-SRPN-1 protein was downloaded from the NCBI database, and the physicochemical properties, structure and antigenic epitopes of the Ss-SRPN-1 protein were predicted using bioinformatics tools, including ExPASy, SWISS-MODEL and Protean. Primers were designed according to the nucleotide sequences of Ss-SRPN-1, and the Ss-SRPN-1 gene was amplified, cloned and sequenced with genomic DNA extracted from the infective third-stage larvae of S. stercoralis as a template. The Ss-SRPN-1 protein sequence was cloned into the pET28a (+) expression vector and transformed into Escherichia coli BL21 (DE) cells for induction of the recombinant Ss-SRPN-1 protein expression. The recombinant Ss-SRPN-1 protein was then purified and identified using Western blotting and mass spectrometry. Bioinformatics analysis showed that the Ss-SRPN-1 protein, which was composed of 372 amino acids and had a molecular formula of C1948H3046N488O575S16, was a stable hydrophilic protein, and the subcellular localization of the protein was predicted to be extracellular. The Ss-SRPN-1 protein was predicted to contain 11 dominant B-cell antigenic epitopes and 20 T-cell antigenic epitopes. The Ss-SRPN-1 gene with a length of 1 119 bp was successfully amplified, and the recombinant plasmid pET28a (+)/Ss-SRPN-1 was constructed and transformed into E. coli BL21(DE) cells. The expressed recombinant Ss-SRPN-1 protein had a molecular weight of approximately 43 kDa, and was characterized as a Ss-SRPN-1 protein. The recombinant Ss-SRPN-1 protein has been expressed successfully, and this recombinant protein may be a potential vaccine candidate against strongyloidiasis.

Effect of Ivermectin on the Expression of P-Glycoprotein in Third-Stage Larvae of Haemonchus contortus Isolated from China.

Haemonchus contortus poses a severe hazard to the healthy development of the sheep industry and threatens the welfare of sheep. Ivermectin is the primary anthelmintic used for the prevention and treatment of H. contortus parasitism. However, the widespread and uncontrolled application of ivermectin has resulted in the development and spread of resistant strains of H. contortus. P-glycoprotein (P-gp) plays important roles in the pharmacology and toxicology of ivermectin, and changes in P-gp expression levels can be used to analyze the resistance of H. contortus to ivermectin. This study aimed to analyze the effects of ivermectin on P-gp expression in H. contortus L3 larvae isolated from China and to evaluate whether changes in P-gp expression levels can be used to analyze resistant H. contortus strains. In the absence of drug treatment, the ivermectin-resistant strains isolated in China showed increased expression of P-gp11 (p < 0.01) compared with sensitive strains from elsewhere, whereas the expressions of P-gp2 and P-gp9.1 were downregulated (p < 0.01). When the same strain was compared before and after drug treatment, obvious differences in expression were observed between the different strains. Ivermectin-induced P-gp expression was found to be very complex among the L3 larvae of different strains. In addition, it was confirmed that using P-gp to determine ivermectin resistance in H. contortus strains from different geographic environments can yield different results.

First Record of the Parasitic Nematode Anisakis simplex in the Intestine and Gonads of Dentex macrophthamus off Syrian Marine Waters

This study aimed to investigate the infection of internal parasites of Dentex macrophthalmus in Syrian marine coastal waters. A total of 409 individuals were collected from Ras Al-Basit in the north to Tartus in the south, from the following research stations: Ras Al-Basit, different areas along the Lattakia beach, Jableh, and Tartous, during the period from January 2019 to December 2020.&#x0D; The internal organs (stomach, intestine, and body cavity) of all fish specimens were examined to detect infection with internal parasites. The third-stage larvae of the parasite Anisakis simplex, which belongs to the nematode worms, was isolated from the stomach and gonad walls of D. macrophthalmus. The number of fish infected with this worm reached 53 out of 409 fish individuals studied, with an infection rate of 12.96% and an infection intensity of 2.70 parasites per fish.&#x0D; This study represents the first record of Anisakis simplex isolated from the abdominal cavity of D. macrophthalmus in Syrian marine waters.

A case of chronic gastric anisakiasis coexisting with early gastric cancer.

Anisakiasis is a parasitic disease caused by the consumption of raw or undercooked fish that is infected with Anisakis third-stage larvae. In countries, such as Japan, Italy, and Spain, where people have a custom of eating raw or marinated fish, anisakiasis is a common infection. Although anisakiasis has been reported in the gastrointestinal tract in several countries, reports of anisakiasis accompanied by cancer are rare. We present the rare case of a 40-year-old male patient with anisakiasis coexisting with mucosal gastric cancer. Submucosal gastric cancer was suspected on gastric endoscopy and endoscopic ultrasonography. After laparoscopic distal gastrectomy, granulomatous inflammation with Anisakis larvae in the submucosa was pathologically revealed beneath mucosal tubular adenocarcinoma. Histological and immunohistochemical investigation showed cancer cells as intestinal absorptive-type cells that did not produce mucin. Anisakis larvae could have invaded the cancer cells selectively because of the lack of mucin in the cancerous epithelium. Anisakiasis coexisting with cancer is considered reasonable rather than coincidental. In cancer with anisakiasis, preoperative diagnosis may be difficult because anisakiasis leads to morphological changes in the cancer.