Ascariasis, caused by both Ascaris lumbricoides and Ascaris suum, is the most prevalent parasitic disease worldwide, affecting both human and porcine populations. However, due to the difficulties of assessing the early events of infection in humans, most studies of human ascariasis have been restricted to the chronic intestinal phase. Therefore, the Ascaris mouse model has become a fundamental tool for investigating the immunobiology and pathogenesis of the early infection stage referred to as larval ascariasis because of the model\'s practicality and ability to replicate the natural processes involved. The Ascaris mouse model has been widely used to explore factors such as infection resistance/susceptibility, liver inflammation, lung immune-mediated pathology, and co-infections and, notably, as a pivotal element in preclinical vaccine trials. Exploring the immunobiology of larval ascariasis may offer new insights into disease development and provide a substantial understanding of key components that trigger a protective immune response. This article focuses on creating a comprehensive guide for conducting Ascaris experimental infections in the laboratory as a foundation for future research efforts. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Acquisition and embryonation of Ascaris suum eggs from adult females Alternate Protocol: Cleaning and purification of Ascaris suum from female A. suum uteri Basic Protocol 2: Preparation of Ascaris suum eggs and murine infection Basic Protocol 3: Measurement of larval burden and Ascaris-larva-induced pathogenesis Basic Protocol 4: In vitro hatching and purification of Ascaris L3 larvae Support Protocol: Preparation of crude antigen from Ascaris infectious stages Basic Protocol 5: Ultrastructure-expansion microscopy (U-ExM) of Ascaris suum larval stages.

Natural killer (NK) cells play a key role in defense against Salmonella infections during the early phase of infection. Our previous work showed that the excretory/secretory products of Ascaris suum repressed NK activity in vitro. Here, we asked if NK cell functionality was influenced in domestic pigs during coinfection with Ascaris and Salmonella enterica serotype Typhimurium. Ascaris coinfection completely abolished the IL-12 and IL-18 driven elevation of IFN-γ production seen in CD16 + CD8α + perforin + NK cells of Salmonella single-infected pigs. Furthermore, Ascaris coinfection prohibited the Salmonella-driven rise in NK perforin levels and CD107a surface expression. In line with impaired effector functions, NK cells from Ascaris-single and coinfected pigs displayed elevated expression of the inhibitory KLRA1 and NKG2A receptors genes, contrasting with the higher expression of the activating NKp46 and NKp30 receptors in NK cells during Salmonella single infection. These differences were accompanied by the highly significant upregulation of T-bet protein expression in NK cells from Ascaris-single and Ascaris/Salmonella coinfected pigs. Together, our data strongly indicate a profound repression of NK functionality by an Ascaris infection which may hinder infected individuals from adequately responding to a concurrent bacterial infection.

Ascaris spp. undergo extensive migration within the body before establishing patent infections in the small intestinal tract of humans and pigs. However, whether larval migration is critical for inducing efficient type 2 responses remains poorly understood. Therefore, we investigated systemic versus local adaptive immune responses along the hepato-tracheal migration of Ascaris suum during primary, single infections in conventionally raised pigs. Neither the initial invasion of gut tissue nor migration through the liver resulted in discernable Th2 cell responses. In contrast, lung-stage larvae elicited a Th2-biased pulmonary response, which declined after the larvae had left the lungs. In the small intestine, we observed an accumulation of Th2 cells upon the arrival of fourth-stage larvae (L4) to the small intestinal lumen. In parallel, we noticed robust and increasing Th1 responses in circulation, migration-affected organs, and draining lymph nodes. Phenotypic analysis of CD4+ T cells specifically recognizing A. suum antigens in the circulation and lung tissue of infected pigs confirmed that the majority of Ascaris-specific T cells produced IL-4 (Th2) and, to a much lesser extent, IL-4/IFN-g (Th2/1 hybrids) or IFN-g alone (Th1). These data demonstrate that lung-stage but not the early liver-stage larvae lead to a locally restricted Th2 response. Significant Th2 cell accumulation in the small intestine occurs only when L4 complete the body migration. In addition, Th2 immunity seems to be hampered by the concurrent, nonspecific Th1 bias in growing pigs. Together, the late onset of Th2 immunity at the site of infection and the Th1-biased systemic immunity likely enable the establishment of intestinal infections by sufficiently large L4 stages and pre-adult worms, some of which resist expulsion mechanisms.

BACKGROUND: Parasitic diseases of pigs are a public and veterinary health problem. Helminths influence pork production, whereas backyard pigs can transmit these parasites.
OBJECTIVE: This work aimed to investigate the prevalence of antibodies against Ascaris suum and Trichinella spiralis in backyard pigs from Jamiltepec, Region de la Costa, Oaxaca, in Southwestern Mexico.
METHODS: Six hundred sixty-four serum samples were obtained from backyard pigs from 23 rural villages distributed in 5 municipalities; samples were taken in a non-probabilistic manner with the owner\'s consent. The presence of serum antibodies against a total extract of A. suum adult worm was determined by ELISA. In contrast, antibodies to the excretion-secretion products of the T. spiralis muscle larva were determined by Western blot.
RESULTS: The global seroprevalence for A. suum was 5.12% and 2.41% for T. spiralis; however, antibodies were only found in 8 villages and distributed in 3 municipalities. The highest frequency of positivity for Ascaris was found in the municipality of Santa Catarina Mechoacán (13.01%), whereas, in Santa María Huazalotitlán, the highest frequency of positivity for Trichinella was found (5.75%). In San Andrés, frequencies were 7.23% and 4.82%, respectively. No statistical differences were observed between populations.
CONCLUSIONS: Our data suggest that helminth transmission is restricted by locality. However, further studies must be conducted to understand the factors limiting this transmission to promote pork meat production in parasite-free zones.

A novel group of biocidal compounds are the Crystal 3D (Cry) and Cytolytic (Cyt) proteins produced by Bacillus thuringiensis (Bt). Some Bt Cry proteins have a selective nematocidal activity, with Cry5B being the most studied. Cry5B kills nematode parasites by binding selectively to membrane glycosphingolipids, then forming pores in the cell membranes of the intestine leading to damage. Cry5B selectively targets multiple species of nematodes from different clades and has no effect against mammalian hosts. Levamisole is a cholinergic anthelmintic that acts by selectively opening L-subtype nicotinic acetylcholine receptor ion-channels (L-AChRs) that have been found on muscles of nematodes. A synergistic nematocidal interaction between levamisole and Cry5B at the whole-worm level has been described previously, but the location, mechanism and time-course of this synergism is not known. In this study we follow the timeline of the effects of levamisole and Cry5B on the Ca2+ levels in enterocyte cells in the intestine of Ascaris suum using fluorescence imaging. The peak Ca2+ responses to levamisole were observed after approximately 10 minutes while the peak responses to activated Cry5B were observed after approximately 80 minutes. When levamisole and Cry5B were applied simultaneously, we observed that the responses to Cry5B were bigger and occurred sooner than when it was applied by itself. It is proposed that the synergism is due to the cytoplasmic Ca2+ overload that is induced by the combination of levamisole opening Ca2+ permeable L-subtype nAChRs and the Ca2+ permeable Cry5B toxin pores produced in the enterocyte plasma membranes. The effect of levamisole potentiates and speeds the actions of Cry5B that gives rise to bigger Ca2+ overloads that accelerates cell-death of the enterocytes.

BACKGROUND: Parasitic infestations have a substantial economic impact on pig production. This study aimed to investigate the gastrointestinal (GI) helminths in pigs and to molecularly characterise two important nematodes, Ascaris and Trichuris species.
METHODS: A total of 500 pig faecal samples were collected from small holder backyard pig farms in five townships within Nay Pyi Taw, Myanmar. Microscopic examination was conducted to estimate the prevalence of GI helminth infestation in the pigs. DNA extraction and PCR were performed on faecal samples that were morphologically positive for Ascaris and Trichuris eggs. Molecular analysis was then conducted to characterise A. suum and T. suis, the most common and zoonotic helminths.
RESULTS: According to microscopic examination, 69.2% (346/500) were positive for GI helminth eggs. The GI helminth species observed were A. suum, Strongyle, Strongyloides spp., T. suis, Metastrongylus spp., Hyostrongylus spp., Fasciolopsis spp., Paragonimus spp., and Schistosoma spp., with occurrences of 34.8%, 29.6%, 21.4%, 20.0%, 4.0%, 1.6%, 1.0%, 1.0%, and 0.4%, respectively. Mixed infections of GI helminths were noted in 31.0% of the samples. Overall, sampled pigs excreted mostly low levels (< 100 EPG) or moderate levels (> 100-500 EPG) of GI helminth eggs. The highest mean EPG for each parasite species was noted in A. suum. The presence of A. suum and T. suis was confirmed molecularly. The sequences of the internal transcribed spacer 1 (ITS1) region of A. suum showed high similarity with previously reported sequences. Likewise, the sequences of T. suis exhibited high similarity with the sequences reported from humans and pigs. Age was noted as an associated factor (P < 0.05) for GI helminth infection status.
CONCLUSIONS: In this report, A. suum and T. suis were molecularly identified for the first time in Myanmar. It is important to extend the information among the farmers to be aware of the necessity of preventing zoonotic parasites by practicing regular deworming, proper use of anthelmintics and maintaining hygienic conditions in their pig farms.

Ascaris suum and Macracanthorhynchus hirudinaceus cause a large loss of yield in farm animals as well as in free-living and captive wild boar herds, thereby causing economic damage. This study compared A. suum and M. hirudinaceus infections in free-ranging and captive wild boars (Sus scrofa) in Hungary. The authors measured the A. suum and M. hirudinaceus infections of a 248-hectare wild boar garden and an 11,893-hectare free-living wild boar herd in the sample area. In all cases, samples were collected from shot wild boars. In total, 216 wild boars were examined from June 2015 to June 2023 in Hungary. Of the 173 dissected wild boars from the wild, 57 (32.9%) were infected with A. suum, while 30 (69.8%) of the 43 individuals from the captive area were infected. The prevalence of M. hirudinaceus in the free-living area population was 9.25% (16 wild boars), while that of the captive population was 34.89% (15 wild boars). In the case of the examined helminths, the captive herd was 36.9% more infected than the herd living in the open area.

OBJECTIVE: Ascariasis caused by the helminth Ascaris suum is the most common parasitosis of swine worldwide and it may involve all age categories of pigs. The present study reports an unusual localization of A. suum worms in the biliary system of a piglet slaughtered for human consumption.
METHODS: The liver was subjected to ultrasound scan and pathological examination. The isolated worms were morphologically examined and the DNA was extracted for the molecular identification of the species involved.
RESULTS: A total of 43 preadult nematodes were found within the gallbladder and the bile ducts. Parasites were morphologically identified as belonging to the genus Ascaris and molecularly as A. suum. At gross examination, the liver was moderately enlarged, with the bile ducts severely dilated. A chronic inflammatory infiltrate was noted, often centered around ectatic bile ducts (up to 5 mm in diameter), lined by hyperplastic epithelium and filled with sections of nematodes. The worm sections showed smooth cuticle, coelomyarian musculature, and an intestinal tract lined by columnar, uninucleated cells within a pseudocoelom. The ex vivo ultrasonographic examination of the liver allowed the visualization of several nematodes in the bile duct lumen and could be suggested for in vivo diagnosis. Unfortunately, the absence of the intestine did not allow to define the pathogenesis of the infection.
CONCLUSIONS: Although, given the unusual nature of this finding, it is difficult to identify predisposing factors for this A. suum localization, it suggests that ascariasis should be considered in the differential diagnosis of pigs with hepatobiliary disease.

Human ascariasis is the most prevalent helminth infection, affecting 445 million people worldwide. To better understand the impact of the immune system on the pathophysiology of individuals infected with Ascaris suum, mice have been used as experimental models. The RT-qPCR technique is a critical auxiliary tool of investigation used to quantify mRNA levels. However, proper normalization using reference genes is essential to ensure reliable outcomes to avoid analytical errors and false results. Despite the importance of reference genes for experimental A. suum infection studies, no specific reference genes have been identified yet. Therefore, we conducted a study to assess five potential reference genes (GAPDH, 18s, ACTB, B2M, and HPRT1) in different tissues (liver, lungs, small and large intestines) affected by A. suum larval migration in C57BL/6j mice. Tissue collection was carried out to analyze parasite burden and confirm the presence of larvae during the peak of migration in each tissue. Upon confirmation, we analyzed different genes in the tissues and found no common gene with stable expression. Our results highlight the importance of analyzing different genes and using different software programs to ensure reliable relative expression results. Based on our findings, B2M was ranked as the ideal reference gene for the liver, while 18S was the most stable gene in the lung and small intestine. ACTB, or a combination of ACTB with GAPDH, was deemed suitable as reference genes for the large intestine due to their stable expression and less variation between the control and infected groups. To further demonstrate the impact of using different reference genes, we normalized the expression of a chemokine gene (CXCL9) in all tissues. Significant differences in CXCL9 expression levels were observed between different groups in all tissues except for the large intestine. This underscores the importance of selecting appropriate reference genes to avoid overestimating target gene expression levels and encountering normalization-related issues that can lead to false results. In conclusion, our study highlights the significance of using reliable reference genes for accurate RT-qPCR analysis, especially in the context of A. suum infection studies in different tissues. Proper normalization is crucial to ensure the validity of gene expression data and avoid potential pitfalls in interpreting results.

The aim of this study was to compare the species composition of gastrointestinal parasites in wild boar feeding in the city of Szczecin with those in its suburban area, as well as to determine the prevalence and intensity of this parasite infection. The intestines and stomachs of 57 wild boars were supplied by a municipal hunter from the city of Szczecin. Both analysed groups of animals were infected with the following parasites: Eimeria debliecki, E. suis, E. polita, E. scabra, Isospora suis, Ascaris suum and Oesophagostomum dentatum. Wild boar from the city were characterised as having a significantly higher prevalence of total Eimeria (p = 0.04) and a lower prevalence of noted species of nematodes (p = 0.15) compared to those from the suburban area. Since the wild boars were mainly infected with Eimeria, it should be assumed that they may pose a real health threat to farm pigs and other farm animals for which Eimeria is a pathogenic parasite. The occurrence of coccidiosis leads to serious health problems and economic losses for breeders. Although the prevalence of A. suum was low, it should be taken into account that this nematode is able to both infect and complete their life cycle in humans.