Changes in porcine nutrient transport physiology in response to Ascaris suum infection.

BACKGROUND: The roundworm Ascaris suum is one of the parasites with the greatest economic impact on pig farming. In this context, lower weight gain is hypothesized to be due to decreased nutrient absorption. This study aims at characterizing the effects of A. suum infection on intestinal nutrient transport processes and potential molecular mechanisms. METHODS: Three groups of six piglets each were infected orally (10,000 embryonated A. suum eggs) in a single dose ("single infection"). Another three groups were infected orally (1000 embryonated eggs) for 10 consecutive days ("trickle infection"). Animals were necropsied 21, 35 and 49 days post-infection (dpi). Three groups served as respective controls. The Ussing chamber technique was applied for the functional characterization of small intestinal tissues [short-circuit currents (Isc) as induced by glucose, alanine and peptides; 3H-glucose net flux rates; tissue conductance (Gt)]. Transcription and expression levels of relevant cytokines and nutrient transporters were evaluated (qPCR/western blot). RESULTS: Peptide- and alanine-induced changes in Isc were significantly decreased in the jejunum and ileum of the trickle-infected group at 49 dpi and in the ileum of the single-infected group at 49 dpi. No significant differences regarding glucose transport were observed between the Ascaris-infected groups and the control group in Ussing chamber experiments. Transcription levels of the glucose and peptide transporters as well as of selected transcription factors (transcription of signal transducer and activator of transcription 6 [STAT6] and hypoxia-inducible factor 1-alpha [Hif-1α]) were significantly increased in response to both infection types after some periods. The transcription of interleukins 4 and 13 varied between decrease and increase regarding the respective time points, as did the protein expression of glucose transporters. The expression of the peptide transporter PepT1 was significantly decreased in the ileal single-infected group at 35 dpi. Hif-1α was significantly increased in the ileal tissue from the single-infected group at 21 dpi and in the trickle-infected group at 35 dpi. The expression levels of Na+/K+-ATPase and ASCT1 remained unaffected. CONCLUSIONS: In contrast to the current hypothesis, these results indicate that the nutrient deprivation induced by A. suum cannot be explained by transcriptional or expression changes alone and requires further studies.

Molecular evidence of hybridization between pig and human Ascaris indicates an interbred species complex infecting humans.

Human ascariasis is a major neglected tropical disease caused by the nematode Ascaris lumbricoides. We report a 296 megabase (Mb) reference-quality genome comprised of 17,902 protein-coding genes derived from a single, representative Ascaris worm. An additional 68 worms were collected from 60 human hosts in Kenyan villages where pig husbandry is rare. Notably, the majority of these worms (63/68) possessed mitochondrial genomes that clustered closer to the pig parasite Ascaris suum than to A. lumbricoides. Comparative phylogenomic analyses identified over 11 million nuclear-encoded SNPs but just two distinct genetic types that had recombined across the genomes analyzed. The nuclear genomes had extensive heterozygosity, and all samples existed as genetic mosaics with either A. suum-like or A. lumbricoides-like inheritance patterns supporting a highly interbred Ascaris species genetic complex. As no barriers appear to exist for anthroponotic transmission of these 'hybrid' worms, a one-health approach to control the spread of human ascariasis will be necessary.

First report on aberrant Ascaris suum infection in a dog, China.

An aberrant Ascaris suum infection in a domestic dog in China in 2019 is described for the first time. This pathogen is a common roundworm of pigs with few reported cases in domestic animals. Our findings suggest a wider infection range with a possible transmission of A. suum to domestic animals that interact with humans.

Pathogen Reduction Potential in Anaerobic Digestion of Organic Fraction of Municipal Solid Waste and Food Waste.

Anaerobic digestion (AD) is a commonly used method of processing waste. Regardless of the type of the used digestate (fertilizer, feedstock in case of solid-state fermentation, raw-material in case of thermal treatment) effective pathogen risk elimination, even in the case of high pathogen concentration is essential. An investigation of the survival time and inactivation rate of the Salmonella Senftenberg W775, Enterococcus spp., and Ascaris suum eggs during thermophilic anaerobic digestion performed on laboratory scale and confirmation of hygienization in full-scale operation were performed in this study. Except for sanitization efficiency, the AD process performance and stability were also verified based on determination of pH value, dry matter content, acidity, alkalinity, and content of fatty acids. The elimination of pathogen was met within 6.06 h, 5.5 h, and about 10 h for the Salmonella Senftenberg W775, Enterococcus spp., and Ascaris suum, respectively in the laboratory trials. The obtained results were confirmed in full-scale tests, using 1500 m3 Kompogas® reactors, operating in MBT Plant located in Poland. Sanitization of the digestate was achieved. Furthermore, the process was stable. The pH value, suspended solids, and ammonium content remained stable at 8.5, 35%, and 3.8 g/kg, respectively. The acetic acid content was noted between almost 0.8 and over 1.1 g/kg, while the concentration of propionic acid was noted at maximum level of about 100 mg/kg. The AD conditions could positively affect the pathogen elimination. Based on these results it can be found that anaerobic digestion under thermophilic conditions results in high sanitation efficiency.

Body fluid from the parasitic worm Ascaris suum inhibits broad-acting pro-inflammatory programs in dendritic cells.

Dendritic cells (DCs) are essential for generating T-cell-based immune responses through sensing of potential inflammatory and metabolic cues in the local environment. However, there is still limited insight into the processes defining the resultant DC phenotype, including the type of early transcriptional changes in pro-inflammatory cues towards regulatory or type 2 immune-based cues induced by a variety of exogenous and endogenous molecules. Here we compared the ability of a selected number of molecules to modulate the pro-inflammatory phenotype of lipopolysaccharide (LPS) and interferon-γ (IFN-γ)-stimulated human monocyte-derived DCs towards an anti-inflammatory or regulatory phenotype, including Ascaris suum body fluid [helminth pseudocoelomic fluid (PCF)], the metabolites succinate and butyrate, and the type 2 cytokines thymic stromal lymphopoietin and interleukin-25. Our data show that helminth PCF and butyrate treatment suppress the T helper type 1 (Th1)-inducing pro-inflammatory DC phenotype through induction of different transcriptional programs in DCs. RNA sequencing indicated that helminth PCF treatment strongly inhibited the Th1 and Th17 polarizing ability of LPS + IFN-γ-matured DCs by down-regulating myeloid differentiation primary response gene 88 (MyD88)-dependent and MyD88-independent pathways in Toll-like receptor 4 signaling. By contrast, butyrate treatment had a strong Th1-inhibiting action, and transcripts encoding important gut barrier defending factors such as IL18, IL1B and CXCL8 were up-regulated. Collectively, our results further understanding of how compounds from parasites and gut microbiota-derived butyrate may exert immunomodulatory effects on the host immune system.

Genomes of parasitic nematodes (Meloidogyne hapla, Meloidogyne incognita, Ascaris suum and Brugia malayi) have a reduced complement of small RNA interference pathway genes: knockdown can reduce host infectivity of M. incognita.

The discovery of RNA interference (RNAi) as an endogenous mechanism of gene regulation in a range of eukaryotes has resulted in its extensive use as a tool for functional genomic studies. It is important to study the mechanisms which underlie this phenomenon in different organisms, and in particular to understand details of the effectors that modulate its effectiveness. The aim of this study was to identify and compare genomic sequences encoding genes involved in the RNAi pathway of four parasitic nematodes: the plant parasites Meloidogyne hapla and Meloidogyne incognita and the animal parasites Ascaris suum and Brugia malayi because full genomic sequences were available-in relation to those of the model nematode Caenorhabditis elegans. The data generated was then used to identify some potential targets for control of the root knot nematode, M. incognita. Of the 84 RNAi pathway genes of C. elegans used as model in this study, there was a 42-53 % reduction in the number of effectors in the parasitic nematodes indicating substantial differences in the pathway between species. A gene each from six functional groups of the RNAi pathway of M. incognita was downregulated using in vitro RNAi, and depending on the gene (drh-3, tsn-1, rrf-1, xrn-2, mut-2 and alg-1), subsequent plant infection was reduced by up to 44 % and knockdown of some genes (i.e. drh-3, mut-2) also resulted in abnormal nematode development. The information generated here will contribute to defining targets for more robust nematode control using the RNAi technology.

The role of Intelectin-2 in resistance to Ascaris suum lung larval burdens in susceptible and resistant mouse strains.

The underlying mechanism of predisposition to Ascaris infection is not yet understood but host genetics are thought to play a fundamental role. We investigated the association between the Intelectin-2 gene and resistance in F2 mice derived from mouse strains known to be susceptible and resistant to infection. Ascaris larvae were isolated from murine lungs and the number of copies of the Intelectin-2 gene was determined in F2 mice. Intelectin-2 gene copy number was not significantly linked to larval burden. In a pilot experiment, the response to infection in parental mice of both sexes was observed in order to address the suitability of female F2 mice. No overall significant sex effect was detected. However, a divergence in resistance/susceptibility status was observed between male and, female hybrid offspring. The responsiveness to Ascaris in mice is likely to be controlled by multiple genes and, despite a unique absence from the susceptible C57BL/6j strain, the Intelectin-2 gene does not play a significant role in resistance. The observed intra-strain variation in larval burden requires further investigation but we hypothesize that it stems from social/dominance hierarchies created by the presence of female mice and possibly subsequent hormonal perturbations that modify the intensity of the immune response.

Immunizing pigs with Ascaris suum haemoglobin increases the inflammatory response in the liver but fails to induce a protective immunity.

To determine whether purified Ascaris suum haemoglobin (AsHb) is a suitable vaccine candidate for the control of Ascaris infections, pigs were vaccinated with AsHb in combination with QuilA adjuvant and challenged with A. suum eggs. The number of liver lesions and worms in the intestine was assessed on day 14, 28 and 56 post-infection (p.i.). No significant differences were found in the number of worms recovered between vaccinated and control pigs on any of these days. However, significantly more white spots were counted on the livers of vaccinated pigs on day 14 (+86%) and day 28 (+118%) p.i. compared with nonvaccinated controls. To investigate whether the increased immunoreactivity against the liver stage L3s in vaccinated pigs was triggered by and directed against AsHb, the transcription and expression of AsHb in this larval life stage was analysed by RT-PCR and immunoblotting. The results showed that neither the AsHb transcript nor protein was detectable in freshly hatched L3. However, the immunoblot analysis showed that vaccination with AsHb resulted in the production of antibodies binding to several other antigens of the L3, suggesting that these might be involved in the increased white spot development.

RNAi-mediated silencing of a novel Ascaris suum gene expression in infective larvae.

In the present study, the potential of RNA interference (RNAi) as a gene silencing tool and the resultant effects on Ascaris suum larval development was examined by targeting a gene (represented by the EST 06G09) specifically expressed in the infective larvae of A. suum. BALB/c mice were infected with RNAi-treated larvae. The results showed that the target gene was silenced after soaking for 72 h, and the survival rate of the RNAi-treated larvae was reduced by 17.25% (P<0.01). A significant difference (P<0.05) was detected in the numbers of larvae collected from the livers and lungs of infected mice 4 days after infection with untreated larvae (164.29 ± 21.51) and RNAi-treated larvae (71.43 ± 14.35). Significant differences (P<0.01) were also found in the body length and width between untreated larvae (480 ± 105.77 µm for length and 23.93 ± 3.72 µm for width) and RNAi-treated larvae (400.57 ± 71.31 µm for length and 20.20 ± 2.43 µm for width). These results show that the gene represented by EST 06G09 may play a role in the development of A. suum larvae.

Fate and transport of zoonotic, bacterial, viral, and parasitic pathogens during swine manure treatment, storage, and land application.

Members of the public are always somewhat aware of foodborne and other zoonotic pathogens; however, recent illnesses traced to produce and the emergence of pandemic H1N1 influenza virus have increased the scrutiny on all areas of food production. The Council for Agricultural Science and Technology has recently published a comprehensive review of the fate and transport of zoonotic pathogens that can be associated with swine manure. The majority of microbes in swine manure are not zoonotic, but several bacterial, viral, and parasitic pathogens have been detected. Awareness of the potential zoonotic pathogens in swine manure and how treatment, storage, and handling affect their survival and their potential to persist in the environment is critical to ensure that producers and consumers are not at risk. This review discusses the primary zoonotic pathogens associated with swine manure, including bacteria, viruses, and parasites, as well as their fate and transport. Because the ecology of microbes in swine waste is still poorly described, several recommendations for future research are made to better understand and reduce human health risks. These recommendations include examination of environmental and ecological conditions that contribute to off-farm transport and development of quantitative risk assessments.

Genetic influence on the kinetics and associated pathology of the early stage (intestinal-hepatic) migration of Ascaris suum in mice.

The generative mechanism(s) of aggregation and predisposition to Ascaris lumbricoides and A. suum infections in their host population are currently unknown and difficult to elucidate in humans and pigs for ethical/logistical reasons. A recently developed, optimized murine model based on 2 inbred strains, putatively susceptible (C57BL/6j) and resistant (CBA/Ca) to infection, was exploited to elucidate further the basis of the contrasting parasite burdens, most evident at the pulmonary stage. We explored the kinetics of early infection, focusing on the composite lobes of the liver and lung, over the first 8 days in an effort to achieve a more detailed understanding of the larval dispersal over time and the point at which worm burdens diverge. Larval recoveries showed a heterogenous distribution among the lobes of the lungs, being higher in the right lung of both strains, and in the susceptible strain larvae accumulating preferentially in 2 (caudal and middle) of the 4 lobes. Total larval burdens in these 2 lobes were largely responsible for the higher worm burdens in the susceptible strain. While total lung larval recoveries significantly differed between mouse strains, a difference in liver larval burdens was not observed. However, an earlier intense inflammatory response coupled with more rapid tissue repair in the hepatic lobes was observed in CBA/Ca mice, in contrast to C57BL/6j mice, and it is possible that these processes are responsible for restricting onward pulmonary larval migration in the resistant genotype.

Ascaris suum infection negatively affects the response to a Mycoplasma hyopneumoniae vaccination and subsequent challenge infection in pigs.

Since their first introduction more than a century ago, vaccines have become one of the most cost-effective tools to prevent and manage infectious diseases in human and animal populations. It is vital to understand the possible mechanisms that may impair optimal vaccine efficacy. The hypothesis posed in this study was that a concurrent Ascaris suum infection of pigs vaccinated with a Mycoplasma hyopneumoniae (Mh) vaccine would modulate the protective immune response to a subsequent challenge infection. Four groups of pigs were either (1) untreated (group C), (2) vaccinated against Mh 3 weeks after the start of the study (group V), (3) given a trickle infection with A. suum throughout the study (group A), or (4) given a trickle infection with A. suum and vaccinated against Mh (group AV). All pigs were subsequently inoculated with live Mh bacteria 4 weeks after the Mh vaccination and necropsied after another 4 weeks. All pigs in group V sero-converted 3 weeks after vaccination (100%), as opposed to only 33% of group AV pigs that were Mh-vaccinated and given A. suum. At the end of the study, only 78% of pigs in group AV had sero-converted. Pigs in group AV had a higher mean percentage of lung pathology and the variation was significantly higher in these pigs compared to pigs in group V. The pattern of gene expression in the lungs and draining lymph nodes indicated a local Th2-skewed response induced by A. suum. Our study indicated that A. suum significantly compromised the effect of Mh vaccination. The impact of reduced vaccine efficacy caused by a common gastrointestinal helminth emphasises the importance of parasite control. More focus should be put into this area of research to outline the practical consequences of this interaction, and to be able to predict, prevent and correct negative interactions.

The dynamics of genetically marked Ascaris suum infections in pigs.

SUMMARY: The genotypes of both host and parasite may influence the outcome of parasitic infections, but few attempts have been made to quantify the effect of parasite genotype on macroparasite infections of socio-economic importance. We examined variation in particular traits during the infection in pigs with the parasitic nematode Ascaris suum. We infected 26 pigs with mixtures of equal proportions of embryonated eggs from 4 single female worms each with a unique mtDNA haplotype--the eggs from each female worm were a mixture of siblings and half-siblings. Pigs were necropsied on days 14, 17 and 28 following inoculation, which corresponded to time-points before, during and after the main immune responses against the nematode. A total of approximately 11,000 worms were recovered at necropsy. The location in the small intestine was recorded for all worms and the length and mtDNA haplotype were determined for about 4200 individual worms. There were significant differences in the distribution and abundance of the 4 individual haplotypes among individual pigs demonstrating strong interactions between parasite and host. We found significant differences in the abundance and position in the small intestine as well as the size of worms among haplotypes. We conclude that both parasite and host effects as well as the interplay between them play important roles in determining the characteristics and outcome of infection.

Effect of aerobic and anaerobic digestion on the viability of Cryptosporidium parvum oocysts and Ascaris suum eggs.

The viability of Cryptosporidium parvum oocysts and Ascaris suum eggs inoculated into aerobic and anaerobic digesters was measured. The digesters were maintained at 37 degrees C, 47 degrees C, and 55 degrees C, with 10-day detention times. Eggs and oocysts were added to each digester in a single spike or in chambers placed in the digesters for varying periods. Oocysts were inactivated very rapidly in all systems as determined by a dye permeability assay, > 99% inactivated after 10 days at 37 degrees C, 4 days at 47 degrees C, and 2 days at 55 degrees C. Eggs were more rapidly inactivated in anaerobic digesters than in aerobic digesters. At 55 degrees C, eggs in both anaerobic and aerobic digesters were > 99% inactivated within 1 h. At 47 degrees C, anaerobic digestion inactivated around 95% eggs in 2 days, but around 25% of the eggs were still viable after 10 days in aerobic digesters. At 37 degrees C, anaerobic digestion inactivated more than 75% of the eggs after 10 days, but in the aerobic digester at 37 degrees C, 10 days of treatment had no effect on viability. The oocysts and eggs added in chambers appeared to behave similarly to these pathogens added directly to the biosolids within the digesters.

Immunohistochemical characterization of hepatic lesions associated with migrating larvae of Ascaris suum in pigs.

This paper describes the histopathological features and the cellular distribution of T lymphocytes (CD3), B cells (CD79a), immunoglobulin (IgG, IgA, IgM)-bearing plasma cells, macrophages (Mac387 and alpha-1-antitrypsin), MHC class II antigen and S-100 protein in hepatic white spots associated with naturally occurring Ascaris suum parasitism in 35 pigs. Hepatic granulomas were observed in 10 pigs, whereas lymphoid proliferation with a diffuse or lymphonodular pattern was the main histopathological lesion in 14 other pigs, and portal fibrosis in a further 11 animals. In lymphonodular lesions, the distribution of immunoreactive cells with all antibodies tested was closely similar to that found in the cortex of lymph nodes. Thus, lymphoid follicles were composed mainly of CD79a(+)B cells and interfollicular tissue was composed mainly of CD3(+)T lymphocytes. The presence of follicular dendritic and interdigitating cells expressing S-100 protein and MHC class II antigen in lymphonodular lesions suggested that these are highly organized structures developed to enhance antigen presentation to B and T cells, and consequently the local immune response against the parasite. The humoral local response was represented mainly by IgG-secreting plasma cells.

Pigs become infected after ingestion of livers and lungs from chickens infected with Ascaris of pig origin.

An experimental infection with Ascaris of pig origin showed that Ascaris suum larvae can migrate extra-intestinally in chickens. Furthermore, after feeding piglets with Ascaris infected chicken liver and lungs, it was possible to recover larvae from their lungs. These observations suggest that the chicken could serve as a paratenic host for Ascaris. There is also the possibility for zoonotic transmission if raw chicken livers are consumed by humans.

Development of patent Ascaris suum infections in pigs following intravenous administration of larvae hatched in vitro.

The normal tissue migration of Ascaris suum in the pig host involves larval development in the liver accompanied by considerable pathological changes. The vast majority of larvae that reach the small intestine are later expelled by unknown mechanisms. We show that when migration through the liver is bypassed by inoculation of pigs with an intravenous dose of larvae hatched in vitro, the larvae not only complete migration and return to the small intestine, but they also seem to have a greater chance of survival to adulthood. This technique offers new possibilities for studies on specific lung involvement in protective immunity, provides valuable information for the understanding of self cure by larval expulsion, and adds to our understanding of the evolution of migration of Ascaris larvae in tissues.

Exposure of sows to Ascaris suum influences worm burden distributions in experimentally infected suckling piglets.

This paper reports on the influence of maternal exposure to Ascaris suum on worm burden distributions in experimentally infected piglets. In the first study, sows were inoculated before and during gestation (6 months, long-term exposure) with 10,000 A. suum eggs twice weekly. In a second study, sows were inoculated during gestation only (3 months, short-term exposure) with increasing doses of eggs (10,000-40,000 eggs twice weekly). Helminth-naive sows served as controls in both studies. The third study used the same design as the short-term exposure study, but piglets from exposed and control sows were cross-suckled within 4 h of birth before colostrum uptake. All piglets were inoculated 2 or 3 times with 50 A. suum eggs on days 4 and 7 (and 14) after birth, and left with the sows. At 10 weeks of age all piglets were necropsied, and liver lesions and worm burdens were recorded. Surprisingly, in piglets born to long-term exposed sows, the prevalence of A. suum infection and the mean worm burden were significantly higher than those in piglets from control sows. In contrast, neither worm burdens nor prevalence were significantly different between piglets from short-term exposed sows compared with their controls. In the cross-suckling experiment, 67% of piglets suckling control sows harboured worms at slaughter, compared with 15% of piglets suckling exposed sows. Maximum likelihood analysis of worm burden distribution and the degree of parasite aggregation showed 3 distinctly different types of overdispersed distributions: worm counts in piglets from control sows, in piglets from short-term exposed sows and in piglets from long-term exposed sows. When the worm burden data were analysed including the cross-suckled piglets by biological mother, it appeared that the control and short-term distributions converged and that only the long-term exposure was significantly different. Overall, the degree of parasite aggregation in piglets infected with A. suum decreased with exposure of the sows. A non-linear relationship was observed between prevalence of infection and mean worm burden, which was different for piglets from exposed and control sows, and similar to relationships of this type that previously have been found in human A. lumbricoides infections. It was concluded that in porcine A. suum infections maternal exposure alters the distribution of worms in their offspring, in which the duration of exposure appeared to be an important influence. The results of the cross-suckling further suggest that maternal factors, e.g. antibodies, are transferred via colostrum.

The morphogenesis of Ascaris suum to the infective third-stage larvae within the egg.

Studies of the morphology of Ascaris suum larvae developing in the egg during embryonation in vitro at room temperature showed that 2 molts take place within the egg. The first larval stage (L1) appeared in the egg after 17-22 days of cultivation, the first molt to the second larval stage (L2) took place from day 22 to day 27, and the second molt to the third larval stage (L3) started on day 27 and continued during the 60-day observation period. Infectivity of the eggs was studied by oral egg inoculation in mice and showed that the L3 are the infective stage for mice. Molting to the L3 stage occurs gradually over a period of 2-6 wk, and it is recommended to have an additional maturation period so the infectivity of an egg batch may reach maximum level.