Strongyloides ratti: Mast cell and goblet cell responses in the small intestine of infected rats

Kinetics of intestinal mast cells and goblet cells were examined in relation to worm localization at various sites in the small intestine of rats infected with 3000 filariform (stage 3) larvae of Strongyloides ratti. The most marked intestinal mastocytosis was observed on Day 20 at the anterior site of the small intestine where the majority of the worms had concentrated. The number of mast cells in the posterior small intestine increased in parallel with the posterior shift of parasites at the later stage of the infection. In contrast to the intestinal mast cell response, the number of goblet cells was not significantly affected by the infection. These results strongly suggest that intestinal mastocytosis is closely related to the presence of the worms and that mast cells may play an important role for the expulsion of S. ratti.     

Peptidases Compartmentalized to the Ascaris suum Intestinal Lumen and Apical Intestinal Membrane

The nematode intestine is a tissue of interest for developing new methods of therapy and control of parasitic nematodes. However, biological details of intestinal cell functions remain obscure, as do the proteins and molecular functions located on the apical intestinal membrane (AIM), and within the intestinal lumen (IL) of nematodes. Accordingly, methods were developed to gain a comprehensive identification of peptidases that function in the intestinal tract of adult female Ascaris suum. Peptidase activity was detected in multiple fractions of the A. suum intestine under pH conditions ranging from 5.0 to 8.0. Peptidase class inhibitors were used to characterize these activities. The fractions included whole lysates, membrane enriched fractions, and physiological- and 4 molar urea-perfusates of the intestinal lumen. Concanavalin A (ConA) was confirmed to bind to the AIM, and intestinal proteins affinity isolated on ConA-beads were compared to proteins from membrane and perfusate fractions by mass spectrometry. Twenty-nine predicted peptidases were identified including aspartic, cysteine, and serine peptidases, and an unexpectedly high number (16) of metallopeptidases. Many of these proteins co-localized to multiple fractions, providing independent support for localization to specific intestinal compartments, including the IL and AIM. This unique perfusion model produced the most comprehensive view of likely digestive peptidases that function in these intestinal compartments of A. suum, or any nematode. This model offers a means to directly determine functions of these proteins in the A. suum intestine and, more generally, deduce the wide array functions that exist in these cellular compartments of the nematode intestine.     

Strongyloides ratti: Acquired resistance in the rat to the preintestinal migrating larvae

Potential sites for expression of acquired resistance to Strongyloides ratti larvae in rats were investigated. In rats immunized by exposure to a single live infection and challenged 30 to 40 days later, 46 to 98% of the challenge larvae failed to reach the small intestine. Multiply immunized rats nearly completely eliminated migrating challenge larvae. This early killing of migrating larvae occurred during the first 48 hr after challenge infection. Resistance to migrating challenge larvae was also induced by repeated injections with heat-killed infective larvae. That the intestine may also serve as an effective site for worm expulsion was confirmed by intestinal transfers of worms from rats with primary infections into resistant rats.     

Ascaris suum: Nutrient absorption,growth, and intestinal pathology in young pigs experimentally infected with 15-day-old larvae

Three-week-old pigs on high (HP) or low (LP) protein diets were infected with 15-day-old Ascaris suum larvae (W). Including noninfected pigs (C), the experimental groups were HPW, LPW, HPC, and LPC. After 8 weeks, worm burden in the intestine averaged 42 in LPW and 31 in HPW. Nitrogen balance during Week 4 showed nonsignificantly less nitrogen absorption and retention in LPW compared to LPC. A similar, nonsignificant decrease in fat absorption was recorded in LPW vs LPC and in HPW vs HPC. The weight of the small intestine was significantly greater in W than C pigs but did not differ because of protein level. The weight correlated positively to worm burden and the increase was due mainly to hypertrophy of the tunica muscularis (muscle layers).     

Strongyloides ratti: Dissociation of the rat's protective immunity into systemic and intestinal components

Analysis of the early stages of a challenge infection with Strongyloides ratti has shown that protection is expressed against the developing third-stage larval worms (L₃) and prevents the maturation to adulthood of most larvae. Challenge after an immunizing infection that was restricted to the parenteral L₃ migratory phase showed that some 10–40% of overall protection could be ascribed to systemic antilarval immunity. Some larvae were trapped in the skin at the site of injection whereas others failed to migrate to the head and lung of immune rats. Larvae arriving in the intestine at Days 3, 4, and 5 did not persist beyond Day 7 and 8. Studies using [⁷⁵Se]methionine-labeled L₃ showed a significant increase in fecal label in rats immunized by a complete infection. This loss did not occur to the same extent in rats immunized only with parenteral larvae. Significant rejection of worms transplanted to the intestine also indicated intestinal protection. The possible existence of large numbers of worms in a state of “arrested development” was excluded by their failure to appear after cortisone treatment and the absence of worm accumulation in radiolabeling studies. It is concluded that at least two responses operate against larval S. ratti, one is systemic and the other operates in the intestine against larvae in a manner that resembles the “rapid expulsion” rejection of Trichinella spiralis in immune rats.     

Bacillus thuringiensis-derived Cry5B Has Potent Anthelmintic Activity against Ascaris suum

Ascaris suum and Ascaris lumbricoides are two closely related geo-helminth parasites that ubiquitously infect pigs and humans, respectively. Ascaris suum infection in pigs is considered a good model for A. lumbricoides infection in humans because of a similar biology and tissue migration to the intestines. Ascaris lumbricoides infections in children are associated with malnutrition, growth and cognitive stunting, immune defects, and, in extreme cases, life-threatening blockage of the digestive tract and aberrant migration into the bile duct and peritoneum. Similar effects can be seen with A. suum infections in pigs related to poor feed efficiency and performance. New strategies to control Ascaris infections are needed largely due to reduced treatment efficacies of current anthelmintics in the field, the threat of resistance development, and the general lack of new drug development for intestinal soil-transmitted helminths for humans and animals. Here we demonstrate for the first time that A. suum expresses the receptors for Bacillus thuringiensis crystal protein and novel anthelmintic Cry5B, which has been previously shown to intoxicate hookworms and which belongs to a class of proteins considered non-toxic to vertebrates. Cry5B is able to intoxicate A. suum larvae and adults and triggers the activation of the p38 mitogen-activated protein kinase pathway similar to that observed with other nematodes. Most importantly, two moderate doses of 20 mg/kg body weight (143 nM/kg) of Cry5B resulted in a near complete cure of intestinal A. suum infections in pigs. Taken together, these results demonstrate the excellent potential of Cry5B to treat Ascaris infections in pigs and in humans and for Cry5B to work effectively in the human gastrointestinal tract.     

Unraveling Ascaris suum experimental infection in humans

Ascaris lumbricoides and Ascaris suum are two closely related parasites that infect humans and pigs. The zoonotic potential of A. suum has been a matter of debate for decades. Here we sought to investigate the potential human infection by A. suum and its immunological alterations. We orally infected five healthy human subjects with eggs embraced by A. suum. The infection was monitored for symptoms and possible respiratory changes, by an interdisciplinary health team. Parasitological, hematological analyses, serum immunoglobulin, cytokine profiles, and gene expression were evaluated during the infection. Our results show that A. suum is able to infect and complete the cycle in humans causing A. lumbricoides similar symptoms, including, cough, headache, diarrhea, respiratory discomfort and chest x-ray alterations coinciding with larvae migration in the lungs. We also observed activation of the immune system with production of IgM and IgG and a Th2/Th17 response with downregulation of genes related to Th1 and apoptosis. PCA (Principal componts analysis) show that infection with A. suum leads to a change in the immune landscape of the human host. Our data reinforce the zoonotic capacity of A. suum and bring a new perspective on the understanding of the immune response against this parasite.     

IL-22 Mediates Goblet Cell Hyperplasia and Worm Expulsion in Intestinal Helminth Infection

Type 2 immune responses are essential in protection against intestinal helminth infections. In this study we show that IL-22, a cytokine important in defence against bacterial infections in the intestinal tract, is also a critical mediator of anti-helminth immunity. After infection with Nippostrongylus brasiliensis, a rodent hookworm, IL-22-deficient mice showed impaired worm expulsion despite normal levels of type 2 cytokine production. The impaired worm expulsion correlated with reduced goblet cell hyperplasia and reduced expression of goblet cell markers. We further confirmed our findings in a second nematode model, the murine whipworm Trichuris muris. T.muris infected IL-22-deficient mice had a similar phenotype to that seen in N.brasiliensis infection, with impaired worm expulsion and reduced goblet cell hyperplasia. Ex vivo and in vitro analysis demonstrated that IL-22 is able to directly induce the expression of several goblet cell markers, including mucins. Taken together, our findings reveal that IL-22 plays an important role in goblet cell activation, and thus, a key role in anti-helminth immunity.     

Intestinal absorption of hexoses in rats infected with Nippostrongylus brasiliensis

The net absorption and accumulation of d-galactose and d-glucose by the small intestine of rats infected with N. brasiliensis were studied in vivo and in vitro. There was no change from control levels in the rate of galactose transfer in vivo by the entire intestine 10 days after infection but fluid transfer was significantly lower at this time. Mucosal galactose transfer in vitro by the entire intestine or by each one-third of the intestine did not change significantly during infection but 10 days after infection mucosal glucose transfer was significantly lower in the infected proximal one-third of the intestine and significantly greater in the distal one-third than in the comparable segments in controls; mucosal glucose transfer by the entire intestine was not affected by infection. Serosal transfer of both hexoses by the proximal two-thirds of the intestine and by the entire intestine was significantly reduced 10 days after infection. Between 10 and 18 days after infection the rate of serosal galactose transfer in vitro was significantly lower than control levels. The difference in response of mucosal and serosal hexose transfer rates to infection appears to be due, in part, to an increase in intestinal glucose metabolism or increased tissue retention of galactose during infection. Mucosal fluid transfer in vitro by the entire intestine was not significantly different from control levels at 10 days of infection when either hexose was used, although there was a significant reduction in the jejunal segment when glucose was used. Mucosal fluid transfer by the entire intestine in the presence of galactose was significantly greater during the rejection phase of the parasite population than in controls.     

Villous atrophy and expulsion of intestinal Trichinella spiralis are mediated by T cells.

The development of villous atrophy and crypt hyperplasia in, and expulsion of nematodes from, the small intestine of the mouse during Trichinella infection is shown to be mediated by T cells. During Trichinella infection, worms initially localise in the anterior half of the small intestine. Their expulsion from here after 6–8 days follows the onset of villous atrophy and crypt hyperplasia in the jejunum and the normal jejunal morphology is restored after complete expulsion of worms from the small intestine at 12–15 days. In thymectomised mice, according to the extent of T-cell depletion, worm localisation is atypical, expulsion is either delayed or absent, and villous atrophy and crypt hyperplasia are either delayed and reduced or absent. The adoptive immunization of infected thymectomised mice with mesenteric lymph node cells (including primed T blasts) from infected donors completely restores the normal host response and enhances the onset of crypt hyperplasia. These findings are discussed in relation to T-cell traffic and delayed-type hypersensitivity in the gut.     

Immune Antibodies and Helminth Products Drive CXCR2-Dependent Macrophage-Myofibroblast Crosstalk to Promote Intestinal Repair

Helminth parasites can cause considerable damage when migrating through host tissues, thus making rapid tissue repair imperative to prevent bleeding and bacterial dissemination particularly during enteric infection. However, how protective type 2 responses targeted against these tissue-disruptive multicellular parasites might contribute to homeostatic wound healing in the intestine has remained unclear. Here, we observed that mice lacking antibodies (Aid^-/-) or activating Fc receptors (Fcrg^-/-) displayed impaired intestinal repair following infection with the murine helminth Heligmosomoides polygyrus bakeri (Hpb), whilst transfer of immune serum could partially restore chemokine production and rescue wound healing in Aid^-/- mice. Impaired healing was associated with a reduced expression of CXCR2 ligands (CXCL2/3) by macrophages (MΦ) and myofibroblasts (MF) within intestinal lesions. Whilst antibodies and helminths together triggered CXCL2 production by MΦ in vitro via surface FcR engagement, chemokine secretion by intestinal MF was elicited by helminths directly via Fcrg-chain/dectin2 signaling. Blockade of CXCR2 during Hpb challenge infection reproduced the delayed wound repair observed in helminth infected Aid^-/- and Fcrg^-/- mice. Finally, conditioned media from human MΦ stimulated with infective larvae of the helminth Ascaris suum together with immune serum, promoted CXCR2-dependent scratch wound closure by human MF in vitro. Collectively our findings suggest that helminths and antibodies instruct a chemokine driven MΦ-MF crosstalk to promote intestinal repair, a capacity that may be harnessed in clinical settings of impaired wound healing.     

STAT6 Expression and IL-13 Production in Association with Goblet Cell Hyperplasia and Worm Expulsion of Gymnophalloides seoi from C57BL/6 Mice

In intestinal helminth infections, Th2 immune respones are generally associated with mucin secretion for worm expulsion from the host intestine. In particular, IL-4 and IL-13 are the important cytokines related with intestinal mucus production via STAT6 signalling in nematode infections. However, this perspective has never been studied in Gymnophalloides seoi infection. The present study aimed to observe the STAT6 signalling and cytokine responses in C57BL/6 mice, a mouse strain resistant to infection with this trematode. The results showed that worm expulsion occurred actively during days 1-2 post-infection (PI), when goblet cells began to proliferate in the small intestine. The STAT6 gene expression in the mouse spleen became remarkable from day 2 PI. Moreover, G. seoi infection induced a significant increase of IL-13 from day 4 PI in the spleen of infected mice. Our results suggested that goblet cell hyperplasia and worm expulsion in G. seoi-infected mice should be induced by STAT6 signalling, in which IL-13 may be involved as a dominant triggering cytokine.     

Nippostrongylus brasiliensis: effects of immunity on the pre-intestinal and intestinal larval stages of the parasite

Nippostrongylus brasiliensis: effects of immunity on the pre-intestinal and intestinal larval stages of the parasite. International journal for Parasitology4: 183–191. Migration of the pre-intestinal larval stages of N. brasiliensis was studied in rats undergoing either primary or challenge infections. In rats undergoing a primary infection, more than 67 percent of larvae successfully migrated from the skin to the oesophagus by 70 h after infection, and subsequently over 90 per cent of these larvae became established in the small intestine as sexually mature adults. In immune rats undergoing a second infection, 46 per cent of larvae completed migration to the oesophagus by 70 h and of these, only 1·6 per cent became established in the intestine to produce eggs. These inhibitory effects on the pre-intestinal and intestinal larval stages were even more pronounced in immune rats undergoing a third or fourth infection and in addition, there was a prolonged sojourn and substantial retention of larvae in their lungs. There was no evidence that the immune response had an adverse effect on oesophageal fourth stags larvae as these organisms (obtained from immune donors) were able to establish and develop to maturity when transferred per os to normal animals.Syngeneic transfer of immune mesenteric lymph node cells to normal recipients, caused expulsion of parasites from the intestine but failed to effect migration of pre-intestinal larval stages. The implications of these findings are discussed in the context of current knowledge of the mechanisms of immunity to helminths.     

Intestinal amino acid absorption in lambs fed fresh Lucerne ( Medicago sativa) during an established Trichostrongylus colubriformis infection

The effects of an established Trichostrongylus colubriformis infection on amino acid (AA) absorption from the small intestine and their availability to other tissues were determined in lambs 48 days post infection. The lambs were fed fresh Lucerne (Medicago sativa; 800 g dry matter (DM)/day) and dosed with 6000 L3 T. colubriformis larvae for 6 days (n = 5) or kept as parasite free controls (n = 6). Faecal egg production was monitored every second day from day 22 to day 48. A nitrogen (N) balance was conducted on days 35 to 43 after infection, and digesta flow and AA concentration measurements were made on day 44. On day 48 after infection, blood was continuously collected from the mesenteric artery and vein, plasma harvested and AA concentrations measured. Faecal egg production peaked on the 26th day after infection (P < 0.001) and intestinal worm burdens on day 48 were greater (P < 0.001) in the infected lambs. Feed intake and liveweight gain were similar (P > 0.10) between control and infected lambs. Digestibility and flow of DM and N through the digestive tract were also unaffected (P > 0.10) by parasite infection. Despite a trend towards higher abomasal AA flux in the parasitised lambs (P < 0.10), apparent AA absorption from the small intestine and AA availability to other tissues were unaffected (P > 0.10) by infection. These results suggest that an established parasite infection had little effect on the intestinal absorption and availability of AA to other tissues in lambs fed fresh Lucerne.     

Trichinella spiralis: acquired immunity in swine

The ability of domestic pigs to develop protective immunity to Trichinella spiralis in response to inoculation with different doses of muscle larvae was assessed. Adult worms developing from the inoculations of 112, 500, and 10,000 larvae were expelled from the intestine about 6 weeks after inoculation. Inoculation with 25,000 larvae, however, resulted in more rapid intestinal worms expulsion, indicating that gut expulsion is dose dependent. Secondary expulsion also tended to be dependent upon primary infection level. Pigs initially inoculated with 500 to 10,000 larvae expelled the challenge infection of adult worms after 22 to 25 days; in contrast, infection by inoculation of only 112 larvae failed to induce significant enchanced gut expulsion of the challenge infection intestinal worms. However, all primary infection levels, including inoculation with 112 larvae, induced nearly absolute resistance to the muscle establishment of larvae from challenge adult worms. The fecundity of female worms recovered from immune pigs was reduced 75% in comparison to controls. These results show that, in contrast to some host species, very rapid gut expulsion does not occur in domestic swine. Yet, immune responses at the gut level are important, perhaps responsible for much of the inhibition reflected as reduction in the establishment of muscle larvae.     

The influence of Trypanosoma brucei infection on local immunoglobulin responses of rats to Nippostrongylus brasiliensis

Wedrychowicz H., Maclean J. M. and Holmes P. H., 1984. The influence of Trypanosoma brucei infection on local immunoglobulin responses of rats to Nippostrongylus brasiliensis. International Journalfor Parasitology14: 453–458. Serum, intestinal and lung immunoglobulin and antibody isotype responses to Nippostrongylus brasiliensis infection were studied in normal and trypanosome-infected Hooded Lister rats. Rats which received trypanosomes 7 days before N. brasiliensis infection had impaired responses of serum IgG and IgA. Bronchial and intestinal mucosal IgG was not reduced whilst IgA concentration in these sites was markedly diminished. Total immunoglobulin M levels in T. brucei parasitised rats were higher in both sera and mucosal sites. However, tests with radiolabelled adult nematode excretory-secretory antigens indicated that specific lung and intestinal IgM responses were reduced. Immunoglobulin A antibody responses were diminished most markedly in sera and lungs and also in the intestine while IgG antibodies were decreased in sera and intestine mucosae T. brucei infected rats had higher worm burdens than rats infected with N. brasiliensis alone but worm expulsion was not delayed. The results indicate that local as well as systemic antibody responses are reduced in trypanosome infected animals.     

The source of antigen in an adult tapeworm

Hopkins C. A. and Barr I. F. 1982. The source of antigen in an adult tapeworm. International Journal for Parasitology12: 327–333. Although a primary infection of Hymenolepis diminuta is not rejected for 9–15 days by a mouse, it has been shown that a primary infection terminated chemically after only 3 days induces as good protection against challenge. This demonstrated that a scolex and 1–2 mm of neck tissue (all that is formed by day 3 post infection) are an adequate source of ‘protective’ antigen. Irradiated (350 Gy) cysticercoids which survive but show little growth immunize as effectively as normal cysticercoids which indicates actively growing neck tissue is not essential and hence the scolex alone is a sufficient source of ‘protective’ antigens. In the rat irradiated cysticercoids were found to establish, double their length over 3–6 days and then slowly shrink, but 14% of the worms were still present 49 days p.i. Although a primary infection of normal worms in a rat markedly depresses growth of a secondary infection administered 7 days after the chemical expulsion of the primary, irradiated scoleces induced no measurable protection. These results are discussed in relation to the source of antigen and the fundamental difference in the protective response of mice (an abnormal host) and rats (a normal host) to the tapeworm H. diminuta in the small intestine.     

Distribution of 14C-l-leucine in organs and tissues of guinea pigs infected with tRichostrongylus colubriformis

Earlier experiments with intestinal nematode infection which had shown changes to skeletal muscle and liver protein metabolism, did not examine the metabolism of the gastrointestinal tract nor attempt to integrate these changes with the whole body. Consequently the distribution of ¹⁴C-l-leucine in all organs and tissues of guinea pigs infected with Trichostrongylus colubriformis was compared with uninfected animals fed either ad libitum or quantitatively reduced rations.There were no differences between experimental groups in total radioactivities recovered, but in the infected animals radioactivity accumulated in the liver, stomach and small intestine, and caecum and large intestine at the expense of the eviscerated carcase and skin. Reducing the ration of uninfected guinea pigs did not affect the distribution of leucine, apart from reducing the fraction in the eviscerated carcase. Incorporation of ¹⁴C-l-leucine and its relationship to protein synthesis in the livers and eviscerated carcases of the three experimental groups is discussed. It was concluded that events in the small and large intestines, which are independent of anorexia, are important components of protein metabolism in intestinal nematode infection.