Resistance against Echinostoma caproni (Trematoda) secondary infections in mice is not dependent on the ileal protein production.

UNLABELLED: Echinostoma caproni (Trematoda: Echinostomatidae) is an intestinal trematode, which has been widely employed to investigate the factors determining the rejection of intestinal helminths. Protein production patterns of intestinal epithelial cells are related to the infection-induced changes that determine the course of E. caproni infections. Herein, we compare the protein production profiles in the ileum of four experimental groups of mice: control; infected; dewormed and reinfected. Worm burdens were significantly lower in secondary infections, confirming the generation of partial resistance to homologous secondary infections in mice. However, quantitative comparison by 2D-DIGE showed that the protein production profile is similar in control and dewormed mice, and after primary and secondary E. caproni infections. These results showed that, unexpectedly, protein production changes in E. caproni infections are not responsible of resistance development. Fifty-one protein spots were differentially produced between control/treated and infected/reinfected mice and 37 of them were identified by mass spectrometry. The analysis of differentially abundant proteins indicate that cell metabolism and the regulation of proliferation and cell death are the most affected processes after primary and secondary E. caproni infections. These results provide new insights into the proteins involved in the regulation of tissue homeostasis after intestinal infection. SIGNIFICANCE: Intestinal helminthiases are highly prevalent parasitic infections with about 1 billion people infected worldwide. In this scenario, better understanding of host-parasite relationships is needed to elucidate the factors that determine intestinal helminth rejection. The intestinal trematode Echinostoma caproni has been broadly employed in this field, with resistance against secondary homologous infections reported in mice. In this paper, new insights are provided in the regulation of tissue homeostasis after intestinal infection. The unexpected lack of an altered pattern of ileal protein production associated to resistance development suggests that this resistance depends on rapid changes, affecting the early establishment of worms, rather than the activation of later effector mechanisms. These results may contribute to the development of new control tools for the management of these parasitic infections.

Animal Models for Echinostoma malayanum Infection: Worm Recovery and Some Pathology.

Echinostomes are intestinal trematodes that infect a wide range of vertebrate hosts, including humans, in their adult stage and also parasitize numerous invertebrate and cold-blooded vertebrate hosts in their larval stages. The purpose of this study was to compare Echinostoma malayanum parasite growth, including worm recovery, body size of adult worms, eggs per worm, eggs per gram of feces, and pathological changes in the small intestine of experimental animals. In this study, 6-8-week-old male hamsters, rats, mice, and gerbils were infected with echinostome metacercariae and then sacrificed at day 60 post-infection. The small intestine and feces of each infected animal were collected and then processed for analysis. The results showed that worm recovery, eggs per worm, and eggs per gram of feces from all infected hamsters were higher compared with infected rats and mice. However, in infected gerbils, no parasites were observed in the small intestine, and there were no parasite eggs in the feces. The volume of eggs per gram of feces and eggs per worm were related to parasite size. The results of histopathological changes in the small intestine of infected groups showed abnormal villi and goblet cells, as evidenced by short villi and an increase in the number and size of goblet cells compared with the normal control group.

Differential alterations in the small intestine epithelial cell turnover during acute and chronic infection with Echinostoma caproni (Trematoda).

BACKGROUND: The intestinal epithelium plays a multifactorial role in mucosal defense. In this sense, augmented epithelial cell turnover appears as a potential effector mechanism for the rejection of intestinal-dwelling helminths. METHODS: A BrdU pulse-chase experiment was conducted to investigate the infection-induced alterations on epithelial cell kinetics in hosts of high (mouse) and low (rat) compatibility with the intestinal trematode Echinostoma caproni. RESULTS: High levels of crypt-cell proliferation and tissue hyperplasia were observed in the ileum of infected mice, coinciding with the establishment of chronic infections. In contrast, the cell migration rate was about two times higher in the ileum of infected rats compared with controls, with no changes in tissue structure, indicating that an accelerated cell turnover is associated with worm expulsion. CONCLUSION: Our results indicate that E. caproni infection induces a rapid renewal of the intestinal epithelium in the low compatible host that may impair the establishment of proper, stable host-parasite interactions, facilitating worm clearance.

Intestinal IFN-γ production is associated with protection from clinical signs, but not with elimination of worms, in Echinostoma caproni infected-mice.

In the present paper, we assess the relationship between the expression of IFN-γ and the development of clinical signs in Echinostoma caproni-infected mice. For this purpose, we studied the course of the infection in three mouse strains: ICR (CD-1®) (a host of high compatibility with E. caproni), BALB/c (a prototypical Th2 strain), and BALB/c deficient for IFN-γ mice (IFN-γ(-/-)). Infection in ICR mice is characterized by the elevated expression of IFN-γ and iNOS in the intestine concomitantly with the lack of clinical signs. In contrast, the infection was more virulent in BALB/c and IFN-γ-deficient mice that developed a severe form of the disease together with the absence of IFN-γ expression. The disease was more severe in IFNγ(-/-) mice in which the disease was lethal during the few first weeks of the infection. The analysis of different parameters of the infection in each host strain showed that most of the features were similar in the three mouse strains, suggesting the IFN-γ plays a central role in that protection against severe disease. Thus, IFN-γ seems to play a dichotomous role in the infection facilitating the parasite establishment, but it may also benefit mice since it protects the mice from morbidity and mortality induced by the parasite.

Control de parásitos gastrointestinales en caballos pura sangre de carrera (Equus Caballus) en el Hipódromo "La Rinconada" Caracas, Venezuela / Control of gastrointestinal parasites in thoroughbred horses (Eqqus Caballus) the Racetrack

Se realizo un estudio coprológico por la técnica de flotación Mc Master (Willis-Molloy), realizando contaje de Huevos por gramo de Heces (HPG), a 100 equinos Pura Sangre de Carreras, durante el periodo de cuarentena Hipodromo “La Rinconada” Caracas Venezuela. Se procedió a la desparasitacióncon una terapéutica a base de Febantel, dosis 6mg/kg, presentación en pasta, vía oral (CALOXBANTEL) Febantel 88.7 mg; Excipientes c.s.p. 1g. A los 7 días post-desparasitación se realizo un estudio coprológico por la técnica de flotación Mc Master (Willis-Molloy), realizando contaje de Huevos por gramo de Heces (HPG). El estudio coprológico evidencio un 60% de infestación (60/100) en los caballos estudiados. El 40% (40/100) fue negativo al examen coprológico. Los resultados post-tratamientos fueron 1% de infestación persistente (01/100) y un 99% (99/100) negativos al examen coprológico. En todos los casos la infestación parasitaria fue por Strongylus sp. La presencia de Strongylus sp. se mantuvo por equino infestado entre un rango de 400-1200 HPG

We study 100 Thoroughbred horses, in the Racetrack “La Rinconada” Caracas, Venezuela, by McMaster flotation technique (Willis-Molloy), making counting of eggs per gram of feces (EPG) before deworming, each of the copies, then proceeded to the parasite with a Febantel based therapy, dose 6mg/kg, pasta presentation, oral (CALOXBANTEL) Febantel 88.7 mg, Excipients 1g. At 7 days post-parasite stool study was conducted by the McMaster flotation technique (Willis- Molloy), by counting eggs per gram of faeces (EPG). The coprology study showed 60% infestation (60/100) in horses studied. The 40% (40/100) was negative. The post-treatment were 1% infestation (01/100) and 99% (99/100) was negative. In all cases, parasite infestation was by Strongylus sp. within the range of 400-1200 HPG

Biochemical and histological responses of Rattus novergicus (Wistar) infected by Echinostoma paraensei (Trematoda: Echinostomatidae).

Tests were performed to evaluate the biochemical alterations in Rattus norvegicus after infection by the intestinal trematode Echinostoma paraensei. The rodents received 150 metacercariae each, serum samples were collected and the parasite load was quantified weekly until the fifth week of infection. The levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALKP), gamma-glutamyl transferase (GGT), bilirubin, glucose, total proteins and fractions and hepatic glycogen were determined. All the animals exposed to the metacercariae were infected in the first week and worms were recovered up to the third week after infection. The levels of AST, ALT, GGT, bilirubin and globulin rose in the first and/or second week and declined thereafter to levels near those of the control group. In contrast, the level of total proteins in the plasma fell significantly in the first week while the ALKP activity went down only in the fourth and fifth weeks in relation to the control group. There was no significant difference in the levels of albumin, glycogen and glucose. Infection by E. paraensei in R. norvegicus causes changes in the hepatic function, possibly resulting from the cholestasis produced by the partial obstruction of the bile duct by the helminths.

Effects of Echinostoma caproni infection on the neutral and polar lipids of intestinal and non-intestinal organs in the BALB/c mouse as determined by high-performance thin-layer chromatography.

The goal of this study was to characterize and quantify the various neutral and polar lipid classes in the BALB/c mouse that are associated with Echinostoma caproni infection. Ten infected mice and 10 uninfected control mice were used for this study (five infected and five uninfected were used for each of the neutral lipid and polar lipid studies). After 3 weeks postinfection, the mice were necropsied and various organs were removed and prepared for lipid class analysis. The organs used were liver, kidney, spleen, colon, cecum, anterior portion of the small intestine (SI), middle portion of the SI, and posterior portion of the SI. Lipids were determined by high-performance thin-layer chromatography (HPTLC) with Analtech 10 x 20 cm HPTLC-HLF silica gel plates. For neutral lipids, petroleum ether-diethyl ether-glacial acetic acid (80:20:1) mobile phase and 5% ethanolic phosphomolybdic acid detection reagent were used to determine the neutral lipids in each organ. Chloroform-methanol-deionized water (65:25:4) mobile phase and 10% cupric sulfate in 8% phosphoric acid detection reagent were used to determine the polar lipids in each organ. The analyzed polar lipids in all organs were phosphatidylcholine (PC), phosphatidylethanolamine (PE), and sphingomyelin (SM). Using HPTLC-densitometry for quantification, PC was found in the greatest amount and SM the smallest of all organs analyzed. The PE in the anterior portion of the SI was determined to be significantly greater (using the Student's t test with P < 0.05), with about twice the amount of PE in mice infected with E. caproni relative to the uninfected mice. No significant differences in any of the neutral lipid classes were found between infected and uninfected samples.

Metabolic profiling of an Echinostoma caproni infection in the mouse for biomarker discovery.

BACKGROUND: Metabolic profiling holds promise with regard to deepening our understanding of infection biology and disease states. The objectives of our study were to assess the global metabolic responses to an Echinostoma caproni infection in the mouse, and to compare the biomarkers extracted from different biofluids (plasma, stool, and urine) in terms of characterizing acute and chronic stages of this intestinal fluke infection. METHODOLOGY/PRINCIPAL FINDINGS: Twelve female NMRI mice were infected with 30 E. caproni metacercariae each. Plasma, stool, and urine samples were collected at 7 time points up to day 33 post-infection. Samples were also obtained from non-infected control mice at the same time points and measured using (1)H nuclear magnetic resonance (NMR) spectroscopy. Spectral data were subjected to multivariate statistical analyses. In plasma and urine, an altered metabolic profile was already evident 1 day post-infection, characterized by reduced levels of plasma choline, acetate, formate, and lactate, coupled with increased levels of plasma glucose, and relatively lower concentrations of urinary creatine. The main changes in the urine metabolic profile started at day 8 post-infection, characterized by increased relative concentrations of trimethylamine and phenylacetylglycine and lower levels of 2-ketoisocaproate and showed differentiation over the course of the infection. CONCLUSION/SIGNIFICANCE: The current investigation is part of a broader NMR-based metabonomics profiling strategy and confirms the utility of this approach for biomarker discovery. In the case of E. caproni, a diagnosis based on all three biofluids would deliver the most comprehensive fingerprint of an infection. For practical purposes, however, future diagnosis might aim at a single biofluid, in which case urine would be chosen for further investigation, based on quantity of biomarkers, ease of sampling, and the degree of differentiation from the non-infected control group.

Development and pathology of Echinostoma caproni in experimentally infected mice.

In the present article, several parasitological features of mice, each experimentally infected with 75 metacercariae of Echinostoma caproni (Trematoda: Echinostomatidae), were studied during the first 12 wk postinfection. Moreover, the early pathological responses also were analyzed and compared with data previously published on other host species of E. caproni to gain further insight into the factors determining worm rejection or establishment of chronic infections. The results obtained show that the pattern of E. caproni infection in mice is consistent with a highly compatible host-parasite system. This combination is characterized by a high worm establishment, high egg output, and long survival of the worms. However, some differences with respect to other highly compatible hosts have been observed, particularly in relation to the survival of the adult worms. Histological studies suggest that the kinetics of goblet cells, mucosal neutrophils, and mononuclear inflammatory cells in the mesentery seem to be essential in determining the course of E. caproni infection in mice.

Echinostoma caproni: intestinal pathology in the golden hamster, a highly compatible host, and the Wistar rat, a less compatible host.

The histopathological changes induced by Echinostoma caproni (Trematoda: Echinostomatidae) in a high (golden hamster) and a low compatible host (rat) were compared at 15 and 30 days post-infection. Infection of rats was characterized by a progressive increase in erosion of villi and elevated numbers of goblet cells, which could be related to the early expulsion of the parasite in a host of low compatibility. In contrast to rats, the number of goblet cell in E. caproni-infected hamsters was low, but increased numbers of neutrophils and mesenteric inflammatory cells were observed. This indicated that local inflammatory responses in hamsters were greater than in rats. An immunohistochemical study using polyclonal IgG anti-E. caproni excretory-secretory antigens demonstrated a greater level of passage of E. caproni antigens through the intestinal mucosa in hamsters than in rats, probably in relation to the greater inflammatory response. Our results indicate the fact that early inflammatory responses could be important for the establishment of E. caproni chronic infections in highly compatible hosts.

Parasite-induced trophic facilitation exploited by a non-host predator: a manipulator's nightmare.

Parasites with complex life cycles, relying on trophic transmission to a definitive host, very often induce changes in the behaviour or appearance of their intermediate hosts. Because this usually makes the intermediate host vulnerable to predation by the definitive host, it is generally assumed that the parasite's transmission rate is increased, and that the modification of the host is, therefore, of great adaptive significance to the parasite. However, in the ecological "real world" other predators unsuitable as hosts may just as well take advantage of the facilitation process and significantly erode the benefit of host manipulation. Here we show that the intertidal New Zealand cockle (Austrovenus stutchburyi), manipulated by its echinostome trematode (Curtuteria australis) to rest on the sediment surface fully exposed to predation from the avian definitive host, is also subject to sublethal predation from a benthic feeding fish (Notolabrus celidotus, Labridae). The fish is targeting only the cockle-foot, in which the parasite preferentially encysts, reducing the infection intensity of manipulated cockles to levels comparable with those in non-manipulated, buried cockles. Based on the frequency and intensity of the foot cropping and predation rates on surfaced cockles by avian hosts, it is estimated that 2.5% of the parasite population in manipulated cockles is transmitted successfully whereas 17.1% is lost to fish. We argue that the adaptive significance of manipulation in the present system depends critically on the feeding behaviour of the definitive host. If cockles constitute the majority of prey items, there will be selection against manipulation. If manipulated cockles are taken as an easily accessible supplement to a diet composed mostly of other prey organisms, behavioural manipulation of the cockle host appears a high risk, high profit transmission strategy. Both these feeding behaviours of birds are known to occur in the field.

Effects of a 100 metacercarial cyst inoculum on the host-parasite relationship of Echinostoma caproni and ICR mice.

The host-parasite relationship of a 100 metacercarial cyst inoculum of Echinostoma caproni in the ICR mouse was examined. Three groups of mice, A, B and C, each with six mice per group were used and all mice were necropsied at 14 days postinfection (p.i.), at which time the worms were ovigerous. Group A consisted of uninfected controls, whereas group B received 25 cysts per mouse (low dose) and group C received 100 cysts per mouse (high dose). There was no significant difference in food consumption between any of the groups from 0 to 14 days p.i. Control mice increased their body weight by 12%, group B by 5%, and group C showed a less than 1% increase in body weight between 0 and 14 days p.i. Echinostome parasitism caused a significant increase in the diameter of the mouse gut, with the gut of group C being more significantly dilated than that of either group A or B. The average worm recovery from group B was 20 worms per host, compared to 72 worms per host from group C. The mean wet and dry weights per worm from group B were 2.4 and 0.4 mg, respectively as compared to 0.6 and 0.2 mg respectively for group C. The mean number of uterine eggs per worm from group B was 180 compared to 125 for worms from group C. Worms from group C were more widely distributed in the small intestine than those from group B. Crowding effects associated with the high dose infection were clearly demonstrated in E. caproni from ICR mice.

Effects of a protein-free diet on worm recovery, growth, and distribution of Echinostoma caproni in ICR mice.

The effects of a protein-free diet on the host-parasite relationship of Echinostoma caproni in ICR mice were studied. The experimental diet was a customized protein-free diet (PFD) in pellet form containing 0% protein. The control diet consisted of a standard laboratory diet containing 23% casein as a source of protein. A total of 24 mice were each infected with 15 metacercarial cysts of E. caproni. Twelve mice were placed on the experimental diet (experimentals) and the remaining mice (controls) were placed on the control diet. Experimental and control mice were necropsied at 2, 3, and 4 weeks postinfection (p.i.). The weight of mice on the PFD was markedly lower than that of mice on the control diet. The length and circumference of the small intestine of infected mice on the PFD were significantly lower than those of the controls at 3 weeks p.i. (Student's t-test; P < 0.05). Worm recoveries from mice on the PFD were significantly lower than those of the controls at 3 weeks p.i. There was a significant decline in worm body area in worms from the mice on the PFD compared with those on the control diet at 2, 3, and 4 weeks p.i. Worm dry weights from mice on the PFD were significantly lower than those on the control diet at 2 weeks p.i. Worms from hosts on the PFD were located more posteriad in the gut than those recovered from mice on the control diet. The findings suggest that the PFD contributes to growth retardation of E. caproni in ICR mice.

Host-parasite relationships between Echinostoma caproni and RAG-2-deficient mice.

The RAG-2-deficient mouse, a strain of genetically altered mice lacking B- and T-lymphocytes, was used as a host for Echinostoma caproni. In all, 12 male RAG mice were exposed to 25 cysts each, and 12 served as uninfected controls. Mice were necropsied at 2 and 3 weeks postinfection (p.i.). The mean number+/-SE (9.7+/-2.4) of worms recovered from infected mice at 2 weeks p.i. was not significantly different from that recovered at 3 weeks p.i. (6.5+/-2.2). The intestinal circumference of infected RAG mice was significantly greater than that of the controls at 2 and 3 weeks p.i. A significant goblet cell hyperplasia occurred at 2 weeks p.i., but the response was not effective in eliminating worms from the RAG mice. The effect of a high cyst burden was examined by exposure of 8 RAG and 8 ICR mice to 100 cysts each. The body length and area and the oral sucker area of worms grown in RAG mice were significantly greater than those of worms grown in ICR mice. Worm recovery at up to 3 months p.i. was examined in RAG mice exposed to 25 cysts and necropsied every 2 weeks p.i. The mean worm recovery recorded at 2 weeks p.i. was significantly greater than that noted at 12 weeks p.i., at which time worm rejection from the RAG mouse host first occurred. The RAG mouse is a useful host for studies on E. caproni in a murine host that lacks B- and T-lymphocytes.

Experimental infection of Rana pipiens tadpoles with Echinostoma trivolvis cercariae.

Studies were done on laboratory-raised Rana pipiens tadpoles experimentally infected with Echinostoma trivolvis cercariae. Tadpoles exposed individually to 250 cercariae died within 24 h. They were edematous at death and their kidneys were heavily infected with metacercarial cysts. Of 20 tadpoles exposed to 100 cercariae each, 9 survived the infection, and their growth was compared for 4 weeks postinfection (p.i.) with that of 20 control tadpoles that had not been exposed to cercariae. There was a significant weekly decline in the total length and body weight of the infected versus control tadpoles. Surviving tadpoles retained their metacercarial infections in the kidneys following metamorphosis to frogs. Following exposure of tadpoles to cercariae, cercarial bodies were first seen in the kidneys by 0.5 h p.i. Metacercariae that were molding their inner and outer cyst walls were first seen at 2.3 h, and by 8.5 h the inner and outer cyst walls were clearly defined. Domestic chicks exposed to cysts aged 2.5 and 4.0 h did not become infected, whereas ovigerous adults of E. trivolvis were recovered from chicks fed 12-h-old cysts. Cercariae aged 6 to 8 h were more infective to tadpoles than were either 1- or 20-h-old cercariae. The E. trivolvis-R pipiens tadpole model is suitable for the study of host-parasite relationships of echinostome larvae in a cold-blooded vertebrate host.

Rapid expulsion of the intestinal trematodes Echinostoma trivolvis and E. caproni from C3H mice by trapping with increased goblet cell mucins.

Echinostoma trivolvis (Cort, 1914) adults were rejected from C3H mice by 15 days post-exposure, corresponding to the increase in the number of goblet cells. Homologous and heterologous infections with the allopatric species E. caproni (Richard, 1964) were used to confirm the effect of increased secretion of goblet cell mucins in rejecting metacercariae of challenge infections of E. trivolvis or E. caproni on days 10, 16 and 20 p.i. after primary infections of E. trivolvis metacercariae. Five-day-old juveniles of E. trivolvis and E. caproni, which were recovered from C3H mice or hamsters, were also used for challenge infections on day 10 p.i. The metacercariae and juveniles, which were challenged homologously and heterologously on day 10 p.i., were almost all expelled. The metacercariae of E. trivolvis, which were challenged homologously on day 16, were completely rejected, but only a few challenged metacercariae of E. caproni in heterologous infection were recovered. Considerable numbers of E. caproni were recovered when challenge infections with the metacercariae were done on day 20 p.i., while only a small number of E. trivolvis was recovered. All controls without primary infections showed a recovery rate of over 50% of the worms. These results indicate that increased secretion of mucins by hyperplastic goblet cells associated with primary infections of E. trivolvis may be responsible for the expulsion of worms challenged homologously with E. trivolvis and heterologously with E. caproni from the mouse host.

Pathogenicity of Artyfechinostomum oraoni in naturally infected pigs.

In an attempt to establish the diarrhoeogenic potential of the newly identified Artyfechinostomum oraoni, which was associated with human diarrhoea in a tribal community near Calcutta, India, two naturally infected domestic pigs of the locality were followed in captivity. Both pigs developed fatal diarrhoea after 5 months. The autopsy revealed a massive infection with the echinostome on a haemorrhagic and oedematous mucosa of the jejunum and duodenum extending up to pyloric end of the stomach. It is suggested that similar pathology might also be operating in the infected man.

The effect of dexamethasone on the course of Echinostoma caproni and E. trivolvis infections in the golden hamster (Mesocricetus auratus).

Golden hamsters (Mesocricetus auratus) were given intramuscular injections of dexamethasone and infected with Echinostoma caproni or E. trivolvis. All animals were necropsied on day 14 postinfection. Dexamethasone treatment at high doses resulted in increased parasite recovery. Decreased total white blood cell counts and decreased relative splenic weights were observed in corticosteroid-treated hamsters. Dexamethasone-treated animals also demonstrated significantly lower mean parasite dry weights for E. caproni. Specific serum IgG against the parasites was not detected in corticosteroid-treated hamsters on day 14 postinfection.

Echinostoma caproni and E. trivolvis alter the binding of glycoconjugates in the intestinal mucosa of C3H mice as determined by lectin histochemistry.

Mouse (C3H) mucosal glycoconjugates were examined in normal small intestines and intestines infected with Echinostoma caproni or E. trivolvis using six different fluorescein-conjugated lectins: Triticum vulgaris agglutinin (WGA), Ulex europaeus agglutinin I (UEA-I), Ricinus communis agglutinin I (RCA-I), Glycine max soybean agglutinin (SBA), Dolichos biflorus agglutinin (DBA), and Arachis hypogaea peanut agglutinin (PNA). The expression of lectin-binding sites and the intensity of the binding of lectins in the mouse small intestines were changed by infection with the echinostomes. Specific differences in the reaction to glycoproteins were clearly observed between the mouse intestines infected with E. caproni and those infected with E. trivolvis. In E. caproni infection, binding of most of the lectins to the villi was remarkably reduced in accord with the villous atrophy and loss of goblet cells. In contrast, in E. trivolvis infection, the binding of WGA, RCA-I and DBA was reduced in the microvillar surfaces, but binding of UEA-I and SBA were unchanged compared to the control intestines. The lectin binding to goblet cells in E. trivolvis-infected mice mostly increased. These observations may reflect the marked increase in goblet cells and the less severe damage in the villi of E. trivolvis infection compared to E. caproni infection. Most of the glycoconjugates were slightly reduced in the hyperplastic crypts except for N-acetyl glucosamine. It is possible that glucose metabolism in the host intestines infected with E. trivolvis was activated, resulting in an increase in the rate of mucin synthesis as well as qualitative changes in mucus, thereby mediating the expulsion of the worms.