Eosinophil and IgE responses of IL-5 transgenic mice experimentally infected with Nippostrongylus brasiliensis.

Eosinophil and IgE responses of interleukin (IL)-5 transgenic and normal C3H/HeN mice were studied after experimental infection with Nippostrongylus brasiliensis (Nb). Intestinal worms were recovered at day 5 post-infection (PI), and numbers of total white blood cells (WBC) and eosinophils, and total serum IgE and anti-hapten (dinitrophenyl) (DNP) specific IgE titers, were measured at days 0, 14 and 21 PI. IL-5 mice appeared resistant to Nb infection showing a significantly lower worm recovery rate than normal mice (P < 0.05). Total WBC and eosinophil counts (/mm3) were significantly increased in Nb infected normal mice (P < 0.05), but unchanged (total WBC) or decreased (eosinophils) in IL-5 mice at day 21 PI. The total serum IgE level remarkably increased in normal mice, but only a little in IL-5 mice at days 14 and 21 PI. Priming with DNP brought about more remarkable increases of the total and anti-DNP specific IgE in normal mice than in IL-5 mice. The results show that IL-5 mice are resistant to Nb infection, and that eosinophil and IgE responses in these mice are not augmented by Nb infection.     

Mucosal mast cell responses in the small intestine of rats infected with Echinostoma hortense

Mucosal mast cell (MMC) responses and worm recovery rates in rats infected with Echinostoma hortense were investigated from day 3 to day 56 post-infection (p.i.). Experimental infected group showed apparently higher number of MMC in each part of the small intestine than that of the control group. The number of MMC in the duodenum increased gradually after the infection and reached a peak on day 35 p.i. Thereafter, the number of MMC continued to decrease at a slow pace. The kinetics of MMC responses in the upper and lower jejunum were similar to that of the duodenum, but the number of MMC in the jejunum was lower. The worm recovery rate decreased with respect to time of which it was markedly reduced on day 49 and 56 p.i. The duration in which a high number of MMC appeared was similar to that in which a low rate in worm recovery was recorded. These results indicate that intestinal mastocytosis may play an important role in the expulsion of E. hortense.     

Strain variation in the susceptibility and immune response to Clonorchis sinensis infection in mice

Mice have shown various susceptibility to infection by Clonorchis sinensis. To compare the intra-specific variation in the host-parasite relationship of C. sinensis, 6 strains of mice (ICR, BALB/c, C57BL/6, DDY, CBA/N, and C3H/HeN) with 3 different haplotypes were evaluated on their susceptibility. The worm recovery rate and immunological responses were observed after 4 and 8 weeks of infection with 30 metacercariae. The highest worm recovery rate was observed as 20.7% in the C3H/HeN strain after 4 weeks of infection along with histopathological changes. The rate was 10.0% in C57BL/6 mice after 8 weeks. ICR, BALB/c, and CBA/N showed elevated levels of IgE at both time points when compared to the rest of the strains. The serum IgG1 and IgG2a levels were elevated in most of the strains; however, the C57BL/6 strain showed a lower level of IgG2a that indicated the IgG1 predominance over IgG2a. The production of IL-4 after concanavalin-A stimulation of splenocytes slightly increased among the mouse strains except C3H/HeN after 4 or 8 weeks of infection, but each strain produced high levels of IFN-γ after 8 weeks, which implied mixed Th1/Th2 responses. ICR, DDY, CBA/N, and C3H/HeN strains showed a significantly increased level of IL-10 after 8 weeks as compared to C57BL/6. All of the strains showed an increased level of IL-13 and suggested fibrotic changes in the mice. In conclusion, mice are insusceptible to infection with C. sinensis; however, the C57BL/6, BALB/c and ICR strains are relatively susceptible after 8 weeks of infection among the six strains. Worm expulsion may be one of the causes of low susceptibility of C3H/HeN mice strain at the 8th week. Elevated IgE, IFN-γ, and IL-13 of infected mice suggest both Th1 and Th2 responses that may be related to the low host susceptibility.     

Nippostrongylus brasiliensis: Increase of sialomucins reacting with anti-mucin monoclonal antibody HCM31 in rat small intestinal mucosa with primary infection and reinfection

Infections with the parasitic helminth, Nippostrongylus brasiliensis, cause changes in rat small intestinal goblet cell mucin, particularly in the peripheral sugar residues of oligosaccharide. These changes may correlate with expulsion. In this study, we examined changes in mucin oligosaccharides caused by primary infection and reinfection with N. brasiliensis, using two monoclonal antibodies, HCM31 and PGM34, that react with sialomucin and sulfomucin, respectively. Enzyme-linked immunosorbent assay of jejunal mucins showed that the relative reactivity of mucins with HCM31, but not PGM34, increased up to 16 days after primary infection and 6 days after reinfection, the times when the worms were expelled from the rats. Immunohistochemical studies confirmed that goblet cells stained with HCM31 greatly increased at the time of worm expulsion. These results indicate that the marked increase observed in HCM31-reactive sialomucins may be related to expulsion of the worms.     

Inflammatory cell stimulation and wound healing in Sphaeridiotrema globulus experimentally infected mallard ducks (Anas platyrhynchos)

Thirty laboratory-reared mallard ducks (Anas platyrhynchos) were experimentally infected with Sphaeridiotrema globulus. Host cell-mediated immunity and wound healing in S. globulus infected ducks were evaluated by gross and histological examination. Establishment, location, and life span of S. globulus differed from previous reports of sphaeridiotremiasis in both naturally and experimentally infected waterfowl. No worms were recovered from the ceca, and worm migration occurred anterior to the ileo-cecal valve with greater dispersion (less worm crowding) at higher rates of infectivity. Parasite death and host lesion resolution were evident at days 8 to 10 postinfection (PI) in ducks fed a moderate dose (200 metacercariae, group A) with a 5% mean parasite recovery rate. Host death occurred at days 3 to 6 in ducks fed a high dose (550 metacercariae, group B) with a 16% mean parasite recovery rate. Mast cells increased significantly (P less than 0.005) in group A ducks from days 4 to 10 PI. Eosinophil proliferation was greater in group B than in group A on day 4 PI, but comparatively fewer eosinophils were identifiable in group B ducks on day 6 PI.     

Increased Intestinal Epithelial Cell Turnover and Intestinal Motility in Gymnophalloides seoi-Infected C57BL/6 Mice

The changing patterns of goblet cell hyperplasia, intestinal epithelial cell turnover, and intestinal motility were studied in ICR and C57BL/6 mice infected with Gymnophalloides seoi (Digenea: Gymnophallidae). Whereas ICR mice retained G. seoi worms until day 7 post-infection (PI), C57BL/6 mice showed a rapid worm expulsion within day 3 PI. Immunosuppression with Depo-Medrol significantly delayed the worm expulsion in C57BL/6 mice. Goblet cell counts were increased in both strains of mice, peaking at day 1 PI in C57BL/6 mice and slowly increasing until day 7 PI in ICR mice. In C57BL/6 mice infected with G. seoi, newly proliferating intestinal epithelial cells were remarkably increased in the crypt, and the increase was the highest at day 1 PI. However, in ICR mice, newly proliferating intestinal epithelial cells increased slowly from day 1 to day 7 PI. Intestinal motility was increased in G. seoi-infected mice, and its chronological pattern was highly correlated with the worm load in both strains of mice. Meanwhile, immunosuppression of C57BL/6 mice abrogated the goblet cell proliferation, reduced the epithelial cell proliferation, and suppressed the intestinal motility. Goblet cell hyperplasia, increased intestinal epithelial cell turnover, and increased intestinal motility should be important mucosal defense mechanisms in G. seoi-infected C57BL/6 mice.     

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.     

Hyperendemicity of Haplorchis taichui Infection among Riparian People in Saravane and Champasak Province,Lao PDR

In this study, we found that Haplorchis taichui, a heterophyid intestinal fluke, is highly prevalent, with heavy worm loads, among riparian people in Saravane and Champasak province, Lao PDR. Fecal specimens were collected from 1,460 people (717 men and 743 women) in 12 riparian (Mekong river) districts and were examined by the Kato-Katz fecal smear technique. The overall helminth egg positive rate was 78.8% and 66.4% in Saravane and Champasak province, respectively. The positive rate for small trematode eggs (STE), which included H. taichui and other heterophyids, Opisthorchis viverrini, and lecithodendriids, was 69.9% and 46.3% in Saravane and Champasak province, respectively. To obtain adult flukes, 30 STE-positive people were treated with 40 mg/kg praziquantel and then purged. Whole diarrheic stools were collected 4-5 times for each person and searched for fluke specimens using a stereomicroscope. Mixed infections with various species of trematodes (H. taichui, Haplorchis pumilio, O. viverrini, Prosthodendrium molenkampi, Centrocestus formosanus, and Echinochasmus japonicus) and a species of cestode (Taenia saginata) were found. However, the worm load was exceptionally high for H. taichui compared with other trematode species, with an average of 21,565 and 12,079 specimens per infected person in Saravane and Champasak province, respectively, followed by H. pumilio (41.9 and 22.5, respectively) and O. viverrini (9.4 and 1.5, respectively). These results show that diverse species of intestinal and liver flukes are prevalent among riparian people in Saravane and Champasak province, Lao PDR, with H. taichui being the exceptionally dominant species.     

Immunity to intestinal parasites: role of mast cells and goblet cells

Nippostrongylus brasiliensis infection of rats and mice is a model for studying immunity at mucosal surfaces. Adult worms are spontaneously expelled from the intestine at the end of the second week of infection. Expulsion from the jejunum requires the presence of immune T lymphocytes and IgG antibodies. Mucosal mast cells (MMCs) are a prominent part of the jejunal inflammatory response. They are derived from a hematopoietic stem cell, possibly the same precursor as basophils. Their differentiation is not absolutely T dependent but their accumulation at the site of infection is. The possible involvement of IgE antibodies and intestinal MMCs through a "leak lesion" is still uncertain. Increased mucus secretion from epithelial goblet cells is also a prominent feature of the inflammatory reaction at the site of infection. Goblet cell numbers increase two to four times at the onset of worm expulsion; this increase is regulated by T lymphocytes and possibly immune serum. The mechanism of mucus secretion in these infections is not clear; it may be a response to mast cell mediators. Together with antiworm antibodies, intestinal mucus may trap worms and prevent them from surviving in the intervillous spaces of the jejunum. Thus, expulsion of this intestinal parasite may occur through a nonspecific process that is induced by specific immune mechanisms.     

Mucosal Immune Responses of Mice Experimentally Infected with Pygidiopsis summa (Trematoda: Heterophyidae)

Mucosal immune responses against Pygidiopsis summa (Trematoda: Heterophyidae) infection were studied in ICR mice. Experimental groups consisted of group 1 (uninfected controls), group 2 (infection with 200 metacercariae), and group 3 (immunosuppression with Depo-Medrol and infection with 200 metacercariae). Worms were recovered in the small intestine at days 1, 3, 5, and 7 post-infection (PI). Intestinal intraepithelial lymphocytes (IEL), mast cells, and goblet cells were counted in intestinal tissue sections stained with Giemsa, astra-blue, and periodic acid-Schiff, respectively. Mucosal IgA levels were measured by ELISA. Expulsion of P. summa from the mouse intestine began to occur from days 3-5 PI which sustained until day 7 PI. The worm expulsion was positively correlated with proliferation of IEL, mast cells, goblet cells, and increase of IgA, although in the case of mast cells significant increase was seen only at day 7 PI. Immunosuppression suppressed all these immune effectors and inhibited worm reduction in the intestine until day 7 PI. The results suggested that various immune effectors which include IEL, goblet cells, mast cells, and IgA play roles in regulating the intestinal mucosal immunity of ICR mice against P. summa infection.     

Trypanosoma evansi: Hematologic changes in experimentally infected cats

This study aimed at evaluating hemogram and erythropoietic changes in cats experimentally infected with Trypanosoma evansi. Thirteen adult female non-breeding Felix catus were separated into two groups: seven animals were infected with 10⁸ trypomastigotes each, and six animals were used as negative controls. Animals were kept in air-conditioned rooms and blood smears were performed daily for 49 days. Blood samples were collected from the jugular vein at days 0, 7, 21, 35 and 49 and stored in blood-collecting tubes containing anticoagulant. Bone marrow was collected from the proximal epiphysis of the right femur at days 14 and 42 post-inoculation (PI). Total erythrocyte count, hematocrit and hemoglobin showed statistical differences among groups from the seventh day PI onwards (P < 0.05). The mean corpuscular volume and the mean corpuscular hemoglobin concentration remained normal, characterizing a normocytic-normochromic anemia. Reticulocyte count increased in the infected group from the 21st day onwards, but remained near normal values suggesting a mild regenerative anemia. Moreover, the myeloid:erythroid ratio significantly reduced at day 42 PI, evidencing a bone marrow hematopoietic response. Based on these results we conclude that cats infected with T. evansi have normocytic, normochromic, regenerative anemia.     

Egg Laying Capacity of Haplorchis taichui (Digenea: Heterophyidae) in Humans

Quantitative fecal egg counts represented as the number of eggs per gram of feces (EPG) are generally a reliable parameter to estimate the worm burden of intestinal and hepatic parasitoses. Although Haplorchis taichui (Digenea: Heterophyidae) is one of the most common minute human intestinal flukes, little is known about the relationship between EPG and the actual worm burden in patients or the severity of the disease. In the present study, fecal samples were collected from 25 villagers in northern Thailand before and after praziquantel treatment. The EPG values of each participant were determined by the modified cellophane thick smear method, and adult worms were collected from the whole stool after the treatment. Eggs per day per worm (EPDPW) of H. taichui were estimated 82 from egg counts and expelled worms. The EPG was not well correlated with the worm burden, and a reverse correlation was observed between the EPDPW and the worm burden.     

Haplorchis taichui, Witenberg, 1930: Development of a HAT-RAPD marker for the detection of minute intestinal fluke infection

Specific primers to determine the presence of an intestinal fluke, Haplorchis taichui, were investigated using the high annealing temperature random amplified polymorphic DNA (HAT-RAPD) PCR, and 18 arbitrary primers (Operon Technologies), to generate different polymorphic DNA profiles. Thirteen kinds of parasites were used to compare fingerprints. A 256 bp HAT-RAPD marker, generated from the OPP-11 primer, was found to be H. taichui-specific, and this marker was cloned, transformed, and sequenced. From the sequence data, a pair of primers were designed with Genetyx-MAC ver.11 and indicated as: Hap-t F 5′-GGC CAA CGC AAT CGT CAT CC-3′ and Hap-t R 5′-GCG TCG GGT TTC AGA CAT GG-3′. These specific primers were tested for efficacy and specificity by amplifying them with all 13 parasites DNAs in PCR reaction. A 256 bp amplicon was generated, which was shown to have a positive result, only for H. taichui DNA. It revealed no cross-reaction with any of the other tested parasite species. The minimum DNA template, needed for detection by PCR, was 0.1 picogram (pg). The successful development of H. taichui-specific primers is expected to be beneficial for epidemiological studies and for prevention and control of these parasitic infections.     

Nippostrongylus brasiliensis: histamine and 5-hydroxytryptamine inhibition and worm expulsion

The daily administration of inhibitors of histamine and 5-hydroxytryptamine to rats infected with Nippostrongylus brasiliensis prevented the onset of the rapid phase of worm expulsion. These findings lend strong support to the importance of amine release and Mast cell discharge in worm expulsion.     

Experimental concurrent infection of Afar breed goats with Oestrus ovis (L1) and Haemonchus contortus (L3): interaction between parasite populations, changes in parasitological and basic haematological parameters

The study was conducted to examine the occurrence and interaction between Oestrus ovis and Haemonchus contortus in experimentally infected Ethiopian Afar breed of goats. Twenty goats were divided into four groups (O, OH, H, and C) of five animals each. Each animal of groups O and OH received weekly infections for 5 weeks with 66 first instar larvae (L₁) of O. ovis. Then animals of groups OH and H were infected with a single dose of 5000 third stage larvae (L₃) of H. contortus. Goats of group C were kept free of any infection as non-infected control. Faecal egg count (FEC), blood cell count, total serum protein level and body weight were recorded weekly throughout the study period. At necropsy worm burden, female worm length, fecundity and larval burden of O. ovis in the nasal-sinus cavities of infected animals were assessed. The results showed that the presence of H. contortus in the abomasum of goats of group OH had no influence on the development of O. ovis. On the contrary, a significant reduction (P < 0.05) in FEC, worm burden, fecundity and female worm length was revealed in group OH animals compared to the mono-infected animals (group H). This was associated with eosinophilia and reduced packed cell volume.     

Susceptibility of Laboratory Rodents to Trichinella papuae

Members of the genus Trichinella are small nematodes that can infect a wide range of animal hosts. However, their infectivity varies depending on the parasite and host species combination. In this study, we examined the susceptibility of 4 species of laboratory rodents, i.e., mice, rats, hamsters, and gerbils to Trichinella papuae, an emerging non-encapsulated Trichinella species. Trichinella spiralis and Trichinella pseudospiralis were also included in this study for comparison. Fifteen animals of each rodent species were infected orally with 100 muscle larvae of each Trichinella species. Intestinal worm burden was determined at day 6 and 10 post-inoculation (PI). The numbers of muscle larvae were examined at day 45 PI. The reproductive capacity index (RCI) of the 3 Trichinella species in different rodent hosts was determined. By day 6 PI, 33.2-69.6% of the inoculated larvae of the 3 Trichinella species became adult worms in the small intestines of the host animals. However, in rats, more than 96% of adult worms of all 3 Trichinella species were expelled from the gut by day 10 PI. In gerbils, only 4.8-18.1% of adult worms were expelled by day 10 PI. In accordance with the intestinal worm burden and the persistence of adults, the RCI was the highest in gerbils with values of 241.5±41.0 for T. papuae, 432.6±48 for T. pseudospiralis, and 528.6±20.6 for T. spiralis. Hamsters ranked second and mice ranked third in susceptibility in terms of the RCI, Rats yielded the lowest parasite RCI for all 3 Trichinella species. Gerbils may be an alternative laboratory animal for isolation and maintenance of Trichinella spp.     

Coproantigen capture ELISA for detection of Clonorchis sinensis infection in experimentally infected rats

The present study investigated the diagnostic value of an ELISA for the detection of Clonorchis sinensis antigen in the feces of experimentally infected rats. A mouse polyclonal IgG antibody against adult C. sinensis crude antigen (CsAg) was used to capture the C. sinensis coproantigen. The detection limit for pure CsAg was 20 ng/ml in sample buffer and 40 ng/ml in uninfected fecal extract. The test was evaluated using a follow-up of five groups of rats experimentally infected with 100, 50, 10, 5 and 1 metacercariae of C. sinensis and an uninfected control group. Coproantigen was detected in all infected groups of rats from 2 weeks of infection, whereas fecal eggs were not observed until 3 weeks of infection. As the infection period progressed, the fecal CsAg concentration increased in all groups of infected rats, even those infected with a single metacercaria. The fecal CsAg concentration was correlated positively with fecal egg counts and worm burden. This coproantigen capture ELISA is highly sensitive for the detection of CsAg in rat feces, and with further development, should be useful for mass screening of human subjects in clonorchiasis-endemic areas.     

Immune expulsion of the trichostrongylid Cooperia oncophora is associated with increased eosinophilia and mucosal IgA

Previous experiments have shown that a primary infection with 100000 infective larvae of the trichostrongylid Cooperia oncophora allows discrimination between different type of responder animals based on the speed by which the parasite is expelled from the host. In most of the animals (intermediate responders) the expulsion occurs 35-42 days after infection. This experiment was carried out to investigate which mechanisms contribute to the clearance of the parasite from the intestine. Sequential necropsy of the animals 14, 28 and 42 days after infection together with a segmental division of the small intestine, allowed us to characterise essential components associated with development of immunity and expulsion of the parasite from its niche. The results show that during the patent phase of the infection the parasite preferentially resides in the proximal gut. Forty-two days after infection ongoing expulsion is characterised by a migration of the worms to the more distal part of the intestine. Expulsion of the adult worm population appears to be mast-cell independent and is associated with a significant increase in parasite-specific mucous IgA and IgG1 as well as with an influx of eosinophils in the intestinal lamina propria. Although we did not observe a specific lymphocyte recruitment into the intestinal mucosa, the accumulation of eosinophils seems to be mediated by CD4+ cells. We measured significant negative correlations between the number of eosinophils and the expulsion rate of the parasite expressed by sex ratio and ratio eggs per gram faeces. Parasite-specific mucosal IgA levels were negatively correlated to the fecundity of the worms, expressed as number of eggs per female worm. Our results describe the involvement of both eosinophils and mucosal IgA in the regulation of C. oncophora expulsion and suggest the development of a Th2 effector immune response.     

Effects of anti-allergic drugs on intestinal mastocytosis and worm expulsion of rats infected with Neodiplostomum seoulense

The effects of anti-allergic drugs on intestinal mastocytosis and the expulsion of Neodiplostomum seoulense were observed in Sprague-Dawley rats, after oral infection with 500 metacercariae. The drugs used were hydroxyzine (a histamine receptor H1 blocker), cimetidine (a H2 blocker), cyclosporin-A (a helper T-cell suppressant), and prednisolone (a T- and B-cell suppressant). Infected, but untreated controls, and uninfected controls, were prepared. Worm recovery rate and intestinal mastocytosis were measured on weeks 1, 2, 3, 5, and 7 post-infection. Compared with the infected controls, worm expulsion was significantly (P < 0.05) delayed in hydroxyzine- and cimetidine-treated rats, despite mastocytosis being equally marked in the duodenum of all three groups. In the cyclosporin-A- and prednisolone-treated groups, mastocytosis was suppressed, but worm expulsion was only slightly delayed, without statistical significance. Our results suggest that binding of histamine to its receptors on intestinal smooth muscles is more important in terms of the expulsion of N. seoulense from rats than the levels of histamine alone, or mastocytosis.