Worm Recovery Research Articles

Experimental model in rats for study on transmission dynamics and evaluation of Clonorchis sinensis infection immunologically, morphologically, and pathologically

This study aims to gain a better insight into the transmission patterns and immunologic profile of Clonorchis sinensis infection and make a headway on the pathogenesis regarding cholangiocarcinoma and hepatic lesions. Experimental models orally infected by C . sinensis metacercariae were constructed in rats. Immunological assays were performed to measure serum level of IgA, IgE, IgG1, IgG2a, IFN-gamma, and IL-4. Infection parameters were assessed by worm recovery rate, eggs per gram faece and worm size. Pathological sections with livers were managed with immunofluorescence, hematoxylin, eosin, and Masson's trichrome staining to evaluate the hepatic pathological changes. Interestingly, rats infected with only one C . sinensis metacercariae even gained a high worm recovery rate of 83.3% compared with rats infected with more metacercariae. Serological changes according to different infection doses indicated that immune response presented a tendency to Th2 type by expressing transient high level of IgG1, IL-4, and IgE. Hepatic tissues appeared inflammatory and fibrotic, revealed by different stainings. Intrahepatic bile ducts displayed cholangiectasis and proliferation with excreted/secreted antigen histologically located. C . sinensis, as a fish-borne zoonosis, presented novel transmission patterns which explained high infection rate in endemic areas; infection rate of C . sinensis was frequency-dependent and dose-related. Humoral immunity played a prevalent role in resisting to C . sinensis based on the rat models. C . sinensis infection played an undoubted role in biliary and hepatic diseases.

The identification of cattle nematode parasites resistant to multiple classes of anthelmintics in a commercial cattle population in the US

Resistance to modern anthelmintics by ruminant nematode parasites is an increasing problem throughout the world. To date the problem has largely been reported in parasites of small ruminants, but there are increasing reports of such resistance in nematodes recovered from cattle. Until now there have been no published reports of drug resistant parasites from cattle in North America. In 2002 a producer in the upper Midwest who backgrounds young cattle acquired from the southeastern US experienced lower than expected weight gain as well as apparent parasitic gastroenteritis in his cattle during the fall. Fecal sample results supported the suspicion that decreased productivity and diarrhea were the result of GI nematode parasitism. The operation used intensive grazing management and practiced strategically timed deworming for >17 year. In 2003, all animals were dewormed the first week of May with Ivomec Plus ®, then with Dectomax ® Injectable on 4 June and 17 July. On 31 July, 10 randomly taken fecal samples showed EPG values from 0 to 55. To assess whether the apparent decreased drug efficacy was the result of drug resistance in the nematode population, on 18 August approximately 150 heads, previously strategic timed dewormed, of 9–11 month old cattle from one pasture were selected for study. The calves were randomly assigned to 1 of 6 treatment groups: untreated (U), ivermectin injectable (I), moxidectin pour-on (M), doramectin injectable (D), eprinomectin pour-on (E), albendazole oral (A). Cattle were weighed prior to treatment and the drug was dosed according to label directions. Seven days later, 3 calves from each group were slaughtered for worm recovery. Fecal samples taken from the remaining animals at 14 days after treatment showed that the reduction of mean fecal EPG value for each group was: U-46%, I-52%, M-72%, D-61%, E-8%, and A-68%. Worm recovery from the slaughter calves showed that all groups harbored significant numbers of Haemonchus placei and H. contortus. In addition, all avermectin-treated groups contained significant numbers of Cooperia punctata, and smaller numbers of C. oncophora and C. spatulata. These results imply that the pastures studied contain substantial numbers of H. contortus resistant to both avermectins and benzimidazoles, and H. placei and Cooperia sp. resistant to all the commonly used avermectin anthelmintics. This is the first report of anthelmintic resistance in American cattle parasites.

Optimization of an Experimental Model for the Recovery of Adult Haplorchis pumilio (Heterophyidae: Digenea)

Recent studies in Vietnam and other Asian countries have shown that fish-borne zoonotic intestinal trematodes (FZT) occur very frequently in humans. The dominant intestinal FZT in Vietnamese fish are species of Haplorchis, in particular H. pumilio. However, basic studies on the biology and pathology of adult H. pumilio are difficult because of the lack of a standardized experimental animal model. The objective of this study was to establish and optimize such an animal-infection model for H. pumilio. Using metacercariae isolated from naturally infected fish, experiments were conducted to identify a suitable experimental animal host species, as well as the optimum metacercariae infection dose, and to determine the post-infection interval for patency. In a series of experiments, mice (Mus musculus) and chickens (Gallus gallus dom.) were infected with different numbers of metacercariae, and worm recoveries were made at varying intervals post-infection (PI). Based on the mean number of adult flukes recovered/number of metacercariae inoculated and the percent of hosts infected, mice were significantly more susceptible to infection than were chickens. The proportion of metacercariae developing to the adult stage increased with dose size. The peak worm recovery (geometric mean) was found to be day 7, although not all recovered flukes were gravid until day 9 PI. These results describe a mouse infection model with good predictability for intestinal flukes, such as H. pumilio, results which could facilitate investigations on important biological and pathological aspects of intestinal fluke infections.

Litomosoides sigmodontis: A simple method to infect mice with L3 larvae obtained from the pleural space of recently infected jirds ( Meriones unguiculatus)

Litomosoides sigmodontis is a filarial nematode that is used as a mouse model for human filarial infections. The life cycle of L. sigmodontis comprises rodents as definitive hosts and tropical rat mites as alternate hosts. Here, we describe a method of infecting mice with third stage larvae (L3) extracted from the pleural space of recently infected jirds ( Meriones unguiculatus). This method enables infection of mice with a known number of L3 larvae without the time-consuming dissection of L3 larvae from mites and results in higher worm recovery and patency rates than conventional methods. Additionally, this method allows for geographical separation of the facility maintaining the L. sigmodontis life cycle from the institution at which mice are infected.

The potential to control Haemonchus contortus in indigenous South African goats with copper oxide wire particles

The high prevalence of resistance of Haemonchus contortus to all major anthelmintic groups has prompted investigations into alternative control methods in South Africa, including the use of copper oxide wire particle (COWP) boluses. To assess the efficacy of COWP against H. contortus in indigenous South African goats, 18 male faecal egg-count-negative goats were each given ca.1200 infective larvae of H. contortus three times per week during weeks 1 and 2 of the experiment. These animals made up an “established” infection group (ESTGRP). At the start of week 7, six goats were each given a 2-g COWP bolus orally; six goats received a 4-g COWP bolus each and six animals were not treated. A further 20 goats constituted a “developing” infection group (DEVGRP). At the beginning of week 1, seven of the DEVGRP goats were given a 2-g COWP bolus each; seven goats were treated with a 4-g COWP bolus each and no bolus was given to a further six animals. During weeks 1–6, each of these DEVGRP goats was given ca. 400 H. contortus larvae three times per week. All 38 goats were euthanized for worm recovery from the abomasa and small intestines in week 11. In the ESTGRP, the 2-g and 4-g COWP boluses reduced the worm burdens by 95% and 93%, respectively compared to controls (mean burden±standard deviation, SD: 23±33, 30±56 and 442±518 worms, P=0.02). However, in the DEVGRP goats, both the 2-g and 4-g COWP treatments were ineffective in reducing the worm burdens relative to the controls (mean burdens±SD: 1102±841, 649±855, 1051±661 worms, P=0.16). Mean liver copper levels did not differ between the ESTGRP goats treated with 2-g COWP, 4-g COWP or no COWP (mean±standard error of the mean, SEM, in ppm: 93.7±8.3; 101.5±8.3; 71.8±8.3, P=0.07) nor did they differ between the DEVGRP goats (mean±SEM, in ppm: 74.1±9.1; 75.4±9.1; 74.9±10.0, P>0.99). The copper values were considered adequate, but not high, for goats. The COWP boluses have the potential to be used in the place of conventional anthelmintics for the control of established H. contortus infections in indigenous South African goats, but their use as part of an integrated approach to control H. contortus in the field must be fully investigated.

The Immunological Effects of Electrolyzed Reduced Water on the Echinostoma hortense Infection in C57BL/6 Mice

Electrolyzed reduced water (ERW) is widely used for drinking by people in Asia. The purpose of this study was to examine the immunological effect of ERW on the immunity of animals by supplying ERW to C57BL/6 mice infected with Echinostoma hortense metacercariae. In the non-infected groups, interleukin (IL)-4 (p < 0.001), IL-5, IL-10, IL-1beta, tumor necrosis factor (TNF)-alpha and immunoglobulin (Ig) A expression of the group fed ERW (ERW group) increased in small intestine compared with the normal control group. In the case of infected groups, the group fed ERW (ERW+E. hortense group) showed the result that IL-4, IL-5, IL-10 and Ig A expression increased, but IL-1beta and TNF-alpha (p < 0.001) decreased, and the number of goblet cells (p < 0.001) and helix pomatia agglutinin (HPA) positive cells increased compared with the group without feeding ERW. However, adult worm recovery rate was markedly increased (p < 0.05). On the other hand, the expression of all the cytokines except IL-10 in spleen was mildly increased but not significant statistically, and there was no significant difference in the numerical changes of white blood cell (WBC). These results indicate that feeding ERW may have influence on the local immune response (Th-1 type cytokines such as IL-1beta, TNF-alpha) in the small intestine but not on the systemic immune response.

Parasitological And Immunopathological Responses Balb/C Mice With Concomitant &lt;italic&gt;Schistosoma Mansoni&lt;/italic&gt; And &lt;italic&gt;Plasmodium Berghei&lt;/italic&gt; Infections

Schistosomiasis and malaria often occur in the same area. This study investigated the effect in BALB/c mice harbouring a schistosomiasis-malaria co-infection. Mice infected with S. mansoni were divided into three groups, which were super-infected at weeks 4, 5, and 6 post-infection with P. berghei. There were uninfected and single-infected controls. Sampling was at day 4 and 7 post-super-infection. The following assays were carried out: parasitaemia, perfusion for worm recovery, IgG ELISA, gross and histopathology. Co-infected mice had: higher malaria parasitemia, higher IgG responses, lower worm counts, smaller granulomas compared to the single-infected mice. This study showed that co-infections with schistosome and malaria parasites aggravated malaria severity, as shown by increased parasitemia and severe gross pathology of liver and spleen in the coinfected mice. In contrast, malaria conferred protection to S. mansoni infection, as demonstrated by reduced worm counts and granuloma sizes and high schistosme-specific IgG levels in the co-infected mice.

Heartworm and Wolbachia: Therapeutic implications

A safer, more effective adulticidal treatment and a safe method for reducing microfilaremia and breaking transmission of heartworm disease early in the treatment are needed. The present study evaluated efficacy of ivermectin (IVM) and doxycycline (DOXY) alone or together (with or without melarsomine [MEL]) in dogs with induced adult heartworm infection and assessed the ability of microfilariae from DOXY-treated dogs to develop to L 3 in Aedes aegypti mosquitoes and subsequently to become reproductive adults in dogs. Thirty beagles were each infected with 16 adult heartworms by intravenous transplantation. Six weeks later, dogs were ranked by microfilarial count and randomly allocated to 6 groups of 5 dogs each. Beginning on Day 0, Group 1 received IVM (6 mcg/kg) weekly for 36 weeks. Group 2 received DOXY (10 mcg/(kg day)) orally Weeks 1–6, 10–11, 16–17, 22–25, and 28–33. Groups 3 and 5 received IVM and DOXY according to doses and schedules used for Groups 1 and 2. At Week 24, Groups 3 and 4 received an intramuscular injection of MEL (2.5 mg/kg), followed 1 month later by two injections 24 h apart. Group 6 was not treated. Blood samples were collected for periodic microfilaria counts and antigen (Ag) testing (and later immunologic evaluation and molecular biology procedures). Radiographic and physical examinations, hematology/clinical chemistry testing, and urinalysis were done before infection, before Day 0, and periodically during the treatment period. At 36 weeks, the dogs were euthanized and necropsied for worm recovery, collection of lung, liver, kidney, and spleen samples for examination by immunohistochemistry and conventional histological methods. All dogs treated with IVM + DOXY (with or without MEL) were amicrofilaremic after Week 9. Microfilarial counts gradually decreased in dogs treated with IVM or DOXY, but most had a few microfilariae at necropsy. Microfilarial counts for dogs treated only with MEL were similar to those for controls. Antigen test scores gradually decreased with IVM + DOXY (with or without MEL) and after MEL. Antigen scores for IVM or DOXY alone were similar to controls throughout the study. Reduction of adult worms was 20.3% for IVM, 8.7% for DOXY, 92.8% for IVM + DOXY + MEL, 100% for MEL, and 78.3% for IVM + DOXY. Mosquitoes that fed on blood from DOXY-treated dogs had L 3 normal in appearance but were not infective for dogs. Preliminary observations suggest that administration of DOXY + IVM for several months prior to (or without) MEL will eliminate adult HW with less potential for severe thromboembolism than MEL alone.

Anticestodal activity of Adhatoda vasica extract against Hymenolepis diminuta infections in rats

Aim of this study Adhatoda vasica Nees has been commonly used in the indigenous system of medicine of Naga tribes in India for curing intestinal worm infections. In this study the anticestodal efficacy of Adhatoda vasica leaf extract was evaluated using Hymenolepis diminuta-rat experimental model. Materials and methods The anticestodal efficacy of leaf extract was determined by monitoring the eggs per gram (EPG) of faeces counts and percentage worm recovery rates following treatment with methanol leaf extract of this plant to different groups of rats harbouring immature and mature Hymenolepis diminuta infections. Results The result indicated 800 mg/kg double dose of extract has profound efficacy against mature worms, where the EPG count was reduced by 79.57% and percentage worm recovery rate by 16.60%. These effects were better than treatment with 5 mg/kg single dose of praziquantel, the standard drug. In case of efficacy against immature worms, the extract showed a significant reduction in worm recovery rate (from 100% in control to 20.00% at 800 mg/kg dose of extract). Conclusion The study shows that the leaf extract of Adhatoda vasica possesses significant anticestodal efficacy and supports its use in the folk medicine.

Haplorchis taichui: Worm recovery rate and immune responses in infected rats ( Rattus norvegicus)

Worm recovery rate, mucosal mast cells (MMCs), eosinophils and serum IgE concentration in rats were investigated after orally feeding 300 Haplorchis taichui metacercariae to male rats. The duodenal, jejunal and ileal tissue sections were stained with 1% alcian blue and 0.5% safranin-O for MMC count. Eosinophil count and the serum IgE concentration assay were measured from cardiac puncture blood. The average worm recovery rates were 20.00%, 13.00%, 0.67%, 1.67% and 0.00% on day 3, 7, 14, 21 and 28 post-infection (PI), respectively. The number of MMCs in the infected rats were significantly higher than in the controls ( P < 0.01), reaching a peak on day 21 PI. They decreased thereafter, with the decline in worm recovery. Eosinophil count and Serum IgE concentration were also increased but not significantly higher than the controls. However, they showed a positive relationship to worm recovery. It could be concluded from the results that MMCs, eosinophils and IgE may play an important role in the expulsion of H. taichui from rat intestine. However, the mechanism by which the MMC result in the helminth expulsion still need to be understood, and it is recommended that other cells such as goblet cells be studied further.

Dissociation of specific and total IgE antibody responses following repeated low-level infections with Nippostrongylus brasiliensis in rats.

IgE, IgG and mast cell responses were studied in rats infected weekly with 10 larvae of Nippostrongylus brasiliensis (NB). Worm recovery at 8 weeks of repeated infections was six-fold greater than that of a single infection with 10 larvae, suggesting the accumulation of worms during the repeated infections. Total serum IgE was increased after 2 weeks of infection, and further increased after repeated infections: at 6 weeks of infection the level was four to six times higher than that after a single infection. Anti-NB IgG1 levels were also significantly higher after repeated infections than after a single infection. On the other hand, there was no significant difference in the level of anti-NB IgE between single and repeated infections, as determined by ELISA, as well as by passive cutaneous anaphylaxis (PCA) reaction. Mastocytosis was induced in the small intestine after both single and repeated infections, but the levels did not differ between the two. These results indicate that total IgE and specific IgG1 production are augmented by repeated helminth infections, but specific IgE and mast cell responses are not. This pattern of response may minimize the development of IgE-dependent hypersensitivity reactions with repeated helminth infections.

The effect of long-term feeding of fresh and ensiled cassava (Manihot esculenta) foliage on gastrointestinal nematode infections in goats

The benefit of long-term feeding of fresh or ensiled cassava foliage on gastrointestinal parasite in goats was evaluated. Eighteen male goats (15.15 +/- 2.83 kg and between 4-6 months) were randomly allocated into three treatments supplemented with 200 g of wheat bran head(-1) day(-1). All groups were fed ad-libitum on either grass (CO), fresh cassava (CaF) or ensiled cassava foliage (CaS). At the beginning of the trial, each goat was inoculated with 3000 L3 containing approximately 50% Haemonchus contortus. Individual LWt, FEC and PCV were measured at weekly intervals for 10 weeks. At the termination of the experiment all goats were slaughtered for worm recovery and enumeration. The goats in CaF and CaS had similar weight gains while those in CO lost weight (p < 0.05) through the trial. FEC in CaF and CaS were lower (p < 0.05) than CO during the patency of parasite infections, but there was no difference between CaF and CaS goats. PCV of all groups decreased from above 30% to around 25% at the end of the trial. The compositions of established worm burdens were mainly H. contortus (19-40%) and Trichostrongylus colubriformis (55-76%). TWB did not differ among the groups, however, CaS significantly reduced H. contortus burdens, as compared to CaF and CO (p < or = 0.005). Thus, ensiled cassava foliage reduced the H. contortus population while the fresh foliage only reduced worm fecundity.

Filarial glutathione- S-transferase: A potential vaccine candidate against lymphatic filariasis

Present report enumerates the vaccine potential of a glutathione- S-transferase purified from Setaria cervi against lymphatic filariasis. In jirds (Meriones unguiculatus) vaccination trial, a very significant 82.75% ( p < 0.005) reduction in adult parasite burden was observed in ScGST immunized group after 90 days post Brugia malayi L3 challenge. An inverse correlation between the antibody level and worm burden was found in ScGST immunized group (Person's correlation r = 0.943, p < 0.05). No recoveries of worms were obtained in heart and lungs of vaccinated group. The Antibodies reactive to ScGST appeared within four weeks of first dose and were able to neutralize the GST activity up to 86%. In an earlier study we have shown vaccine potential of ScGST against B. malayi by ADCC. Evaluation of cytokine profile in T-cells isolated from BALB/c mice immunized with ScGST were also showed predominance of Th2 response which further maintained the humoral immunity generated by ScGST administration in mice. The overall observations prompted us to envisage ScGST as a potential vaccine candidate against lymphatic filariasis.

Schistosoma japonicum: Protective Immunity Induced by Schistosomulum-Derived Cells in a Mouse Model

We previously reported that immunization with intact live cells from schistosomula of Schistosoma japonicum (S.j) partially protected the Kunming strain of mice from challenge infection. In the present work, 2 immune protective experiments were designed to further validate the protective effect induced by this type of vaccine and to optimize the immunization protocol, including the number of inoculations and parasite stages from which immunogenic cells were derived. Three antigens derived from 18-day-old postinfection live (LLC) and dead (DLC) larval worm cells and from dead 42-day-old postinfection adult worm cells (DAC) were used as immunogens. Our results demonstrate that live cells from 18-day-old worms are capable of inducing significant protection in mice using a murine-Sj challenge model as shown by reduction rates of worm recoveries and egg burdens. The development of adult worms was stunted. A Th1-biased immune response was reflected in the protected groups as evidenced by the ratio of IgG2a/IgG1. A 38-kDa polypeptide was recognized by sera from LLC immunized animals. We demonstrate that live parasite cells are a source of novel protective antigens that can be exploited for vaccine development.

Combined ivermectin and doxycycline treatment has microfilaricidal and adulticidal activity against Dirofilaria immitis in experimentally infected dogs

There is still a pressing need for effective adulticide treatment for human and animal filarial infections. Like many filarial nematodes, Dirofilaria immitis, the causative agent of canine heartworm disease, harbours the bacterial endosymbiont Wolbachia, which has been shown to be essential for worm development, fecundity and survival. Here the authors report the effect of different treatment regimens in dogs experimentally infected with adult D. immitis on microfilariemia, antigenemia, worm recovery and Wolbachia content. Treatment with ivermectin (IVM; 6μg/kg per os weekly) combined with doxycycline (DOXY; 10mg/kg/day orally from Weeks 0–6, 10–12, 16–18, 22–26 and 28–34) resulted in a significantly faster decrease of circulating microfilariae and higher adulticidal activity compared with either IVM or DOXY alone. Quantitative PCR analysis of ftsZ (Wolbachia DNA) and 18S rDNA (nematode DNA) absolute copy numbers showed significant decreases in Wolbachia content compared with controls in worms recovered from DOXY-treated dogs that were not, however, associated with worm death. Worms from IVM/DOXY-treated dogs, on the other hand, had Wolbachia/nematode DNA ratios similar to those of control worms, suggesting a loss of both Wolbachia and nematode DNA as indicated by absolute copy number values. Histology and transmission electron microscopy of worms recovered from the IVM/DOXY combination group showed complete loss of uterine content in females and immunohistochemistry for Wolbachia was negative. Results indicate that the combination of these two drugs causes adult worm death. This could have important implications for control of human and animal filarial infections.

Comparative efficacies of albendazole and the Chinese herbal medicine long-dan-xie-gan-tan, used alone or in combination, in the treatment of experimental eosinophilic meningitis induced byAngiostrongylus cantonensis

Angiostrongylus cantonensis, the rat lungworm, is the principal cause of human eosinophilic meningitis or meningoencephalitis world-wide. In the present study, the efficacies of early-stage treatment with the Chinese herbal medicine long-dan-xie-gan-tan (LDXGT) and albendazole, used alone or in combination, were evaluated in BALB/c mice with A. cantonensis-induced dysfunction of the blood-central-nervous-system barrier and eosinophilic meningo-encephalitis. Indicators of the therapeutic effect included worm recovery, histopathological scores for the meningitis, assays of tissue-type plasminogen activator (PA), urokinase-type PA and matrix metalloproteinase-9 (MMP-9) in the brain, the ratio between albumin concentrations in the cerebrospinal fluid (CSF) and serum, and counts of eosinophils in the CSF. Combined treatment with albendazole and LDXGT gave better results than monotherapy based on either drug, apparently inhibiting eosinophilic meningitis via antagonists of the PA/MMP-9 system. LDXGT may have a therapeutic role in reducing inflammatory reaction in the subarachnoid space. Monotherapy with such an anti-inflammatory drug may relieve the symptoms of mild infection and the host's immune responses to A. cantonensis larvae. In severe infection, however, co-therapy with an anthelmintic (to kill the larvae) and an anti-inflammatory agent (to provide symptomatic relief) is probably a better approach. The therapeutic strategy should be tailored to the severity of the illness and the numbers of eosinophils in the CSF.

Factors in the resistance of rats to re-infection and super-infection by Clonorchis sinensis

Rats develop resistance to re-infection by Clonorchis sinensis while humans do not. We investigated factors involved in the development of resistance to re-infection and super-infection in rats. Sprague-Dawley rats were infected by C. sinensis metacercariae and treated with praziquantel, then re-infected after immune modulation. The rats were also subjected to super-imposed infection after primary infection. Resistance to re-infection was observed by lowered rates of worm recovery after various durations from treatment; 1% at 5days and 24.4% at 11months vs 56.2% in the control. Similar significant resistance was observed in the super-infected rats after 3weeks of primary infection. The re-infected or super-infected worms grew very slowly and remained immature. Resistance disappeared in immune-suppressed and nude rats but not in splenectomized rats. Immunization with antigens of C. sinensis produced no resistance. Local tissue reaction and immune response in the infected bile duct may play an important role in the resistance, although the crowding effect may also be implicated in resistance in super-infected rats.

Fasciola gigantica cathepsin L proteinase‐based synthetic peptide for immunodiagnosis and prevention of sheep fasciolosis

Sheep fasciolosis is a devastating burden for the livestock industry. We herein report on immunodiagnosis of fasciolosis, and significant protection of sheep against challenge infection with Fasciola gigantica following immunization with a peptide based on the H-Asp(110)-Lys-Ile-Asp-Trp-Arg-Glu-Ser-Gly-Tyr-Val-Thr-Glu-Val(123)-OH (Fas14p) sequence of F. gigantica cathepsin L-cysteine proteinase. This sequence was synthesized in three different forms: as N(alpha) acetylated (Ac-Asp(110)-Lys-Ile-Asp-Trp-Arg-Glu-Ser-Gly-Tyr-Val-Thr-Glu-Val(123)-OH, FasAc14p), bearing at the amino-terminus an N(alpha) acetylated cystein (Ac-Cys-Asp(110)-Lys-Ile-Asp-Trp-Arg-Glu-Ser-Gly-Tyr-Val-Thr-Glu-Val(123)-OH, FasAcCys14p), and conjugated to sequential oligopeptide carrier Ac-[Lys-Aib-Gly](4)-OH (Ac-SOC(4)) through an amide bond formed between Val(123) carboxylic group of the epitope and the lysine N(epsilon) groups of the carrier (Ac-[Lys(Fas14p)-Aib-Gly](4)-OH). Ac-[Lys(Fas14p)-Aib-Gly](4)-OH failed to readily discriminate between naïve and infected sheep. In contrast, the free peptides reproducibly differentiated between parasite-free sheep, sheep infected with parasites other than Fasciola, and experimentally Fasciola-infected sheep. The data together indicated that the peptides might be of considerable use for discriminating between early and late, and low and high burden, sheep infection with F. gigantica. FasAc14p was chosen to determine whether a peptide based on a critical enzymatic site of cathepsin L proteinase may induce protection against challenge infection. Sheep immunization with FasAc14p peptide induced significant expression of interleukin-4 mRNA, and humoral antibodies that bound to molecule(s) on the intact surface membrane of newly excysted juvenile worms, and mediated their attrition. The immune responses were associated with significant (P < 0.02) decrease of 23.1% in worm recovery, but with no decrease in the size or maturation of worms recovered.

Antibody Responses in Sera of Different Mouse Strains Experimentally Infected with Neodiplostomum seoulense

To examine humoral immune responses in the host, we measured serum antibody levels in different strains of mice (ICR, BALB/c, and C3H) experimentally infected with Neodiplostomum seoulense. Specific IgG antibody levels were increased remarkably with little difference among 3 strains of mice infected with N. seoulense from day 7 to 35 post-infection. More target proteins of adult parasites reacted with IgG at the time when the worm recovery decreased compared with other times. More than 20 protein bands, from 14 kDa to 94 kDa in size, were separated from the crude antigen of N. seoulense adults by SDS-PAGE, and among them 26, 30, 35, 43, 54, 67, and 94 kDa proteins were the major antigenic proteins. The results suggest that significant IgG antibody responses occur against N. seoulense in mice and this may be related with expulsion of worms.

Centrocestus formosanus (Digenea: Heterophyidae) Encysted in the Freshwater Fish, Puntius brevis, from Lao PDR

The metacercariae of Centrocestus formosanus, a minute intestinal trematode of mammals and birds, were detected in the freshwater fish, Puntius brevis, from Vientiane Municipality, Lao PDR. The metacercariae were experimentally fed to mice, and adult flukes were recovered in their small intestines 7 days later. The adult flukes were morphologically characterized by having 32 (rarely 34) circumoral spines arranged in 2 alternative rows, a large bipartite seminal vesicle, an oval-shaped ovary, and an X-shaped excretory bladder. Based on these characters, the adults were identified as Centrocestus formosanus (Nishigori, 1924). The taxonomic significance of C. formosanus, in relation to a closely related species, C. caninus (Leiper, 1913), is briefly discussed. It has been first verified by adult worm recovery that C. formosanus is prevalent in Vientiane areas of Lao PDR, taking the freshwater fish, P. brevis, as a second intermediate host.