Distribution of Cryptosporidium parvum subgenotypes in pre-weaned calves in Germany.

Cryptosporidium parvum is a main cause of severe diarrhea in young calves leading to economic loss and animal suffering. Little is known about the epidemiology of the genetic subtypes that may differ in their infectiousness and zoonotic potential. The present study analyzes the distribution of Glycoprotein 60 (GP60) variants in C. parvum positive samples obtained from young calves in Germany where individual fecal samples of 441 pre-weaned calves collected from 98 German dairy herds were evaluated for cryptosporidiosis (Göhring et al., 2014). DNA was extracted for Restriction Fragment Length Polymorphism (RFLP) analysis from 268 samples that were positive for Cryptosporidium by coproantigen EIA originating from 91 farms confirming C. parvum as the only species occurring. GP60 subtype analysis revealed 12C. parvum subtypes, all belonging to the zoonotic allele family IIa. The most widespread subtype was IIaA15G2R1 (71.4%). Other subtypes occurred sporadically: IIaA16G2R1, IIaA16G3R1 and IIaA17G2R1 (6.2%), IIaA13G1R2 (3.5%), IIaA14G1R1 (3.1%), IIaA14G2R1 (1.3%), IIaA17G1R1 (0.9%) and IIaA13G2R1, IIaA16G1R2, IIaA17G1R2 and IIaA24G1R1 (0.4%). Altogether a high diversity of subgenotypes was found with IIaA15G2R1 clearly dominating irrespective of geographic region. All subgenotypes are considered zoonotic underscoring that pre-weaned calves are likely to be a reservoir for zoonotic C. parvum and thus may be a risk for animal-to-human transmission.

Eimeria tenella oocysts attenuated by low energy electron irradiation (LEEI) induce protection against challenge infection in chickens.

In vitro and in vivo studies were performed to assess whether Eimeria tenella (E. tenella) oocysts, exposed to low energy electron irradiation (LEEI), might be considered potential vaccine candidates against cecal coccidiosis. Sporulated oocysts were exposed to LEEI of 0.1 kGy to 10.0 kGy. Reproduction inhibition assays (RIA) were performed in MDBK cells to assess infectivity of sporozoites excysted from irradiated and non-irradiated oocysts. LEEI of 0.1 kGy or 0.5 kGy resulted in 73.2% and 86.5% inhibition of in vitro reproduction (%IRIA), respectively. Groups of 12 one day old (D1) chicken were orally inoculated with Paracox®-8 (G1), 2.0 × 103 non-irradiated oocysts (G2) or 1.0 × 104 irradiated oocysts exposed to LEEI of 0.1 kGy (G3, G4) or 0.5 kGy (G5). Chicken of groups G1, G2, G4 and G5 were challenged 3 weeks later (D21) by a single inoculation of 7.5 × 104 non-attenuated oocysts of the same strain while G3 remained unchallenged. All chickens were subject to necropsy 7 days after challenge (D28) to estimate lesion scores (LS) and oocyst index (OI). A positive control (PC, non-vaccinated, challenged) and a negative control (NC, non-vaccinated, non-challenged) were kept in parallel. Chicken of group G5 had similar weight gain as the Paracox®-8 group (G1) after challenge and higher weight gains as compared to the other vaccinated groups. Feed conversion ratio (FCR) did not differ between chickens inoculated with oocysts irradiated with 0.5 kGy (G5) and negative control (NC) before challenge (1.25-1.52). After challenge FCR was 1.99 (G5) to 2.23 (G4) in the vaccinated chicken compared to 1.76 in group NC. LS and OI were significantly lower in all vaccinated groups as compared to group PC. Progeny oocysts collected from the feces of chickens following vaccination with irradiated oocysts exhibited lower in vitro infectivity/reproduction in MDBK cells with %IRIA of 89.7% and 82.4% for progeny of oocysts irradiated with 0.5 kGy and 0.1 kGy, respectively, suggesting hereditary attenuation by LEEI treatment. Seroconversion was demonstrated by ELISA before challenge (D21) in all vaccinated groups, however, chicken inoculated with irradiated oocysts displayed higher antibody levels than those inoculated with precocious oocysts (G1). In Western blot analysis chicken vaccinated with virulent (G2) or 0.1 kGy-irradiated E. tenella oocysts (G3, G4) showed more protein bands compared to G5 (0.5 kGy). We conclude that LEEI could be a promising technology for production of attenuated oocyst vaccines.

Passive immunization with Eimeria tenella gametocyte antigen 56 (EtGAM56) specific antibodies and active immunization trial with the epitope containing peptide.

Eimeria species cause avian coccidiosis leading to substantial economic losses in the poultry industry. Hence, anticoccidial drugs and vaccines have been used to combat this devitalizing disease. An effective vaccine based on gametocyte recombinant proteins would be very useful in terms of cost, labor and ethics (no animal experimentation). A mouse derived monoclonal antibody against Eimeria tenella gametocyte antigen 56 (EtGAM56) was used to immunize peritoneally E. tenella infected chicken a reduction of oocyst shedding by up to 78% was observed. The epitope recognized by the monoclonal antibody was mapped, recombinant expressed and used to immunize chicken (rEtGAM56N). After rEtGAM56N intramuscular immunization and parasite challenge, clinical parameters like faecal oocyst output, body weight gain, lesion score, feed conversion rate and serum antibody response were assessed to test the efficacy of vaccination against experimental infection with E. tenella. Chicken immunized with rEtGAM56N and challenged with E. tenella oocysts showed a robust antibody response against the rEtGAM56N peptide but no considerable effects on oocyst output and clinical parameters (weight gain, lesion score, feed conversion rate) compared to the mock control group. This study demonstrates the complexity of an effective vaccination. The immunoprotective epitope might be a conformational epitope that was recognized by the monoclonal mouse antibody but only weakly by the antibodies produced against the linear peptide leading to a divergent outcome between the passive and active immunization.

Improvement of in vitro evaluation of chemical disinfectants for efficacy on Cryptosporidium parvum oocysts.

Cryptosporidium parvum has been suggested as a suitable target for in vitro efficacy testing of disinfectants. To improve validity of a method based on exposure of HCT-8 monolayers to C. parvum oocysts we here critically evaluate and we propose certain procedural steps needed for the validation of disinfectants. Within a range of 0.02% to 0.4%, sodium taurocholate at 0.2% stimulated infection most efficiently while preserving host cell integrity. The course of invasion was monitored for periods of 30-240min post infection (p.i.). FACS analysis revealed that the proportion of sporozoites liberated from oocysts in the presence of 0.2% sodium taurocholate increased within 120min of incubation but remained constant thereafter. Maximum invasion of cells measured by qPCR was reached 180min p.i. and therefore set as invasion endpoint. As monolayers harvested 24h or 48h p.i. did not differ in the quantity of parasite hsp70 gene copies, DNA extraction can be performed as early as 24h p.i. Incubation of oocysts with 20% H2O2 for 2h resulted in inactivation of more than 99.5% both at room temperature and 10°C and appeared thus suitable as positive chemical treatment control. Four washing procedures considered to remove potentially toxic residual disinfectant from oocyst suspensions were tested. An application of a combination of DMSO (Dimethylsulfoxid), Tween20 and WSH (water of standardized hardness) appeared most efficient without deleterious effect of disinfectant residuals on the cell monolayer viability when oocysts accordingly washed were applied. In conclusion, for standardized in vitro evaluation of chemical disinfectants in C. parvum infected HTC-8 monolayers. (i) excystation medium should contain 0.2 % sodium taurocholate. (ii) excystation medium should be replaced by growth medium after 180 min. (iii) monolayers should be harvested 24 h p.i. for DNA preparation. (iv) ocysts exposed to 20 % H2O2 should be included as positive controls. (v) disinfected oocysts should be washed with DMSO/Tween20/WSH before they are transferred to monolayers.

Molecular characterization of bovine Cryptosporidium isolated from diarrheic calves in the Sudan.

Cryptosporidiosis is a common protozoan infection causing morbidity and mortality in young cattle and may be zoonotically transmitted to humans. So far, there is no data available on the presence of Cryptosporidium spp. in the Sudan. The aim of this study was to isolate, identify, and genotype Cryptosporidium oocysts sampled from diarrheic calves housed at different farms in three different municipalities in Khartoum State (Khartoum, Khartoum North, Omdurman). A total of 149 fecal samples were evaluated microscopically for the presence of Cryptosporidium oocysts using the modified Ziehl-Neelsen staining method and 87 (58.3%) samples tested positive. Positive and negative samples were further analyzed by nested PCR targeting the SSU rRNA region. Positive samples were subjected to restriction enzyme analysis of PCR amplicons (PCR-RFLP). Nested PCR identified Cryptosporidium DNA in 53 samples (35.5%); restriction digestion of the PCR products revealed the presence of C. parvum (73.5%), C. ryanae (13.2%), C. andersoni (7.5%), and C. bovis (1.8%). Species distribution was clearly related to age with C. parvum being the predominant species in dysenteric pre-weaned calves. Sequencing of three genes (SSU rRNA, COWP, and GP60) for three C. parvum isolates originating from the three different municipalities showed that all belong to C. parvum subtype family IId. Based on data obtained by GP60, sequencing the two C. parvum isolates from Khartoum and Omdurman represent subtype IIdA18G1, whereas oocysts isolated in Khartoum North belong to subtype IIdA19G1. The observed genotypes are zoonotic and thus C. parvum in calves is potentially a health risk to humans in Khartoum State, Sudan. To the best of our knowledge, this is the first reported attempt to characterize Cryptosporidium isolated from cattle in the Sudan.

Nodular presentation of Dirofilaria repens infection in a cat mimicking a fibrosarcoma.

A cat with multiple subcutaneous nodules suggesting a soft tissue sarcoma by physical and computed tomographic examination was diagnosed as being affected by subcutaneous filariosis based on cytologic and ultrasonographic assessments. Nodules were surgically removed and extracted nematodes were identified by PCR as Dirofilaria repens. Furthermore, DNA of Dipetalonema dracunculoides (syn. Acantocheilonema dracunculoides) was detected by PCR, with no evidence of circulating microfilariae. To the best of the authors' knowledge, this represents the first report describing adults of D repens in multiple subcutaneous nodules in a cat. Cytopathologic examination allowed characterization of the parasitic nature of the nodules. Veterinary practitioners should be aware of the possible nodular presentation of D repens in cats and should include D repens in the differential diagnosis of subcutaneous neoformations in the cat.

Inactivation of Cryptosporidium parvum under laboratory conditions.

The aim of the present study was to evaluate alternatives for inactivating Cryptosporidium parvum under experimental conditions. Disinfectants against this protozoan are usually based on cresols and often difficult to handle in laboratories. Four different substances (ethanol, denatured ethanol, sodium hypochlorite and peroxide) at different concentrations were tested for several exposure times (30 min, 2 h, 4 h, 12 h and 24 h). The results show an inactivation over 99% by using 10% H2O2 at an exposure time over 2 h as well as 3 and 6% NaOCl after 12 h of exposure. Furthermore, the ability of UV-C light to inactivate oocysts on smooth surfaces (e.g., laminar flow) was evaluated. To mimic laboratory conditions, oocysts were given on germ carriers. Best results (>99%) were achieved at an exposure time of 30 min (100.8 mJ/cm(2)).

NADPH oxidase, MPO, NE, ERK1/2, p38 MAPK and Ca2+ influx are essential for Cryptosporidium parvum-induced NET formation.

Cryptosporidium parvum causes a zoonotic infection with worldwide distribution. Besides humans, cryptosporidiosis affects a wide range of animals leading to significant economic losses due to severe enteritis in neonatal livestock. Neutrophil extracellular trap (NET) formation has been demonstrated as an important host effector mechanism of PMN acting against several invading pathogens. In the present study, C. parvum-mediated NET formation was investigated in human and bovine PMN in vitro. We here demonstrate that C. parvum sporozoites indeed trigger NET formation in a time-dependent manner. Thereby, the classical characteristics of NETs were demonstrated by co-localization of extracellular DNA with histones, neutrophil elastase (NE) and myeloperoxidase (MPO). A significant reduction of NET formation was measured following treatments of PMN with NADPH oxidase-, NE- and MPO-inhibitors, confirming the key role of these enzymes in C. parvum-induced NETs. Additionally, sporozoite-triggered NETosis revealed as dependent on intracellular Ca(++) concentration and the ERK 1/2 and p38 MAPK-mediated signaling pathway. Moreover, sporozoite-triggered NET formation led to significant parasite entrapment since 15% of the parasites were immobilized in NET structures. Consequently, PMN-pre-exposed sporozoites showed significantly reduced infectivity for epithelial host cells confirming the capability of NETs to prevent active parasite invasion. Besides NETs, we here show that C. parvum significantly up-regulated CXCL8, IL6, TNF-α and of GM-CSF gene transcription upon sporozoite confrontation, indicating a pivotal role of PMN not only in the bovine and human system but most probably in other final hosts for C. parvum.

A novel CDPK1 inhibitor--a potential treatment for cryptosporidiosis in calves?

Cryptosporidium parvum is a zoonotic agent that infects humans and animals occasionally causing severe, watery diarrhoea. In immunocompetent hosts, cryptosporidiosis is self-limiting but can have a fatal outcome in immunocompromised individuals. Cryptosporidium is one of the most common causes of waterborne diseases (recreational water and drinking water) in humans, a leading cause of moderate to severe childhood diarrhoea, and a major agent of diarrhoea in calves leading to high economic losses and up to 10% lethality. So far, available treatment options are insufficient for both veterinary and human clinical disease cases. Here, we report for the first time that the novel bumped kinase inhibitor (BKI) 1294 targeting the calcium-dependent protein kinase 1 (CDPK1) of Cryptosporidium is able to reduce the oocyst shedding of C. parvum by calves--its natural host--without obvious side effects.

Establishment of a germ carrier assay to assess disinfectant efficacy against oocysts of coccidian parasites.

Parasites are a common threat to human and animal health. One option to combat parasites that produce infective environmental stages is to inactivate them by chemical disinfection. Standardised laboratory assays that enable proper evaluation of products suspected to be efficient are highly desirable to allow prudent selection and use of such potentially hazardous agents. Here, we present a newly developed in vitro germ carrier assay to evaluate inactivation of oocysts of the model organism Cryptosporidium parvum by chemical disinfectants. Stainless steel discs were used as carrier to mimic surface contamination by C. parvum oocysts. The germ carriers were incubated with approved chemical disinfectant for the specified time (2 h) and rinsed thereafter to remove the disinfectant and recover the exposed oocysts. Recovered oocysts were transferred to HCT-8 monolayers, and 48 h later, genomic DNA was extracted and quantified by real-time PCR targeting the hsp70 gene to estimate parasite reproduction. A panel of commercially available and approved disinfectants were examined and data compared with those of suspension assays and historical data obtained from efficacy assays based on infection of chicken with oocysts of Eimeria tenella. Altogether, data achieved by these divergent assays allowed similar conclusions although the sensitivity of the in vitro assay was higher. Consequently, a threshold of 99.5% inactivation is proposed to evaluate disinfectants in vitro using C. parvum as model organism as compared to the E. tenella animal infection assay (95%).

Efficacy of an anticoccidial live vaccine in prevention of necrotic enteritis in chickens.

Necrotic enteritis (NE) is an important disease in poultry caused by Clostridium perfringens combined with predisposing factors, mainly eimeriosis. In the present study, we investigated the protective effect of a commercial attenuated anticoccidial live vaccine against NE in a clinical infection model using 60 day-old chicks. Vaccination was performed on study day (SD) 1 with natural booster-infections for 4 weeks from Eimeria spp. oocysts present in litter. On SD 28, five groups were formed (n=12): group V+/C-E- (vaccinated, uninfected), group V+/C-E+ (vaccinated, infected with Eimeria spp.), group V+/C+E+ (vaccinated, infected with clostridia and Eimeria spp.), group V-/C+E+ (unvaccinated, infected with clostridia and Eimeria spp.), and group NC (negative control). Efficacy was measured by clinical parameters, pathogen multiplication, and pathological parameters assessed during two necropsies on SD 34 and SD 40, respectively. Additionally, cytokine expression was measured in gut and spleen tissues at necropsy. Clinical signs of NE were observed only in the coinfected groups, mainly in group V-/C+E+. Accordingly, lowest body weight gain was observed in group V-/C+E+ (301.8 g from SD 28 to SD 40; group NC: 626.2 g). Oocyst excretion varied significantly (P<0.01) between all Eimeria spp. infected groups and was highest in group V-/C+E+, followed by V+/C+E+, and lowest in group V+/C-E+. NE typical intestinal lesions showed only in groups V+/C+E+ and V-/C+E+. The intestinal mucosa featured partly severe lesions in the jejunum, C. perfringens colonization was histologically visible. Upregulation of IFN-γ, was observed in the jejunal tissue of group V-/C+E+ (P<0.01 (SD 34) or P<0.05 (SD 40) compared to all other groups). IL-10 and IL-12 were upregulated in group V-/C+E+, IL-10 also in group V+/C+E+ (SD 40) while IL-2 expression remained unaltered. In conclusion, vaccination against coccidiosis was effective in preventing NE in a mixed infection comparable to field situations.

CDPKs of Cryptosporidium parvum--stage-specific expression in vitro.

Cryptosporidium parvum is a zoonotic agent that bears a high risk for the health of particularly immunocompromised humans and animals. As currently available drugs and therapies against cryptosporidiosis do not turn out satisfactory, more intensive research on the control of this parasite is necessary. The genus Cryptosporidium is unique within the phylum Apicomplexa as its localisation is intracellular but extracytoplasmatic. Infection of host cells is initially a parasite-driven process, but the signalling events and their downstream actions within Cryptosporidium are poorly understood. Calcium-dependent protein kinases (CDPKs) are probably involved in the regulation of invasion and egress. Previously described in plants, algae and other Apicomplexa, CDPKs are not found in vertebrates. They are thus promising targets for pharmaceutical intervention. While CDPK1 is well characterised in Toxoplasma gondii (TgCDPK1) and Plasmodium falciparum (PfCDPK1), only little information exists about the expression and function of CDPK in C. parvum. Here, we describe results of the in silico analysis of seven CpCDPKs. Five CpCDPKs contain potential sites for N-myristoylation and N-palmitoylation. In a nested 3' rapid amplification of cDNA ends (RACE)-PCR, expression of six CpCDPKs resulted in distinct bands in infected cell cultures and extracts of freshly excysted sporozoites. The length of the 3' untranslated region (3' UTR) is described as well. Our results indicate CDPK expression to be stage specific on the mRNA level.

Cryptosporidium infections: molecular advances.

Cryptosporidium host cell interaction remains fairly obscure compared with other apicomplexans such as Plasmodium or Toxoplasma. The reason for this is probably the inability of this parasite to complete its life cycle in vitro and the lack of a system to genetically modify Cryptosporidium. However, there is a substantial set of data about the molecules involved in attachment and invasion and about the host cell pathways involved in actin arrangement that are altered by the parasite. Here we summarize the recent advances in research on host cell infection regarding the excystation process, attachment and invasion, survival in the cell, egress and the available data on omics.

Evaluation of putative anti-cryptosporidial drugs in an in vitro culture system.

Cryptosporidium species are major pathogens severely affecting the health of neonate animals, in particular calves, but also cause life-threatening infection of immunocompromised humans. Currently, only halofuginone is approved for prophylactic and metaphylactic treatment of calves but this drug suffers from its limited safety margins. For treatment of immunodeficient human patients, only nitazoxanide and paramomycin are used but data regarding their efficacy are controversial. Aim of the present study was to test a substituted benzimidazole (BAY-AF76184) and a heterocyclic substituted 1,2,4-triazin (BAY-AB24992), both drugs with known anti-protozoal activity, for their potential ability to interfere with Cryptosporidium parvum development in vitro. Development of C. parvum in HCT-8 cells treated with these compounds was compared to negative controls and parasites treated with paromomycin or halofuginone using real-time PCR targeting the 18S rDNAs of parasites and host cells as template. Potential cytotoxic and anti-proliferative effects of drugs were analysed using a lactate dehydrogenase and a WST-1 assay, respectively. BAY-AF76184 and paromomycin dose-dependently inhibited development of C. parvum with EC50 values of 2.37 µM and 69.5 µM, respectively. Although high concentrations of halofuginone and BAY-AB24992 also significantly inhibited parasite development, effects of lower concentrations were very heterogeneous preventing calculation of EC50 values. Halofuginone and BAY-AF76184 dose-dependently interfered with host cell proliferation (EC50 values of 0.35 µM and 9.07 µM, respectively) and the latter also had direct cytotoxic effects (EC50=48.78 µM). However, drug concentrations required for cytopathic were higher than those for effects against C. parvum. Therefore, both BAY-AB24992 and BAY-AF76184 should be considered for further evaluation, e.g. using in vivo models.

Embryonated chicken eggs as an alternative model for mixed Clostridium perfringens and Eimeria tenella infection in chickens.

The chorioallantoic membrane (CAM) of chicken embryo eggs is a suitable model for viral and bacterial infections. In the present study, a new approach for testing the pathogenesis and virulence of Clostridium perfringens and Eimeria tenella dual infections as a model using the CAM of embryonated chicken eggs was developed. For this purpose, 24 specific pathogen-free (SPF) embryonated chicken eggs were divided into four groups (n = 6) and designated group E, group CP, group CPE, and NC. Sporozoites of E. tenella (20,000 sporozoites) were inoculated into 10-day-old embryonated SPF chicken eggs (groups E and CPE) via allantoic sac route. At 15-day-old, eggs of groups CP and CPE were infected with 10 (4)  cfu C. perfringens via the same route. Assessment of pathogenicity was assessed using gross and histopathological lesions. Embryo mortality reached 17 % after mono-infection with C. perfringens and/or E. tenella and 50 % in the mixed-infected group. Lesions in the CAMs were most numerous and most severe in co-infected eggs (group CPE), reaching the maximum score of 3 in 50 % of the inoculated eggs (P < 0.01). In Eimeria spp.-infected eggs (group E), lesions of score were between 1 and 2. Mono-infection with C. perfringens did not lead to a significant occurrence of lesions. Histopathological investigations of the CAM revealed clusters of Gram-positive bacteria, infiltration with leukocytes, lymphocytes, and developmental stages of E. tenella in the co-infected group. These data suggest that embryonated eggs could be an in ovo model for studying the pathogenesis of mixed infection with Eimeria and C. perfringens.

Inactivation of exogenous endoparasite stages by chemical disinfectants: current state and perspectives.

Chemical disinfection is common practice and inevitable to achieve sufficient control over parasites particularly in intensive animal housing systems. To identify suitable chemicals, reliable data on antiparasitic efficacy of disinfectants are required. This review summarizes recently published experience with procedures applied to evaluate the viability of a variety of endoparasites following physical or chemical stress. It is concluded that laboratory models used to assess antiparasitic efficacy of e.g. commercial disinfectants should consider the most resistant stages of both helminths and protozoa, i.e. ascarid eggs and coccidia oocysts. To ensure reproducibility and transparency, standardized protocols are pivotal. Such protocols are established on a national level (e.g. DVG guidelines in Germany); however, internationally accepted certification procedures are currently lacking.

[Cryptosporidiosis--an update]. / Kryptosporidiose--ein Update.

Cryptosporidia are protozoan parasites which have emerged as important causative agents of diarrhea in man and animals. Because of their small size and their tenacity, water- and foodborne outbreaks remain to be of significance in public health. Furthermore Cryptosporidium may cause up to 5-10% of economical losses in calf rearing. This review aims to give a short update on the current state of knowledge concerning species delimitation, prevalence, disease control in animal husbandry and zoonotic potential.

A matter of timing: early, not chronic phase intestinal nematode infection restrains control of a concurrent enteric protozoan infection.

Infections with parasitic worms are often long lasting and associated with modulated immune responses. We analyzed the influence of the nematode Heligmosomoides polygyrus bakeri dwelling in the small intestine on concurrent protozoan infection with Eimeria falciformis residing in the cecum. To dissect the effects of a nematode infection in the early versus chronic phase, we infected animals with E. falciformis 6 or 28 days post H. p. bakeri infection. Only a concurrent early nematode infection led to an increased replication of the protozoan parasite, whereas a chronic worm infection had no influence on the control of E. falciformis. Increased protozoan replication correlated with the reduced production of IFN-γ, IL-12/23, CCL4, CXCL9 and CXCL10, reduced migration of T cells and increased expression of Foxp3 at the site of protozoan infection. This was accompanied by a stronger nematode-specific Th2 response in gut-draining LN. Protection of mice against challenge infections with the protozoan parasite was not altered. Hence, the detrimental effect of a nematode infection on the control of a concurrent protozoan infection is transient and occurs only in the narrow time window of the early phase of infection.

Attempts to establish RNA interference in the parasitic nematode Heligmosomoides polygyrus.

To analyze the potential of RNA interference (RNAi) in the intestinal nematode Heligmosomoides polygyrus, we delivered double-stranded RNA (dsRNA) of tropomyosin to various life stages of the parasite. Three different methods were examined for their potential use. First, feeding of recombinant bacteria that expressed dsRNA did neither result in phenotypical changes of H. polygyrus nor in a significant reduction of tropomyosin mRNA levels. In contrast, feeding of such bacteria to Caenorhabditis elegans elicited the expected phenotypes. Quantification of bacteria ingested by C. elegans and H. polygyrus larvae (L1) revealed that the parasitic worms took up only a fraction of the bacteria ingested by C. elegans. Second, electroporation of L1 failed to transport siRNA through the cuticle and was lethal to the larvae. However, the cuticle of adult worms was penetrated by dye-labeled RNA, but no systemic spreading was observed. Third, soaking of adult H. polygyrus in tropomyosin dsRNA led to a higher proportion of worms showing symptoms of ageing, such as a disintegrated gut and ovaries, but did not induce reduction of tropomyosin mRNA levels. Database analysis revealed that orthologous proteins involved in dsRNA-uptake and -systemic spread in C. elegans are missing in the parasitic nematodes Brugia malayi and Haemonchus contortus, whereas proteins responsible for dsRNA-processing, -amplification and mRNA-regulation are present. Thus, our data indicate that the study of gene function by RNAi in H. polygyrus is limited, possibly due to deficiencies of genes involved in RNA-uptake and spread.