Cystoisospora suis merozoite development assay for screening of drug efficacy in vitro.

Cystoisospora suis is a common diarrheal pathogen of piglets and typically controlled by metaphylactic toltrazuril application. Recently, toltrazuril resistance has been reported in the field; however, both evaluation of toltrazuril efficacy against field isolates and the anticoccidial drug development for pigs is hampered by costs and labor of animal experimentation. Therefore an in vitro merozoite development assay was developed to evaluate the efficacy of compounds against C. suis in vitro. Monolayers of IPEC-1 cells were infected with sporozoites derived from oocysts of defined C. suis laboratory strains and the optimal infection dose as well as concentration, time point and duration of treatment were evaluated by quantitative real-time PCR. Cell cultures were treated with bumped kinase inhibitor (BKI) 1369 at different time points to evaluate the possibility to delineate effects on different developmental stages in vitro during invasion and early infection, and to determine different inhibitory concentrations (IC50, IC95). BKI 1369 had an IC50 of 35 nM and an IC95 of 350 nM. Dose- and duration-dependent efficacy was seen when developing stages were treated with BKI 1369 after infection (days 0-1, 2-3 and 2-5) but not when sporozoites were pre-incubated with BKI 1369 before infection. Efficacies of further BKIs were also evaluated at 200 nM. BKI 1318, 1708, 1748 and 1862 had an efficacy comparable to that of BKI 1369 (which is also effective in vivo). BKI 1862 showed a more pronounced loss of efficacy in lower concentrations than BKI 1369, signifying pharmacokinetic differences of similar compounds detectable in vitro. In addition, the effects of toltrazuril and its metabolites, toltrazuril sulfoxide and toltrazuril sulfone, on a toltrazuril sensitive and a resistant strain of C. suis were evaluated. Inhibition of merozoite growth in vitro by toltrazuril and its metabolites was dose-dependent only for toltrazuril. Clear differences were noted for the effect on a toltrazuril-sensitive vs. a resistant strain, indicating that this in vitro assay has the capacity to delineate susceptible from resistant strains in vitro. It could also be used to evaluate and compare the efficacy of novel compounds against C. suis and support the determination of the optimal time point of treatment in vivo.

Current anthelmintic treatment is not always effective at controlling strongylid infections in German alpaca herds.

BACKGROUND: Endoparasites are considered a major health problem of South American camelids as shown in a recent survey among German and Austrian camelid owners. Although prophylactic and therapeutic measures such as application of anthelmintics are commonly used, treatment efficacy is usually not assessed. Owners have expressed significant concerns regarding the effect of antiparasitic therapy, so this study aimed to evaluate the outcome of anthelmintic treatment in German alpaca herds with different drugs. RESULTS: Overall, 617 samples from 538 clinically healthy alpacas > 1 year-old from 27 farms (n = 11-157 animals/herd) were examined. The most common parasites detected by flotation were Eimeria spp. (75.1%) followed by strongylids (55.0%), Nematodirus spp. (19.3%), cestodes (3.1%) and Trichuris (2.7%). After initial coproscopical examination by flotation and strongylid egg quantification by the McMaster technique, positive animals excreting at least 150 eggs per gram of faeces were included in a faecal egg count reduction test (FECRT) using fenbendazole (n = 71 samples), moxidectin (n = 71) or monepantel (n = 66). Pre-treatment larval cultures (n = 23 positive pooled farm samples) revealed Haemonchus (87% of the farms), Cooperia (43.5%), Trichostrongylus (21.7%), Ostertagia (13.0%), Nematodirus and Oesophagostomum (4.3% each). Fenbendazole treatment reduced egg excretion by 45%, moxidectin by 91% and monepantel by 96%. On the farm level, 13/18 farms that used fenbendazole, 6/6 farms that used moxidectin and 2/5 farms that used monepantel had individual FECR values < 90% (fenbendazole) or < 95% (moxidectin, monepantel). Haemonchus and Cooperia were overrepresented on the farms with reduced treatment efficacy. CONCLUSIONS: Gastrointestinal strongylids are common in German alpacas and fenbendazole in particular was not sufficiently effective to reduce strongylid egg excretion. Although the FECRT could not unambiguously determine anthelmintic resistance in the present study, the finding that small ruminant strongylids, especially Haemonchus, are common in alpacas indicates that determination of effective anthelmintic doses, monitoring of efficacy and adapted (selective) treatment regimens must be implemented as part of sustainable deworming practices in this species in accordance with recommendations for ruminants.

In vitro inhibitory effects of plant-derived by-products against Cryptosporidium parvum.

Disposal of organic plant wastes and by-products from the food or pharmaceutical industries usually involves high costs. In the present study, 42 samples derived from such by-products were screened in vitro against Cryptosporidium parvum, a protozoan parasite that may contaminate drinking water and cause diarrhoea. The novel bioassay was previously established in the microtitre plate format. Human ileocaecal adenocarcinoma (HCT-8) cell cultures were seeded with C. parvum oocysts and parasite development was monitored by an indirect fluorescent antibody technique (IFAT) and microscopic assessment for clusters of secondary infection (CSI). Minimum inhibitory concentrations (MICs) and potential detrimental effects on the host cells were determined. An ethanolic extract from olive (Olea europaea) pomace, after oil pressing and phenol recovery, reproducibly inhibited C. parvum development (MIC = 250-500 µg mL(-1), IC50 = 361 (279-438) µg mL(-1), IC90 = 467 (398-615) µg mL(-1)). Accordingly, tyrosol, hydroxytyrosol, trans-coniferyl alcohol and oleuropein were selected as reference test compounds, but their contributions to the observed activity of the olive pomace extract were insignificant. The established test system proved to be a fast and efficient assay for identifying anti-cryptosporidial activities in biological waste material and comparison with selected reference compounds.

Oocyst-Derived Extract of Toxoplasma Gondii Serves as Potent Immunomodulator in a Mouse Model of Birch Pollen Allergy.

INTRODUCTION: Previously, we have shown that oral infection with Toxoplasma gondii oocysts prevented type I allergy in mice. Here we investigated whether the application of a T. gondii oocyst lysate antigen (OLA) could also reduce allergy development. BALB/c mice were immunised twice with OLA followed by sensitisation with the major birch pollen (BP) allergen Bet v 1 and an aerosol challenge with BP extract. METHODS: First, we tested OLA in vitro. Stimulation of splenocytes and bone marrow-derived dendritic cells (BMDC) with OLA led to the production of pro-inflammatory and regulatory cytokines such as IL-6, IFN-γ and IL-10. Moreover, BMDC exposed to OLA upregulated the maturation markers CD40, CD80, CD86, and MHCII. Furthermore, OLA was recognised by TLR2-transfected human embryonic kidney cells. RESULTS: Immunisation of mice with OLA induced high levels of Toxoplasma-specific IgG antibodies in sera along with increased production of IFN-γ and IL-10 in Toxoplasma-antigen restimulated splenocytes. OLA reduced allergic airway inflammation as manifested by significant reduction of eosinophils in bronchoalveolar fluids, decreased cellular infiltrates and mucus production in the lungs. Accordingly, Bet v 1-specific IgE was decreased in OLA-pretreated mice. The reduced allergic immune responses were accompanied by increased numbers of CD4+CD25highFoxp3+ regulatory T cells in spleens as well as by increased numbers of granulocytic myeloid-derived suppressor cells in lungs when compared to sensitised controls suggesting that these two cell populations might be involved in the suppression of the allergic immune responses. CONCLUSION: Our data demonstrate that pretreatment with the oocyst extract can exert anti-allergic effects comparable to T. gondii infection. Thus, the immunomodulatory properties of the parasite extract indicate that this extract and in the future defined molecules thereof might serve as immunomodulatory adjuvants in allergy treatment and prophylaxis.

Practical and low cost whole-organism motility assay: A step-by-step protocol.

Here, we provide a step-by-step protocol for a practical and low cost whole-organism assay for the screening of chemical compounds for activity against parasitic worms. This assay has considerable advantages over conventional methods, mainly in relation to ease of use, throughput, time and cost. It is readily suited to the screening of hundreds to thousands of compounds for subsequent hit-to-lead optimisation, and should be applicable to many different parasites and other organisms commensurate with the size of wells in the microtiter plates used for phenotypic screening.

Llamas and alpacas in Europe: Endoparasites of the digestive tract and their pharmacotherapeutic control.

There are distinctive specifications for veterinary medical care of South American camelids (SACs), namely, llamas, alpacas, vicunas and guanacos. Camelids are classified as food-producing animals, but as veterinary medicinal products are often only licensed for domestic food-producing species such as horses, goats, sheep and cattle, treatment of SACs generally requires off-label use of drugs. Endoparasitism is a major health concern in camelids and can result in severe clinical diseases and economic losses. There is still a lack of work on the pharmacokinetics, safety and efficacy for most antiparasitic drugs used in SACs. Even when choosing an appropriate route of administration, several aspects must be considered such as the fact that pour-on formulations are largely ineffective in camelids due to the unique features of llama and alpaca skin and hair that result in extremely low drug bioavailability. This review focuses on the main endoparasites of the digestive tract in llamas and alpacas in Europe and pharmacotherapeutic options based on current knowledge.

Low cost whole-organism screening of compounds for anthelmintic activity.

Due to major problems with drug resistance in parasitic nematodes of animals, there is a substantial need and excellent opportunities to develop new anthelmintics via genomic-guided and/or repurposing approaches. In the present study, we established a practical and cost-effective whole-organism assay for the in vitro-screening of compounds for activity against parasitic stages of the nematode Haemonchus contortus (barber's pole worm). The assay is based on the use of exsheathed L3 (xL3) and L4 stages of H. contortus of small ruminants (sheep and goats). Using this assay, we screened a panel of 522 well-curated kinase inhibitors (GlaxoSmithKline, USA; code: PKIS2) for activity against H. contortus by measuring the inhibition of larval motility using an automated image analysis system. We identified two chemicals within the compound classes biphenyl amides and pyrazolo[1,5-α]pyridines, which reproducibly inhibit both xL3 and L4 motility and development, with IC50s of 14-47 µM. Given that these inhibitors were designed as anti-inflammatory drugs for use in humans and fit the Lipinski rule-of-five (including bioavailability), they show promise for hit-to-lead optimisation and repurposing for use against parasitic nematodes. The screening assay established here has significant advantages over conventional methods, particularly in terms of ease of use, throughput, time and cost. Although not yet fully automated, the current assay is readily suited to the screening of hundreds to thousands of compounds for subsequent hit-to-lead optimisation. The current assay is highly adaptable to many parasites of socioeconomic importance, including those causing neglected tropical diseases. This aspect is of major relevance, given the urgent need to deliver the goals of the London Declaration (http://unitingtocombatntds.org/resource/london-declaration) through the rapid and efficient repurposing of compounds in public-private partnerships.

Superinfection of sows with Cystoisospora suis ante partum leads to a milder course of cystoisosporosis in suckling piglets.

Cystoisospora (syn. Isospora) suis is a leading cause of diarrheal disease in neonatal piglets. To address the possibility of maternal immunization against C. suis infection six non-naïve pregnant sows were superinfected with 100,000 oocysts 2 weeks ante partum and compared to non-superinfected animals. Their piglets were infected with 1000 oocysts on the third day of life. Clinical and parasitological parameters as well as antibody titers in colostrum/milk and blood of sows and in the blood of piglets were evaluated by IFAT against sporozoites and merozoites from 2 weeks ante partum until the 35th day after birth. For IFAT two different invasive stages of C. suis were used to find possible differences between the immune response against the initially infectious stages (sporozoites) and later occurring asexual developmental stages (merozoites), which might be responsible for persisting/extraintestinal infections. IFN-γ production of PBMC and piglet splenocytes was determined by ELISPOT. Maternal superinfection resulted in increased titers of IgA, IgM and IgG in colostrum and milk as well as in the blood of sows and their piglets. Oocyst shedding and diarrhea were observed in the offspring of both groups, but piglets of superinfected sows showed significantly reduced oocyst shedding and less diarrhea. This protective effect was correlated with increased titers of antibodies, especially IgA, in colostrum, milk and blood serum of sows and piglets, and with the reactivity of splenocytes to parasite antigen. Superinfection of sows ante partum could partially protect piglets against the clinical outcome of experimental infection. Both colostrum and milk contain maternal protective substances as the effect of protection was highly correlated with antibody titers during the first 2 weeks of life. IgA in different substrates may serve as a marker for the level of protection against clinical cystoisosporosis.

Transfer of Cystoisospora suis-specific colostral antibodies and their correlation with the course of neonatal porcine cystoisosporosis.

Cystoisospora suis is the most pathogenic species of coccidia in suckling piglets, affecting them predominantly within their first three weeks of life. The clinical signs of neonatal cystoisosporosis include watery diarrhea and wasting, leading to significant economic losses for the farmer. Since neonatal piglets have an immature immune system, colostral transfer of maternal factors such as immune cells or antibodies is essential for controlling infections at that age. However, the role of C. suis-specific antibodies transferred from the sow to the piglets and possible correlations between antibody levels in the piglets acquired from colostrum with the clinical outcome of disease are currently not understood. To address this issue, 12 non-infected piglets and 14 piglets experimentally infected with C. suis on the third day of life were examined during their first four weeks of life. IgG, IgA, and IgM titers in the blood serum specific for sporozoites and merozoites of C. suis were evaluated, along with oocyst excretion and fecal consistency. Additionally, the antibody content in the colostrum and milk of three mother sows was determined. A transfer of naturally acquired C. suis-specific antibodies from sows to piglets with the colostrum could be demonstrated. Maternal antibodies in piglets' blood sera did not persist for longer than 14-21 days except for IgG which was present in high titers until the end of the study. Within 2-3 weeks after birth the onset of endogenous antibody production was noticed. Titers in blood serum showed a correlation with the severity of diarrhea which was positive for IgG and IgM (possibly due to increased consumption or loss of these antibodies) and negative for IgA. C. suis-specific mucus antibodies isolated from infected and non-infected piglets (n=6/group) on the 28th day of life were present in both groups, showing significantly higher titers of IgA and IgM in infected piglets. Maternally transferred antibodies acquired by natural infections of sows as observed in this study did not provide protection against the clinical manifestation of disease. The level and effect of transferrable maternal factors necessary for protection still need to be elucidated. However, correlations between antibody titers and fecal consistency in the piglets indicate that C. suis-specific antibodies might be useful markers for the expectable clinical severity of cystoisosporosis.

Efficacy and safety of oral praziquantel against Dicrocoelium dendriticum in llamas.

Dicrocoelium dendriticum can cause severe pathological changes of the liver and bile system in camelids, and therapeutic options for treatment are limited. To address this problem, the efficacy of two different dose rates of praziquantel was investigated in llamas suffering from natural D. dendriticum infections. 53 llamas were examined under field conditions on two occasions: before and two weeks after treatment. At the beginning of the study, the animals were weighed, randomly allocated to one of the treatment groups (n=21 each) or the control group (n=11) and dosed orally using a praziquantel-containing paste (250 mg/ml) at a dose of either 25 mg (group 1) or 50 mg (group 2) per kg of body weight. Criteria for efficacy were faecal egg count reduction (FECR) and extensity effect. Animals treated with 25 mg/kg of body weight showed a FECR of 85%. Therapy with 50 mg/kg led to a FECR of 91%. Almost twice the number of animals of group 1 (33%) still shed eggs two weeks after treatment compared with group 2. The results of this study indicate that 50 mg/kg oral praziquantel is required for efficacious dosing and that this dose rate is safe in llamas and thus is recommended for the treatment of camelids naturally infected with D. dendriticum.

Oesophagostomum dentatum extract modulates T cell-dependent immune responses to bystander antigens and prevents the development of allergy in mice.

One third of the human population is currently infected by one or more species of parasitic helminths. Certain helminths establish long-term chronic infections resulting in a modulation of the host's immune system with attenuated responsiveness to "bystander" antigens such as allergens or vaccines. In this study we investigated whether parasite-derived products suppress the development of allergic inflammation in a mouse model. We show that extract derived from adult male Oesophagostomum dentatum (eMOD) induced Th2 and regulatory responses in BALB/c mice. Stimulation of bone marrow-derived dendritic cells induced production of regulatory cytokines IL-10 and TGF-beta. In a mouse model of birch pollen allergy, co-administration of eMOD with sensitizing allergen Bet v 1 markedly reduced the production of allergen-specific antibodies in serum as well as IgE-dependent basophil degranulation. Furthermore, eMOD prevented the development of airway inflammation, as demonstrated by attenuation of bronchoalveolar lavages eosinophil influx, peribronchial inflammatory infiltrate, and mucus secretion in lungs and IL-4 and IL-5 levels in lung cell cultures. Reduced secretion of Th2-related cytokines by birch pollen-re-stimulated splenocytes and mesenteric lymph node cells was observed in eMOD-treated/sensitized and challenged mice in comparison to sensitized and challenged controls. The suppressive effects of eMOD were heat-stable. Immunization with model antigens in the presence of eMOD reduced production of antibodies to thymus-dependent but not to thymus-independent antigen, suggesting that suppression of the immune responses by eMOD was mediated by interference with antigen presenting cell or T helper cell function but did not directly suppress B cell function. In conclusion, we have shown that eMOD possesses immunomodulatory properties and that heat-stable factors in eMOD are responsible for the dramatic suppression of allergic responses in a mouse model of type I allergy. The identification and characterization of parasite-derived immune-modulating molecules might have potential for designing novel prophylactic/therapeutic strategies for immune-mediated diseases.

In vivo and in vitro efficacy of sainfoin (Onobrychis viciifolia) against Eimeria spp in lambs.

The effect of sainfoin (Onobrychis viciifolia) against ovine coccidia was evaluated in vivo and in vitro. In 3 in vivo trials weaned lambs were allocated into two treatment groups receiving diets with either lucerne (Medicago sativa) or sainfoin. During the trials, which lasted for 7 (trial 1) or 8 weeks (trials 2 and 3), oocysts per gram of faeces (OPGs), faecal scores and weight gain were recorded. In two of the experiments (trials 1 and 3) a reduction in the mean oocyst excretion rates was observed, starting three to four weeks after sainfoin hay feeding. This reduction ranged between 21.3% (trial 1) and 61.7% (trial 3) compared to the control values. As a result, a decrease in the total number of oocysts excreted (expressed as the mean area under the curve of the OPG) was observed from week 4 to the end of the two trials, respectively (trial 1: 42.6% reduction, p=0.05; trial 3: 52.4% reduction, p=0.06). The results did not show any significant diet effect on lamb growth rates and faecal scores. In the in vitro experiments the effect of 39 sainfoin extracts were tested in an oocyst sporulation inhibition assay. The Eimeria oocysts sporulation inhibition throughout the experiments did not exceed 10.7%, showing that extracts of this forages do not have a significant inhibitory effect on Eimeria oocyst sporulation. This was an initial attempt to investigate a possible anticoccidial effect of sainfoin and further studies are needed in order to better understand its mode of action against Eimeria.

In vitro determination of anticryptosporidial activity of phytogenic extracts and compounds.

Cryptosporidiosis caused by Cryptosporidium spp. is an important diarrhoeal disease observed in farm animals and humans, especially in young or immunocompromised individuals. A novel cell culture assay for testing extracts and pure compounds against Cryptosporidium parvum in 96-well microplate format was established and evaluated. It is based on previously described indirect fluorescent antibody techniques and was optimised for higher sample throughput. Rapid assessment of minimal inhibitory concentrations (MICs) was done by checking each well microscopically for the presence or absence of parasite stages. As a novelty, parasite development was quantified by enumeration of clusters of secondary infection (CSI), which typically appeared upon infection with a distinct parasite inoculum after a defined incubation time. Host cell (HCT-8) viability was measured by an integrated non-destructive water-soluble tetrazolium salt assay (WST-1), which facilitated discrimination of antiparasitic activity from possible cytotoxic effects of a test compound against the host cells. Host cell viability was regarded unimpaired when cultures had 75% or more viability when compared to control cultures without test substance. In this study, a maximum density of distinguishable CSI was obtained when cultures were infected with 2.5 × 10(3) oocysts and incubated for 48 h. The applicable inoculum has to be optimised for each batch of oocysts and before each experimental series. Parasite development was inhibited completely by monensin at 134 nM and silymarin at 50 mg/mL. These concentrations were non-toxic to the host cells and comparable to literature data. The percentages of parasite inhibition were determined for monensin and a 50% inhibitory concentration (IC(50)) of 36.6 nM (27.4-45.5) and a 90% inhibitory concentration of 65.9 nM (54.8-90.2) were calculated. The introduced assay is economic because relatively low parasite numbers may be used. If MICs are determined, evaluation is fast, as each well is viewed only briefly under the fluorescence microscope for presence or absence of CSI. Furthermore it is highly critical because only full parasite inhibition is assessed. Counting of CSI is more laborious and time-consuming, but it allows calculation of parasite inhibition rates and parameters like the half maximal inhibitory concentration (IC(50)). This assay shall be used to assess anticryptosporidial activities of various plant waste materials and by-products from the food and the pharmaceutical industries in the course of the EU project SAFEWASTES. Comparison with in vivo models should be performed to further corroborate the results. Automated evaluation by flow cytometry might facilitate higher sample throughput and reduce operator bias.

Immunoregulation by Toxoplasma gondii infection prevents allergic immune responses in mice.

Toxoplasma gondii is a ubiquitous intracellular parasite affecting most mammals including humans. In epidemiological studies, infection with T. gondii and allergy development have been postulated to be inversely related. Using a mouse model of birch pollen allergy we investigated whether infection with T. gondii influences allergic immune responses to birch pollen. BALB/c mice were infected with T. gondii oocysts either before or at the end of sensitisation with the major birch pollen allergen Bet v 1 and thereafter aerosol challenged with birch pollen extract. During the acute phase of infection, clinical signs correlated with increased levels of serum TNF-alpha, IL-6, IFN-gamma and anti-Toxoplasma-IgM. In the chronic phase, Toxoplasma-specific serum IgG, brain tissue cysts and high IFN-gamma production in spleen cell cultures were detected. Mice infected prior to allergic sensitisation produced significantly less allergen-specific IgE and IgG1, while IgG2a levels were markedly increased. IL-5 levels in spleen cell cultures and bronchoalveolar lavage fluid were significantly reduced, and airway inflammation was prevented in these mice. Notably, in mice infected at the end of the allergic sensitisation process, systemic and local immune responses to the allergen were markedly reduced. T.gondii infection was associated with up-regulation of Toll-like receptor 2 (TLR2), 4, 9 and 11, as well as T-bet (a differentiation factor for Th1 cells) mRNA expression in splenocytes; moreover, enhanced TGF-beta, IL-10 and Foxp3 mRNA expression in these cells suggested that regulatory mechanisms were involved in suppression of the allergic immune response. Kinetic studies confirmed the induction of Foxp3(+)CD4(+)CD25(+) regulatory T cells preferentially during the chronic phase of T. gondii infection. Our data demonstrate that T. gondii exhibits strong immunomodulating properties which lead to prevention of allergic immune responses and thereby support the hygiene hypothesis.