Correction: A novel cell-free method to culture Schistosoma mansoni from cercariae to juvenile worm stages for in vitro drug testing.

[This corrects the article DOI: 10.1371/journal.pntd.0006590.].

Identification of hit compounds with anti-schistosomal activity on in vitro generated juvenile worms in cell-free medium.

BACKGROUND: Anthelminthic treatment options against schistosomiasis are limited. The current treatment relies almost exclusively on a single drug, praziquantel (PZQ). As a consequence, the development of resistance to PZQ and limited activity of PZQ against earlier development stages are respectively a risk and a limitation to achieving the goals of the new WHO roadmap towards elimination. For the discovery of new chemical starting points, the in vitro drug screening on Schistosoma mansoni (S. mansoni) against newly transformed schistosomula (NTS) is still the most predominant approach. The use of only NTS in the initial screening limits sensitivity to potential new compounds which are predominantly active in later developmental stages. Using our recently described highly standardized, straightforward and reliable culture method that generates high rates of juvenile worms, we aimed to repurpose a subset of the National Center for Advancing Translational Sciences (NCATS) Pharmaceutical Collection (340 compounds) to identify new hits with an in vitro worm culture assay. METHODOLOGY/PRINCIPAL FINDINGS: Cercariae were mechanically transformed into skin-stage (SkS) schistosomula and continuously cultured for 3-6 weeks to the liver stage (LiS). A commercial source of serum was identified, and decrease of NTS/well along with optimal drug testing conditions was established to test compounds on early and late LiS worms. The library was screened in 96-well format assays using praziquantel (PZQ) as a positive control. Primary screening allowed a 5.9% hit rate and generated two confirmed hits on adult worms; a prophylactic antianginal agent and an antihistaminic drug. CONCLUSION: With this standardized and reliable in vitro assay, important S. mansoni developmental stages up to LiS worms can be generated and cultured over an extended period. When exposed to a subset of the NCATS Pharmaceutical Collection, 3 compounds yielded a defined anti-schistosomal phenotype on juvenile worms. Translation of activity on perfused adult S. mansoni worms was achieved only for perhexiline (a prophylactic antianginal agent) and astemizole (an antihistaminic drug).

Host immune responses during Taenia solium Neurocysticercosis infection and treatment.

Taenia solium cysticercosis and taeniasis (TSCT), caused by the tapeworm T. solium, is a foodborne and zoonotic disease classified since 2010 by WHO as a neglected tropical isease. It causes considerable impact on health and economy and is one of the leading causes of acquired epilepsy in most endemic countries of Latin America, Sub-Saharan Africa, and Asia. There is some evidence that the prevalence of TSCT in high-income countries has recently increased, mainly due to immigration from endemic areas. In regions endemic for TSCT, human cysticercosis can manifest clinically as neurocysticercosis (NCC), resulting in epileptic seizures and severe progressive headaches, amongst other neurological signs and/or symptoms. The development of these symptoms results from a complex interplay between anatomical cyst localization, environmental factors, parasite's infective potential, host genetics, and, especially, host immune responses. Treatment of individuals with active NCC (presence of viable cerebral cysts) with anthelmintic drugs together with steroids is usually effective and, in the majority, reduces the number and/or size of cerebral lesions as well as the neurological symptoms. However, in some cases, treatment may profoundly enhance anthelmintic inflammatory responses with ensuing symptoms, which, otherwise, would have remained silent as long as the cysts are viable. This intriguing silencing process is not yet fully understood but may involve active modulation of host responses by cyst-derived immunomodulatory components released directly into the surrounding brain tissue or by the induction of regulatory networks including regulatory T cells (Treg) or regulatory B cells (Breg). These processes might be disturbed once the cysts undergo treatment-induced apoptosis and necrosis or in a coinfection setting such as HIV. Herein, we review the current literature regarding the immunology and pathogenesis of NCC with a highlight on the mobilization of immune cells during human NCC and their interaction with viable and degenerating cysticerci. Moreover, the immunological parameters associated with NCC in people living with HIV/AIDS and treatments are discussed. Eventually, we propose open questions to understand the role of the immune system and its impact in this intriguing host-parasite crosstalk.

A novel cell-free method to culture Schistosoma mansoni from cercariae to juvenile worm stages for in vitro drug testing.

BACKGROUND: The arsenal in anthelminthic treatment against schistosomiasis is limited and relies almost exclusively on a single drug, praziquantel (PZQ). Thus, resistance to PZQ could constitute a major threat. Even though PZQ is potent in killing adult worms, its activity against earlier stages is limited. Current in vitro drug screening strategies depend on newly transformed schistosomula (NTS) for initial hit identification, thereby limiting sensitivity to new compounds predominantly active in later developmental stages. Therefore, the aim of this study was to establish a highly standardized, straightforward and reliable culture method to generate and maintain advanced larval stages in vitro. We present here how this method can be a valuable tool to test drug efficacy at each intermediate larval stage, reducing the reliance on animal use (3Rs). METHODOLOGY/PRINCIPAL FINDINGS: Cercariae were mechanically transformed into skin-stage (SkS) schistosomula and successfully cultured for up to four weeks with no loss in viability in a commercially available medium. Under these serum- and cell-free conditions, development halted at the lung-stage (LuS). However, the addition of human serum (HSe) propelled further development into liver stage (LiS) worms within eight weeks. Skin and lung stages, as well as LiS, were submitted to 96-well drug screening assays using known anti-schistosomal compounds such as PZQ, oxamniquine (OXM), mefloquine (MFQ) and artemether (ART). Our findings showed stage-dependent differences in larval susceptibility to these compounds. CONCLUSION: With this robust and highly standardized in vitro assay, important developmental stages of S. mansoni up to LiS worms can be generated and maintained over prolonged periods of time. The phenotype of LiS worms, when exposed to reference drugs, was comparable to most previously published works for ex vivo harvested adult worms. Therefore, this in vitro assay can help reduce reliance on animal experiments in search for new anti-schistosomal drugs.

Temperature dependent embryonic development of Trichuris suis eggs in a medicinal raw material.

The therapeutic potential of infective pig whipworm eggs, Trichuris suis ova (TSO), is currently tested in several clinical trials on immune-mediated diseases. This paper studied the embryonic development of TSO in a medicinal raw product, where the parasite eggs were suspended in sulphuric acid (pH1). Unembryonated T. suis egg batches were stored at 5, 10, 15, 20, 25, 30, and 40°C (±1°C) and examined at 2, 4, 8, and 14 weeks. Subsequently, sub-batches from each temperature were allowed to embryonate for additional 14 weeks at 25°C, and selected samples were tested for infectivity in Göttingen minipigs. Both male and female pigs were used to evaluate eventual gender specific infectivity. Storage at 30°C up to 14 weeks and subsequent embryonation for 14 weeks at 25°C did not significantly reduce the overall larval establishment in minipigs, as compared to storage at 5°C and subsequent embryonation at 25°C. As marked impairment of egg development was observed during storage at 40°C, a second set of unembryonated egg batches were incubated at 30, 32, 34, 36, 38, and 40°C (±1°C) for 1-8 weeks. The development of the eggs was repeatedly examined by manual light microscopy, multispectral analysis (OvaSpec), and an egg hatching assay prior to the final testing in minipigs (Trial 1). These methods showed that the development started earlier at higher temperatures, but the long-term storage at higher temperature affected the egg development. The present study further documents tolerance of the TSO to storage at temperature 5-15°C, at which temperature development of larvae is not initiated.

Bacteria-induced egg hatching differs for Trichuris muris and Trichuris suis.

BACKGROUND: Eggs of the porcine whipworm Trichuris suis are currently explored in human clinical trials as a treatment of immune-mediated diseases. In this context, only the infective, embryonated eggs, constitute the Active Pharmaceutical Ingredient (API). The rodent whipworm, Trichuris muris is commonly used as a laboratory model to study Trichuris biology. The embryonated eggs (containing a fully developed larva) are biologically active and will invade the large intestinal mucosa of the host. This study aims to assess the in vitro hatching of T. muris and T. suis eggs in various bacterial cultures as a measure for their biological activity. METHODS: Eggs of T. muris and T. suis were incubated with Escherichia coli strain (BL-21) at three concentrations in a slightly modified in vitro egg hatching assay previously developed for T. muris. Additionally, E. coli strains (M15, SG13009, PMC103, JM109, TUNER, DH5alpha, TOP10) and five Gram-positive bacteria (Enterococcus caccae, Streptococcus hyointestinalis, Lactobacillus amylovorus, L. murinus, and L. reuteri) were tested as a hatching stimulus for T. muris and T. suis eggs. RESULTS: Whereas T. muris eggs hatched, T. suis did not, even when exposed to different concentrations and strains of E. coli after 4 and 24-hour incubation. When incubated with Gram-positive bacteria, only T. muris eggs showed noticeable hatching after 20 h, although with high variability. CONCLUSIONS: The observed difference in hatching of T. muris and T. suis eggs incubated with selected bacteria, indicate significant biological differences which may reflect specific adaptation to different host-specific gut microbiota.

In vitro hatching of Trichuris suis eggs.

Eggs of the pig whipworm, Trichuris suis ova (TSO), are currently tested in human clinical trials for their potential immunomodulatory capacity. The biological potency of TSO (egg viability and infectivity) is traditionally assessed in Göttingen minipigs as the establishment of intestinal larvae after inoculation with a known number of eggs. To minimize testing in animal models, development of an in vitro egg hatching assay is proposed as a reliable, cost-effective, and a faster alternative to test the egg viability. The present study aimed to investigate the influence of different chemical, physical, and biological factors on egg hatching. Thus, in a series of experiments and in different combinations, the eggs were stimulated with glass beads, artificial gastric juice, bile salt and trypsin solution, fermentation gut medium, or stimulated with mucosal scrapings from the ileum and the large intestine of the infected and uninfected Göttingen minipig. Mechanical stimulation with glass beads presented a simple and reproducible method for egg hatching. However, incubation of eggs with mucosal scrapings from the ileum, caecum, and colon for 24 h at 38 °C significantly increased hatching.

Dose-dependent establishment of Trichuris suis larvae in Göttingen minipigs.

Embryonated eggs of the pig whipworm Trichuris suis (TSOee) constitute the active pharmaceutical ingredient (API) in a medicinal product explored in human clinical trials against several immune-mediated diseases. The measurement of TSO biological potency (hatchability and infectivity) is a requirement for the assessment of TSO's pharmacological potency in human clinical trials. The present study aims to validate the dose-dependent establishment of T. suis larvae in Göttingen minipigs and eventual clinical implication of a dose range (1000-10,000 TSO). Four groups of 5 minipigs were inoculated with doses of 1000, 2500, 7500, and 10,000 TSOee, respectively, to evaluate a range of concentrations of TSOee in a minipig infectivity model. Unembryonated eggs (TSOue) were added to keep the total egg number in the inoculum constant at 10,000 eggs. Two groups received 2500 and 7500 TSOee per pig without the addition of TSOue as controls. The intestinal larval establishment at 21 days post inoculation (dpi) demonstrated a clear positive linear dose-response relationship between numbers of inoculated TSOee and recovered larvae. There was a low level of variation in larval counts in all study groups. Thus, the infectivity model in minipigs within the tested dose range offers a reliable, sensitive and accurate assay for testing biological potency of TSO.

OvaSpec - A vision-based instrument for assessing concentration and developmental stage of Trichuris suis parasite egg suspensions.

BACKGROUND: OvaSpec is a new, fully automated, vision-based instrument for assessing the quantity (concentration) and quality (embryonation percentage) of Trichuris suis parasite eggs in liquid suspension. The eggs constitute the active pharmaceutical ingredient in a medicinal drug for the treatment of immune-mediated diseases such as Crohn׳s disease, ulcerative colitis, and multiple sclerosis. METHODS: This paper describes the development of an automated microscopy technology, including methodological challenges and design decisions of relevance for the future development of comparable vision-based instruments. Morphological properties are used to distinguish eggs from impurities and two features of the egg contents under brightfield and darkfield illumination are used in a statistical classification to distinguish eggs with undifferentiated contents (non-embryonated eggs) from eggs with fully developed larvae inside (embryonated eggs). RESULTS: For assessment of the instrument׳s performance, six egg suspensions of varying quality were used to generate a dataset of unseen images. Subsequently, annotation of the detected eggs and impurities revealed a high agreement with the manual, image-based assessments for both concentration and embryonation percentage (both error rates <1.0%). Similarly, a strong correlation was demonstrated in a final, blinded comparison with traditional microscopic assessments performed by an experienced laboratory technician. CONCLUSIONS: The present study demonstrates the applicability of computer vision in the production, analysis, and quality control of T. suis eggs used as an active pharmaceutical ingredient for the treatment of autoimmune diseases.

Culicoides obsoletus (Diptera: Ceratopogonidae) in Bosnia and Herzegovina-first report.

The first occurrence of bluetongue disease in Bosnia and Herzegovina was registered in 2002 in the area of Kalesija municipality. Entomological investigation of the presence of Culicoides species in that area was conducted in 2007. The aim of the research was to establish the presence of the main vector of bluetongue virus. Collections and analyses of Culicoides midges were performed in accordance with the protocols of the National Reference Centre for Exotic Diseases (Centro Studi Malattie Esotiche) in Teramo, Italy. Traps for capturing midges were placed next to four sheep farms. During the investigation, a total of 2,256 Culicoides midges were collected and only one species was identified, Culicoides obsoletus Meigen, 1818.