Toxocara is a genus of nematodes, which infects a variety of hosts, principally dogs and cats, with potential zoonotic risks to humans. Toxocara spp. larvae are capable of migrating throughout the host tissues, eliciting eosinophilic and granulomatous reactions, while surviving for extended periods of time, unchanged, in the host. It is postulated that larvae are capable of altering the host\'s immune response through the release of excretory-secretory products, containing both proteins and extracellular vesicles (EVs). The study of EVs has increased exponentially in recent years, largely due to their potential use as a diagnostic tool, and in molecular therapy. To this end, there have been multiple isolation methods described for the study of EVs. Here, we use nanoparticle tracking to compare the yield, size distribution, and % labelling of EV samples acquired through various reported methods, from larval cultures of Toxocara canis and T. cati containing Toxocara excretory-secretory products (TES). The methods tested include ultracentrifugation, polymer precipitation, magnetic immunoprecipitation, size exclusion chromatography, and ultrafiltration. Based on these findings, ultrafiltration produces the best results in terms of yield, expected particle size, and % labelling of sample. Transmission electron microscopy confirmed the presence of EVs with characteristic cup-shaped morphology. These findings can serve as a guide for those investigating EVs, particularly those released from multicellular organisms, such as helminths, for which few comparative analyses have been performed.

OBJECTIVE: We compared the effect of 12 weeks lesser mealworm-based (Alphitobius diaperinus) protein supplementation to whey protein and placebo supplementation on Exercise-Induced Muscle Damage (EIMD) after long-distance walking in older adults.
METHODS: in this randomized controlled trial, 70 physically active older adults (>60 years) were randomly allocated to the following groups: I) lesser mealworm protein, II) whey protein or III) iso-caloric placebo. Participants received supplements 11 weeks before and 1 week during a 3-day long-distance walking challenge (30-50 km per day). Blood concentrations of creatinine kinase (CK) and lactate dehydrogenase (LDH), handgrip strength and muscle soreness were measured pre-exercise and directly after each walking bout.
RESULTS: Significant elevations of CK concentrations (103 [76-161] U/l to 758 [342-1104] U/l, p < 0.001) and LDH concentrations (202 [175-220] to 283 [252-339] U/l, p < 0.001) were observed following 7h45 min ± 11 min of walking exercise per day, but the magnitude of this effect did not differ among suppletion groups. Hand grip strength decreased significantly (p < 0.001) while muscle soreness increased (p = 0.002) after the first walking day compared to pre-exercise, with no group differences.
CONCLUSIONS: 12-weeks of lesser mealworm-based protein supplementation (30 g/day) does not attenuate exercise induced muscle damage in older adults following three days of prolonged walking exercise in comparison to placebo or whey protein.

Fasciola hepatica is a trematode parasite that infects animals and humans causing fasciolosis, a worldwide-distributed disease responsible for important economic losses and health problems. This disease is of growing public health concern since parasite isolates resistant to the current treatment (triclabendazole) have increasingly been described. F. hepatica infects its vertebrate host after ingestion of the encysted parasite (metacercariae), which are found in the water or attached to plants. Upon ingestion, newly excysted juveniles of F. hepatica (FhNEJ) emerge in the intestinal lumen and cross the intestinal barrier, reach the peritoneum and migrate to the biliary ducts, where adult worms fully develop. Despite the efforts made to develop new therapeutic and preventive tools, to date, protection against F. hepatica obtained in different animal models is far from optimal. Early events of host-FhNEJ interactions are of paramount importance for the infection progress in fasciolosis, especially those occurring at the host-parasite interface. Nevertheless, studies of FhNEJ responses to the changing host environment encountered during migration across host tissues are still scarce. Here, we set-up an ex vivo model coupled with quantitative SWATH-MS proteomics to study early host-parasite interaction events in fasciolosis. After comparing tegument and somatic fractions from control parasites and FhNEJ that managed to cross a mouse intestinal section ex vivo, a set of parasite proteins whose expression was statistically different were found. These included upregulation of cathepsins L3 and L4, proteolytic inhibitor Fh serpin 2, and a number of molecules linked with nutrient uptake and metabolism, including histone H4, H2A and H2B, low density lipoprotein receptor, tetraspanin, fatty acid binding protein a and glutathione-S-transferase. Downregulated proteins in FhNEJ after gut passage were more numerous than the upregulated ones, and included the heath shock proteins HSP90 and alpha crystallin, amongst others. This study brings new insights into early host-parasite interactions in fasciolosis and sheds light on the proteomic changes in FhNEJ triggered upon excystment and intestinal wall crossing, which could serve to define new targets for the prevention and treatment of this widespread parasitic disease.

BACKGROUND: An accurate test for the diagnosis and post-treatment follow-up of patients with schistosomiasis is needed. We assessed the performance of different laboratory parameters, including the up-converting reporter particle technology lateral flow assay to detect circulating anodic antigen (UCP-LF CAA), for the post-treatment follow-up of schistosomiasis in migrants attending a dedicated outpatient clinic in a non-endemic country.
METHODS: Routine anti-Schistosoma serology results and eosinophil counts were obtained of patients with positive urine/stool microscopy and/or PCR (confirmed cases) or only positive serology (possible cases), and at least one follow-up visit at 6 (T6) or 12 (T12) months after praziquantel treatment. All sera samples were tested with the UCP-LF CAA assay.
RESULTS: Forty-eight patients were included, 23 confirmed and 25 possible cases. The percentage seropositivity and median antibody titers did not change significantly during follow-up. UCP-LF CAA was positive in 86.9% of confirmed and 20% of possible cases. The percentage positivity and median CAA levels decreased significantly post-treatment, with only two patients having positive CAA levels at T12.
CONCLUSIONS: The UCP-LF CAA assay proved useful for the diagnosis of active infection with Schistosoma spp. and highly valuable for post-treatment monitoring in migrants, encouraging the development of a commercial test.

The objective of this study was to analyze the mRNA transcription levels of ten functional genes of P-glycoproteins (P-gp) in free life stages, eggs and infective larvae (L3) and in endoparasitic stages, fourth larval stage (L4) and adult males of two native isolates of Haemonchus contortus: resistant and susceptible to IVM. The IVM resistant isolate was obtained from sheep naturally infected with H. contortus, and the susceptible isolate (with no pressure to IVM) conserved since 1990. The lethal effect of IVM was evaluated under in vitro conditions, which showed significant differences between susceptible and resistant H. contortus L3 isolates (P < 0.01). The IVM susceptible isolate revealed a lethal effect of 79.22% at 11.42 mM, whereas that resistant isolate showed no lethal effect at any of the four assessed concentrations (1.43, 2.85, 5.71 and 11.42 mM) of IVM. The expression levels of ten Hco-pgp genes (1, 2, 3, 4, 9, 10, 11, 12, 14, and 16) were evaluated in the resistant isolate of H. contortus and compared to the susceptible isolate (as control), using two constitutive genes (GAPDH and β-tubulin). Up-regulation at two statistical significant values (P ≤ 0.05, 0.1) was the criterion to associate IVM resistance with the free life and endoparasitic stages of H. contortus. The expression levels in H. contortus adult nematodes showed 5.64 to 127.56-fold increase for Hco-pgp genes 1, 9, 12, 14, and 16, followed by an increase for Hco-pgp-2 (49.75-fold) and Hco-pgp-10 (106.40-fold) in L4, and for Hco-pgp-16 (2.90-fold) in eggs (P ≤ 0.05). In addition, high expression levels with P < 0.1 were detected in H. contortus L3, L4, and adults for Hco-pgp genes 1, 4, 11, 12, and 16, with changes ranging from 2.17 to 29.72-fold. In conclusion, the highest expression was observed in the adult stage of H. contortus, and the most frequent gene with a significant P-value was Hco-pgp-16, revealing it plays an important role in IVM resistance.

Cystic echinococcosis (CE) is one of the most important zoonotic parasitic diseases caused by the larval stage of Echinococcus granulosus. Based on molecular studies and DNA sequencing, E. granulosus has been classified into 10 different genotypes (G1 to G10). Two neighboring countries, Turkey and Iran, are considered the two main foci of CE in the Middle East. The current study is aimed at examining the genotype diversity of E. granulosus isolated from human clinical samples in Turkey and Iran. Surgically removed human hydatid cysts were collected from East Azerbaijan and Fars provinces in Iran and Van province in Turkey. After extracting DNA, performing PCR, targeting the cox1 gene, the PCR products were purified from the gel and were sequenced from both directions. The sequences were aligned and compared, using BioEdit and also the BLAST program of GenBank. The maximum likelihood tree was constructed based on the Tamura-Nei model, using the MEGAX software. Phylogenetic analysis showed that the human isolated samples were classified into two major clades: G1 (from Iran and Turkey) and G3 (5 samples from northwestern Iran and one sample from Turkey). The mean and degree of genetic divergence (K2P) between the two major clades, G1 and G3, were 0.2% and 0.7 ± 0.4%, respectively. The findings of the current study revealed that the sheep strain (G1) and the less important strain G3 have major roles in the transmission cycle of CE in two neighboring countries, Iran and Turkey. Therefore, it is necessary to interpose the life cycle of this parasite and reduce the disease burden in livestock and humans by adopting common regional preventive and control policies.

Preventive chemotherapy with praziquantel (PZQ) is the cornerstone of schistosomiasis control. However, a single dose of PZQ (40 mg/kg) does not cure all infections. Repeated doses of PZQ at short intervals might increase efficacy in terms of cure rate (CR) and intensity reduction rate (IRR). Here, we determined the efficacy of a single versus four repeated treatments with PZQ on Schistosoma mansoni infection in school-aged children from Côte d\'Ivoire, using two different diagnostic tests.
An open-label, randomized controlled trial was conducted from October 2018 to January 2019. School-aged children with a confirmed S. mansoni infection based on Kato-Katz (KK) and point-of-care circulating cathodic antigen (POC-CCA) urine cassette test were randomly assigned to receive either a single or four repeated doses of PZQ, administered at two-week intervals. The primary outcome was the difference in CR between the two treatment arms, measured by triplicate KK thick smears 10 weeks after the first treatment. Secondary outcomes included CR estimated by POC-CCA, IRR by KK and POC-CCA, and safety of repeated PZQ administration.
During baseline screening, 1,022 children were assessed for eligibility of whom 153 (15%) had a detectable S. mansoni infection, and hence, were randomized to the standard treatment group (N = 70) and the intense treatment group (N = 83). Based on KK, the CR was 42% (95% confidence interval (CI) 31-52%) in the standard treatment group and 86% (95% CI 75-92%) in the intense treatment group. Observed IRR was 72% (95% CI 55-83%) in the standard treatment group and 95% (95% CI 85-98%) in the intense treatment group. The CR estimated by POC-CCA was 18% (95% CI 11-27%) and 36% (95% CI 26-46%) in the standard and intense treatment group, respectively. Repeated PZQ treatment did not result in a higher number of adverse events.
The observed CR using KK was significantly higher after four repeated treatments compared to a single treatment, without an increase in adverse events. Using POC-CCA, the observed CR was significantly lower than measured by KK, indicating that PZQ may be considerably less efficacious as concluded by KK. Our findings highlight the need for reliable and more accurate diagnostic tools, which are essential for monitoring treatment efficacy, identifying changes in transmission, and accurately quantifying the intensity of infection in distinct populations. In addition, the higher CR in the intense treatment group suggests that more focused and intense PZQ treatment can help to advance schistosomiasis control.
www.clinicaltrials.gov NCT02868385.

An original cytogenetic study combining classical karyotype analysis and modern fluorescence in situ hybridization using telomeric (TTAGGG)n and ribosomal sequences (18S rDNA) was performed in Khawia abbottinae (Cestoda, Caryophyllidea), a parasite of Chinese false gudgeon (Abbottina rivularis) from China. Analyses based on conventional Giemsa staining, DAPI, YOYO-1 dye, and silver (Ag) staining were also carried out. The karyotype is composed of eight pairs of metacentric and telocentric chromosomes (2n = 16, n=5m + 3t). Constitutive heterochromatin was mainly positioned at pericentromeric regions, and telomeric sequences (TTAGGG)n were restricted to the end of all chromosomes. In mitotic preparations stained with Giemsa, both homologues of chromosome pair 4 showed a distinct secondary constriction. FISH with rDNA probe confirmed that this secondary constriction contains a nucleolar organizer region (NOR). The process of spermatocyte meiosis and the dynamics of nucleolus degradation in dividing cell were scrutinized. The present study and its results enhance the limited knowledge on basic karyotype characteristics and 18S rDNA clusters location in caryophyllidean tapeworms.

The point-of-care strip assay for the detection of the schistosome Circulating Cathodic Antigen (POC-CCA) in urine has shown to be a user-friendly and sensitive alternative to stool microscopy for the diagnosis of Schistosoma mansoni infections. However, visual scoring of the test is by definition observer dependent and leads to discussion about the qualitative interpretation, in particular in low intensity infections when test lines tend to be weak. In order to standardise visual scoring, an innovative approach for semi-quantitative interpretation of the POC-CCA cassettes, called G-scores, was developed and evaluated. Urines (n = 110) from a S. mansoni endemic area were used to evaluate this new approach. Test lines of the POC-CCA were visually compared against the G-scores, i.e. a series of artificial cassettes containing inkjet-printed strips of different intensities in order to grade the POC-CCA test line on a scale of 1 to 10. A significant positive correlation (Spearman 0.660, p < 0.001) was observed between G-scores and eggs per gram of faeces. This proof-of-concept study demonstrates the usefulness of the G-scores for standardising the visual scoring of the POC-CCA urine strip assay. Several research groups have already indicated an interest in the G-scores for their field work. Further distribution of the cassettes, in particular when provided in combination with reference standards, will assist the wider schistosomiasis community in dealing with issues like batch-to-batch differences and interpretation of trace readings.

Schistosomiasis remains a serious zoonotic disease in China and the Philippines. Water buffalo and cattle account for the majority of transmission. Vaccination of water buffalo is considered a key strategy to reduce disease prevalence. Previously, we showed that vaccination of water buffalo with SjC23 or SjCTPI plasmid DNA vaccines, induced 50% efficacy to challenge infection. Here, we evaluated several parameters to determine if we can develop a two dose vaccine that maintains the efficacy of the three dose vaccine. We performed four trials evaluating: (1) lab produced vs. GLP grade vaccines, (2) varying the time between prime and boost, (3) the influence of an IL-12 adjuvant, and (4) a two dose heterologous (DNA-protein) prime-boost. We found the source of the DNA vaccines did not matter, nor did increasing the interval between prime and boost. Elimination of the IL-12 plasmid lowered homologous DNA-DNA vaccine efficacy. A major finding was that the heterologous prime boost improved vaccine efficacy, with the prime-boost regimen incorporating both antigens providing a 55% reduction in adult worms and 53% reduction in liver eggs. Vaccinated buffalo produced vaccine-specific antibody responses. These trials suggest that highly effective vaccination against schistosomes can be achieved using a two dose regimen. No adjuvants were used with the protein boost, and the potential that addition of adjuvant to the protein boost to further increase efficacy should be evaluated. These results suggest that use of these two schistosome vaccines can be part of an integrated control strategy to reduce transmission of schistosomiasis in Asia.