Anisakis pegreffii is a sibling species within the A. simplex (s.l.) complex requiring marine homeothermic (mainly cetaceans) and heterothermic (crustaceans, fish, and cephalopods) organisms to complete its life cycle. It is also a zoonotic species, able to accidentally infect humans (anisakiasis). To investigate the molecular signals involved in this host-parasite interaction and pathogenesis, the proteomic composition of the extracellular vesicles (EVs) released by the third-stage larvae (L3) of A. pegreffii, was characterized.
Genetically identified L3 of A. pegreffii were maintained for 24 h at 37°C and EVs were isolated by serial centrifugation and ultracentrifugation of culture media. Proteomic analysis was performed by Shotgun Analysis.
EVs showed spherical shaped structure (size 65-295 nm). Proteomic results were blasted against the A. pegreffii specific transcriptomic database, and 153 unique proteins were identified. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis predicted several proteins belonging to distinct metabolic pathways. The similarity search employing selected parasitic nematodes database revealed that proteins associated with A. pegreffii EVs might be involved in parasite survival and adaptation, as well as in pathogenic processes. Further, a possible link between the A. pegreffii EVs proteins versus those of human and cetaceans\' hosts, were predicted by using HPIDB database. The results, herein described, expand knowledge concerning the proteins possibly implied in the host-parasite interactions between this parasite and its natural and accidental hosts.

UNASSIGNED: When Angiostrongylus cantonensis develops from the third and fourth stage, it needs to change its host from the middle host, snail to the final host, rat. However, the mechanism involved in this change remains unclear.
UNASSIGNED: The transcriptome differences of the third and fourth stages of A. cantonensis were explored by next-generation Illumina Hiseq/Miseq sequencing in China, in 2018.
UNASSIGNED: Overall, 137 956 488 clean reads and 20 406 213 373 clean bases of the two stages larvae were produced. Based on the queries against the Gene Ontology (GO), NCBI non-redundant protein sequences (Nr), Swissprot, and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, 14 204 differentially expressed genes (DEGs) were predicted. GO enrichment analysis revealed 5660 DEGs with the top s categories as followings: biological process (GO:0008150, related to 5345 DEGs), cellular component (GO:0005575, related to 5297 DEGs), molecular function (GO:0003674, related to 5290 DEGs). In KEGG enrichment analysis, 116 genes were related to oxidative phosphorylation and 49 genes involved in the glycolytic process.
UNASSIGNED: Metabolism changes, especially oxidative phosphorylation and glycolysis, might play a key role in A. cantonensis infection of its final rat host. Many other pathways might also contribute to the transcriptome changes between these two life stages. Overall, additional studies are needed for further details.

Anthelmintics of the macrocyclic lactone (ML) drug class are widely used as preventives against the canine heartworm (Dirofilaria immitis). Over the past several years, however, reports of ML lack of efficacy (LOE) have emerged, in which dogs develop mature heartworm infection despite the administration of monthly prophylactics. More recently, isolates from LOE cases have been used to infect laboratory dogs and the resistant phenotype has been confirmed by the establishment of adult worms in the face of ML treatment at normally preventive dosages. Testing for and monitoring resistance in D. immitis requires a validated biological or molecular diagnostic assay. In this study, we assessed a larval migration inhibition assay (LMIA) that we previously optimized for use with D. immitis third-stage larvae (L3). We used this assay to measure the in vitro ML susceptibilities of a known-susceptible laboratory strain of D. immitis and three highly suspected ML-resistant isolates originating from three separate LOE cases; progeny from two of these isolates have been confirmed ML-resistant by treatment of an infected dog in a controlled setting. A nonlinear regression model was fit to the dose-response data, from which IC50 values were calculated. The D. immitis LMIA yielded consistent and reproducible dose-response data; however, no statistically significant differences in drug susceptibility were observed between control and LOE parasites. Additionally, the drug concentrations needed to paralyze the L3 were much higher than those third- and fourth-stage larvae would experience in vivo. IC50 values ranged from 1.57 to 5.56μM (p≥0.19). These data could suggest that ML resistance in this parasite is not mediated through a reduced susceptibility of L3 to the paralytic effects of ML drugs, and therefore motility-based assays are likely not appropriate for measuring the effects of MLs against D. immitis in this target stage.

A quantitative real-time polymerase chain reaction (qPCR) based on hydrolysis (TaqMan) probes is described for robust and sensitive detection of the infection levels with eggs and third stage larvae (L3) of Cooperia oncophora and Ostertagia ostertagi isolated from cattle faeces. The current microscopic method for identification of strongyle nematodes in cattle faeces is labour-intensive where reliable species determination also requires trained expertise, which is increasingly lacking. The goal of this study was to develop a sustainable non-labour intensive diagnostic qPCR assay to detect and determine the levels of infection with the two most common gastro-intestinal nematodes (GIN) in cattle faeces targeting the second internal transcribed spacer of nuclear ribosomal DNA (ITS2) region (rDNA). According to our results, this procedure allows to reliably detect the relative proportions of eggs and L3 for each of the two species. This assay produced consistent results when mixtures with known numbers of L3 of both species were tested, although it was also demonstrated that the calculated copy numbers of ITS2 between single L3 sometimes varied very much. In addition, a positive correlation (r(2)=0.23) between the proportion of eggs and L3 in different paired samples collect in the field was observed for both species. Thus, for the first time a qPCR assay is reported, which can discriminate between the two most important cattle nematode parasites in temperate regions. This is of major importance to the livestock sector as it can be used with great precision to demonstrate strategic treatment efficacy that is important for the detection of anthelmintic resistance (AR).

Recent experiments on the effects of rainfall and/or soil moisture (SM) on development of sheep gastro-intestinal nematodes to infective L3 stage have used soil of relatively low moisture content in small experimental samples that dry out faster than field soil. To determine whether higher and more sustained SM content modulates the effects of rainfall amount and timing on faecal moisture (FM) and development of H. contortus and T. colubriformis to infective third stage larvae (L3), a climate-controlled chamber experiment was conducted. It was designed to test the effects of rainfall amount (0, 12 and 24 mm), rainfall timing (days -1, 0 and 3 relative to faecal deposition) and soil moisture maintained at 10, 20 and 30% on these variables. Total recovery of L3 14 days after faecal deposition was significantly affected by SM, rainfall timing and their interaction (P<0.01), but not by rainfall amount or species or other two-way interactions. Recovery of L3 was maximal (28%) with a SM treatment of 30% and simulated rainfall on day 3. Faecal moisture was significantly affected by collection day, SM treatment, rainfall amount and rainfall timing with significant interaction between many of these effects (P<0.05). A positive linear association between FM and total L3 recovery was strongest on day 4 after faecal deposition (R(2)=0.64, P<0.001) for H. contortus and day 6 (R(2)=0.78, P<0.001) for T. colubriformis. Overall the results show that SM is able to modulate the effects of rainfall timing and amount with increased SM acting to broaden the window of opportunity for the free-living stages to respond to post deposition rainfall to complete development to L3. If SM is maintained in the range 10-30%, the reported benefits of early rainfall (days -1 and 0) of up to 24 mm appear to be negated with later rainfall (day 3) proving more beneficial. These results require field confirmation.

Two climate chamber experiments were conducted to determine the effect of varying initial soil moisture (0, 10 and 15%), simulated rainfall amount (0, 12 and 24 mm) and simulated rainfall timing (days -1, 0 and 3 relative to faecal deposition) on development (day 14) of Haemonchus contortus and Trichostrongylus colubriformis to the third stage larvae (L3) and faecal moisture (FM). Increasing initial soil moisture content from 0 to 10 or 15% led to higher recovery of total L3 (P<0.001). Total L3 recovery increased with each level of simulated rainfall (P<0.001) in the ascending order of 0, 12 and 24 mm. There was an interaction between the effects of initial soil moisture and simulated rainfall amount on the recovery of total L3, showing that the benefit of increased simulated rainfall lessened with increasing soil moisture. Simulated rainfall on the day of deposition resulted in higher recovery of L3 (P<0.001) than simulated rainfall on other days. FM on day 3 relative to faecal deposition was best associated with recovery of total H. contortus and T. colubriformis L3 (R(2)=0.32-0.46), reinforcing the importance of sufficient moisture soon after faecal deposition. The effects of initial soil moisture, and the amount and timing of simulated rainfall on development to L3 were largely explained by changes to FM and soil moisture values within 4 days relative to faecal deposition. These results highlight the influence of soil moisture and its interaction with rainfall on development of H. contortus and T. colubriformis to L3. Consequently we recommend that soil moisture be given greater importance and definition in the conduct of ecological studies of parasitic nematodes, in order to improve predictions of development to L3.