Regulatory effect of host miR-101b-3p on parasitism of nematode Angiostrongylus cantonensis via superoxide dismutase 3.

MicroRNA plays a vital role in the regulation of host-parasite interaction. In recent years, genomic and transcriptomic resources have become increasingly available for many helminths, but only a limited number of reports in this area are on the regulatory effects of host microRNAs on parasitic nematodes. In this work, we screened increased expression of host microRNAs after nematode infection from miRNA-seq data and predicted target genes by combined bioinformatics analysis and transcriptional profiling. We elucidated regulatory effects of one host miRNA on nematode infection using miRNA inhibitor and adeno-associated virus (AAV)-based TuD miRNA inhibitor. Using AAV-based TuD miRNA inhibitor, we showed that stable blockade of mmu-miR-101b-3p could alleviate the pathological damages of Angiostrongylus cantonensis, a parasitic nematode. Data from a luciferase report assay showed that mmu-miR-101b-3p targeted the extracellular superoxide dismutase 3 (Acsod3). Increased Acsod3 expression in larvae and alleviated oxidative damages were seen in the groups receiving mmu-miR-101b-3p inhibitor treatment in vitro and AAV-based TuD miRNA inhibitor injection in vivo. Results of this study demonstrate that murine miR-101b-3p inhibits the expression of antioxidant enzyme in A. cantonensis to strengthen host oxidative responses to nematodes. This work expands our knowledge of interspecies regulation of nematode gene expression by of host miRNAs.

Intermediate hosts of Angiostrongylus cantonensis in Tenerife, Spain.

The nematode Angiostrongylus cantonensis is the causative agent of human angiostrongyliasis, the main clinical manifestation of which is eosinophilic meningitis. Although this parasite has been found recently in its definitive rat host in Tenerife (Canary Islands, Spain), showing a widespread distribution over the north-east part of the island, there are no available data regarding which snail and/or slug species are acting as intermediate hosts on this island. Consequently, the objective of this work was to determine the possible role of three mollusc species, Plutonia lamarckii, Cornu aspersum and Theba pisana, as intermediate hosts of A. cantonensis in Tenerife. Between 2011 and 2014, 233 molluscs were collected from five biotopes where rats had been found previously to harbor either adult worms or antibodies against A. cantonensis, and the identification was carried out on the basis of morphological features and a LAMP technique. The prevalence of A. cantonensis larvae in the mollusc samples, based on morphological identification, was 19.3%, whereas 59 out of the 98 individuals (60.2%) analyzed by LAMP were positive. Positive results were obtained for the three mollusc species analyzed and two of the positive samples, both obtained from P. lamarckii, were confirmed as positive by 18S rRNA and ITS1 PCR. Sequence analysis of 18S rRNA PCR products showed 100% similarity with previously published A. cantonensis sequences. These results may be relevant from a public health point of view, since all the biotopes from which the samples were obtained were in inhabited areas or areas with human activity, but it is also important from the perspective of a possible transmission to other accidental hosts, such as dogs and horses, animals that are present in some of the areas analyzed.

Angiostrongylus cantonensis: tegumental and hypodermic alterations of the fourth-stage larvae following administration of tribendimidine in vivo and in vitro.

Angiostrongylus cantonensis is a parasitic pathogen whose forth-stage larvae (L4) parasitize in the central nervous system (CNS) of the human cause severe eosinophilic encephalitis or meningoencephalitis. Previous study indicated an impressive anthelmintic efficacy of tribendimidine (TBD) against CNS parasitized L4 of A. cantonensis. Tegument of the larvae is the first physical barrier to protect them from attack by the host immune system. In the present study, tegumental and hypodermic alterations were observed by scanning electron microscopy and transmission electron microscopy after administration of TBD. During treatment of TBD in vivo, L4 presented wizened side sensor, disappearance of mastoids and longitudinal grain, prominent surface coat, heterogeneous tegumental layers, incompact hypodermic cell junctions, blurred myotube, and small scale of vacuole in a basal layer. After incubation with TBD in vitro, L4 exhibited a swollen side sensor and mastoids disappearance in head end. Abundant tegumental blebs and obvious deformation of both cross-grain and longitudinal grain were detected on the surface, and shrinkage of all tegumental layers, chaotic cell junction, turbid muscle cell, disappearance of myotubes, and vacuole-like changes were visible under the electron microscope. The results implied the potential mechanism of the anthelmintic effect of tribendimidine against L4 of A. cantonensis by direct damages to tegumental and hypodermic.

Angiostrongylus cantonensis: scanning electron microscopic observations on the cuticle of moulting larvae.

Angiostrongylus cantonensis is a parasitic nematode that needs to develop in different hosts in different larval stages. Freshwater snails, such as Pomacea canaliculata, are the intermediate host, and rats are the definitive host. Periodic shedding of the cuticle (moulting) is an important biological process for the survival and development of the parasite in the intermediate and definitive hosts. However, there are few studies on the cuticle alterations between different stages of this parasite. In this study, we observed the ultrastructural appearance and changes of the cuticle of the 2nd/3rd stage larvae (L2/L3) and the 3rd/4th stage larvae (L3/L4) using a scanning electron microscope. We also first divided L2/L3 into late L2 and early L3. The late L2 lacked alae, but possessed a pull-chain-like fissure. Irregular alignment of spherical particles on the cuticle were noted compared to the L3. Alae appeared in the early L3. The old cuticle turned into a thin film-like structure which adhered to the new cuticle, and spherical particles were seen regularly arranged on the surface of this structure. Regular rectangular cavities were found on the surface of L3/L4. The caudal structure of L3/L4 was much larger than that of L3, but caudal inflation, such as seen in L4, was not observed. These results are the first to reveal the ultrastructural changes of the cuticle of A. cantonensis before and after moulting of L2/L3 and L3/L4.

Light and electron microscopic studies on two nematodes, Angiostrongylus cantonensis and Trichuris muris, differing in their mode of nutrition.

The morphological characteristics of the adult heteroxenous blood nematode Angiostrongylus cantonensis and the adult monoxenous intestinal nematode Trichuris muris were compared with special regard to the ultrastructure of their digestive systems. The small circular mouth of A. cantonensis appears sucker like. The very narrow mouth of T. muris is surrounded by three lips covered by the cuticle that extends into the buccal space. In the buccal cavity of A. cantonensis, a single tooth occurs opposite to a cutting plate, while no teeth are present in T. muris. The lumen of the well-developed muscular pharynx of A. cantonensis shows a trifurcated star-like cross-section. The anterior segment of the bipartite pharynx presumably functions as a pump. The lumen of the bipartite pharynx and esophagus of T. muris exhibits a very narrow oval cross-section and possesses no musculature. It is composed of a long column of stichosome cells. The esophagus region is lined inside by bands of bacillary cells as well as outside by two longitudinal rows of funnel-like papillae. These structures may be involved in the uptake of nutrients by T. muris. The gland cells might excrete digestive exoenzymes, while the bacillary cells take up the predigested nutrients. The presence of many vesicles suggests a vesicular transport of the material into the pharynx. The intestinal epithelium of A. cantonensis is densely covered with short microvilli. The lumen itself is filled with red blood cells originating from host blood. The intestine of T. muris has a thick epithelium being placed on a basal lamina and shows long thin microvilli. The intestinal lumen is very narrow and free from particles or granules. This again suggests that T. muris lives on low molecular nutrients resorbed from the environment. The epithelium cells of the intestine of T. muris contain glycogen and electron light granules but are lacking mitochondria. This finding may indicate that the epithelium cells have an anaerobic energy metabolism. This statement fits with the fact that the habitat of the worm, the cecum, is largely anaerobic.

The growth effects of gamma-ray irradiation on third-stage larvae of Angiostrongylus cantonensis in snail.

In order to clarify growth effects of gamma-ray irradiation on the third-stage larvae of Angiostrongylus cantonensis, snails naturally infected with larvae of A. cantonensis were irradiated with a cobalt-60 source, and the total dosage of exposure was 0.1 kGy. Third-stage larvae were collected from each group of irradiated and non-irradiated snails and then administered to rats. A significant reduction was observed in the number of recovery worms from rats sacrificed at 45 days after infection compared with the control group. Moreover, there was an increase in the percentage of female worms, and the body length of the irradiated group's worms was significantly shorter than that of the control group. In ultrastructural observations by transmission electron microscope, the cuticle was thickened and the subcuticle showed partial vacuolization and separation from muscle in the irradiated group. In addition, the muscles myofibril became shorter, the microvilli of intestinal mucosa were shorter and irregular, the terminal web disappeared, and the phagocytic vesicles, mitochondria, lysosomes and secretion granules decreased in number, respectively. Focusing on the mitochondria of the epithelial cells of the intestine, there were degenerative changes in the irradiated group showing vacuolization. The gonads of female worms recovered from the irradiated group revealed degenerative vacuolization, and the gonads of male worms could not be found.

Third report of ocular parastrongyliasis (angiostrongyliasis) from Sri Lanka.

A further case of ocular parastrongyliasis has been seen in a patient from Sri Lanka. As it is a juvenile female worm it could not be identified to the species, but it is probably Parastrongylus (= Angiostrongylus) cantonensis which is the commonly reported species in the country. This is the third authentic case of such an infection in Sri Lanka in recent years.