Angiostrongylus cantonensis: identification and characterization of microRNAs in male and female adults

Angiostrongylus cantonensis causes eosinophilic meningitis and eosinophilic pleocytosis in humans and is of significant socio-economic importance globally. microRNAs (miRNAs) are endogenous small non-coding RNAs that play crucial roles in gene expression regulation, cellular function and defense, homeostasis and pathogenesis. They have been identified in a diverse range of organisms. The objective of this study was to determine and characterize miRNAs of female and male adults of A. cantonensis by Solexa deep sequencing. A total of 8861,260 and 10,957,957 high quality reads with 20 and 23 conserved miRNAs were obtained in females and males, respectively. No new miRNA sequence was found. Nucleotide bias analysis showed that uracil was the prominent nucleotide, particularly at positions of 1, 10, 14, 17 and 22, approximately at the beginning, middle and the end of the conserved miRNAs. To our knowledge, this is the first report of miRNA profiles in A. cantonensis, which may represent a new platform for studying regulation of genes and their networks in A. cantonensis.     

The discovery of Angiostrongylus cantonensis as a cause of human eosinophilic meningitis

The theoretical and subsequent confirmation in 1961 of the rat lungworm, Angiostrongylus cantonensis, as the causative agent o f eosinophilic meningitis is one o f the remarkable parasitological findings of the twentieth century. Here, Joseph Alicato briefly summarizes the early history and his epidemiological studies on the relationship between the parasite and the epidemics o f encephalitis that swept through Oceania after the Second World War.     

Studies on the cryopreservation of eggs of Angiostrongylus cantonensis

The possibility of cryopreserving the eggs of Angiostrongylus cantonensis collected from the uterus of female worms was investigated. Eggs were cultured in NCTC 109 medium containing 50% rat serum, and various growth stages, from one-cell eggs to embryonated eggs, were used in this study. As a cryoprotective agent, dimethylsulphoxide (Me2SO) was added to the medium at a final concentration of 1 M. Eggs suspended in 0.2 ml of the medium at 37 degrees C were cooled to 0 degrees C at a rate of 1 degree C min-1, then an equal volume of 2M-Me2SO solution was added. After equilibration for 15 min, the freezing procedures were started. In the freezing procedures, the effectiveness of (i) a seeding process, (ii) different cooling and warming rates and (iii) the relationship between the growth stages of the eggs and their tolerance to freezing at -20 degrees C were investigated. It was found the highest level of survival could be obtained with 32-cell eggs cooled at a rate of 0.3 degrees C min-1 or more slowly with seeding at -4 degrees C and warming at a rate of 5 degrees C min-1. Survival was influenced more by cooling rate than by warming rate. Using these optimum conditions, the survival of eggs was then investigated following cooling to various temperatures. While more than 50% of eggs were found to survive cooling to -30 degrees C, extremely low survival was noted from lower temperatures.     

The role of eosinophils in Angiostrongylus cantonensis infection

Angiostrongylus cantonensis is the causative agent of human eosinophilic meningoencephalitis in the Pacific Islands and Southeast Asia. Prominent eosinophilia in the cerebrospinal fluid (CSF) of the patients has been used as one of the diagnostic criteria for the disease but the role(s) of the CSF eosinophils has remained to be elucidated. In this article, Kentaro Yoshimura, Hiroko Sugaya and Kazuto Ishido discuss the involvement of CSF eosinophils in the killing of intracranial worms and the damage of the central nervous system of the hosts, and consider why eosinophils in A. cantonensis infection play a more important role in nonpermissive hosts (including humans) than in the permissive rat host.     

Acquired immunity in rats against Angiostrongylus cantonensis infection

Acquired immunity against Angiostrongylus cantonensis was induced by immunizing rats with somatic antigens from fifth-stage larvae and adult worms and live third-stage larvae. Rats immunized twice had significantly fewer worms than rats immunized three times. Fewer worms were recovered from rats immunized with 200 live third-stage larvae than from any other groups. Rats immunized with somatic antigens had higher enzyme-linked immunosorbent assay (ELISA) antibody levels than rats immunized with live larvae. Rats immunized with live third-stage larvae of Angiostrongylus cantonensis were more strongly protected against challenge infections (62-92%) than rats immunized with antigens extracted from fifth-stage larvae (0-30%) and adult worms (11-24%).     

Seroprevalence of Angiostrongylus cantonensis infection in humans in China

A seroepidemiological survey was carried out in China during 2009-2010 to determine the extent of circulating antigens (CAg) for Angiostrongylus cantonensis in the Chinese population using the gold immunochromatographic assay, with the objective of elucidating the nationwide prevalence of angiostrongyliasis in China. A total of 1,730 blood samples was collected and assayed from the general adult population (the "general group"), and those involved in aquaculture or processing of snails Achatina fulica and Pomacea canaliculat (the "occupational group") from 5 provinces (Fujian, Hunan, Guangdong, Guangxi, and Zhejiang) and 1 municipal city (Beijing). The overall seroprevalence for the "occupational group" was 7.4% (40/540), which was significantly higher (P < 0.001) than that of the "general group" (0.8%, 9/1,190). The seroprevalence in males (9.5%) was significantly higher than in females (4.2%) (P < 0.05). These results demonstrate that angiostrongyliasis represents a significant zoonotic disease in China, requiring the strengthening of food safety for control of this food-borne disease.     

Public health importance of Angiostrongylus cantonensis and its relatives

Species of Angiostrongylus are fairly common nematode parasites of a variety of small mammals, but some species - particularly A. cantonensis and A. costaricensis - are increasingly seen as human infections. The worms make use of a range of aquatic, amphibious or terrestrial gastropod molluscs as intermediate hosts, which become infected by ingestion of eggs shed in the faeces of infected mammals. Porotenic hosts, including crustaceans, amphibians and reptiles, are known for most Angiostrongylus species, and can also be a source of the infective third-stage larvae that initiate mammalian infections.     

Observations on the transmission of Angiostrongylus cantonensis from snail to rodent

A survey of Angiostrongylus cantonensis was carried out to investigate the mode of transmission from mollusc to rat in a fixed study area of Yoron Island from 1979 to 1982. Rattus rattus was found to be infected with a small number of worms in spite of heavy infection with third-stage larvae in Achatina fulica and an abundance of this snail in the area. Natural infection and/or susceptibility with A. cantonensis were confirmed in three small snail species. Bradybaena circulus, Fruticicola despecta and Luchuena reticulata. Young A. fulica was found to be infected with fewer third-stage larvae than mature A. fulica. It was concluded that molluscs which were infected with a small number of third-stage larvae of A. cantonensis play an important role in maintaining the life cycle of A. cantonensis. The percentage of rat stomachs containing mollusc tissue was relatively low, and the incidence and infection was low in rats. Infection with A. cantonensis did not occur very often in R. rattus in nature.     

Seroprevalence of Angiostrongylus cantonensis in wild rodents from the Canary Islands

Background

Angiostrongylus cantonensis is a lungworm of rats (Muridae) that is the causative agent of human cerebral angiostrongyliasis. The life cycle of A. cantonensis involves rats and mollusks as the definitive and intermediate hosts, respectively. This study was designed to increase the knowledge about the occurrence and distribution of A. cantonensis in its definitive host in the Canary Islands, using parasitological and serological analysis in different areas and age groups.

Methodology/Principal Findings

Between 2009 and 2010, 54 black rats (Rattus rattus) from Tenerife were captured from six human-inhabited areas and sera samples were obtained. The lung nematodes were identified by morphological and molecular tools as A. cantonensis. The 31-kDa glycoprotein antigen was purified from A. cantonensis adult worms by electrophoresis and electroelution. Of the 54 tested rodents, 30 showed IgG antibodies against A. cantonensis 31-kDa antigen by ELISA. Therefore, the overall seroprevalence was 55.6% (95% CI: 42.4–68). Seroprevalent rodents were found in all the 6 areas. This 31-kDa antigen was not recognized by some sera of rats infected by other helminth species (but not A. cantonensis). Seroprevalence of IgG antibodies against A. cantonensis and prevalence based on the presence of adult worms showed significant correlation (R² = 0.954, p<0.05).

Conclusions/Significance

The present results could indicate a high prevalence of A. cantonensis in Tenerife and suggest the inclusion of two new zones in the distribution area of the parasite. The commonness and wide distribution of A. cantonensis in rats implies the presence of intermediate hosts, indicating that humans may be at risk of getting infected.     

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.     

Experimental infection routes of Angiostrongylus cantonensis in mice.

Stomach intubation is the most common method used in the experimental infection of animals with Angiostrongylus cantonensis. In order to compare the effectiveness of other possible transmission methods, groups of BALB/c mice were given infective third-stage larvae of A. cantonensis by different routes including intraperitoneal or subcutaneous injections, and penetration of anal mucosa, vaginal mucosa, conjunctival mucosa, lacerated skin, unabraded skin, foot pad and tail skin, while stomach intubation was used as control. Recovery of fifth-stage larvae was higher in mice inoculated with third-stage larvae subcutaneously. Successful infections were established through all experimental transmission routes except tail skin penetration. This study suggests that oral infection may not be the only route for the transmission of human angiostrongyliasis, and subcutaneous infection may be a better method for experimental infection.     

Characterization of Angiostrongylus cantonensis excretory-secretory proteins as potential diagnostic targets

Angiostrongyliasis results from infections with intra-arterial nematodes that accidentally infect humans. Specifically, infections with Angiostrongylus cantonensis cause eosinophilic meningitis and Angiostrongylus costaricensis infections result in eosinophilic enteritis. Immunological tests are the primary means of diagnosing infections with either pathogen since these parasites are usually not recoverable in fecal or cerebrospinal fluid. However, well-defined, purified antigens are not currently available in sufficient quantities from either pathogen for use in routine immunodiagnostic assays. Since A. costaricensis and A. cantonensis share common antigens, sera from infected persons will recognize antigens from either species. In addition to their potential use in angiostrongyliasis diagnosis, characterization of these proteins that establish the host-parasite interphase would improve our understanding of the biology of these parasites. The main objective of the present work was to characterize A. cantonensis excretory-secretory (ES) products by analyzing ES preparations by two-dimensional gel electrophoresis coupled with immunoblotting using pools of positive sera (PS) and sera from healthy individuals (SC). Protein spots recognized by PS were excised and analyzed by electrospray ionization (ESI) mass spectrometry. MASCOT analysis of mass spectrometry data identified 17 proteins: aldolase; CBR-PYP-1 protein; beta-amylase; heat shock protein 70; proteosome subunit beta type-1; actin A3; peroxiredoxin; serine carboxypeptidase; protein disulfide isomerase 1; fructose-bisphosphate aldolase 2; aspartyl protease inhibitor; lectin-5; hypothetical protein F01F1.12; cathepsin B-like cysteine proteinase 1; hemoglobinase-type cysteine proteinase; putative ferritin protein 2; and a hypothetical protein. Molecular cloning of these respective targets will next be carried out to develop a panel of Angiostrongylus antigens that can be used for diagnostic purposes and to further study host-Angiostrongylus interactions.     

Infectivity and development of the gamma-irradiated first stage larvae of Angiostrongylus cantonensis

Effects of gamma-ray irradiation on the first-stage larvae of Angiostrongylus cantonensis were studied. There was a significant reduction in the infectivity of the 10,000 R-irradiated larvae to intermediate host snails compared to other groups. In the final host infection, both the worm populations and worm body length of irradiated groups differed markedly from those of the non-irradiated control.