Transplantation of young adult Angiostrongylus cantonensis into the rat pulmonary vessels and its application to the assessment of acquired resistance

Yoshimura K., Aiba H. and Oya H. 1979. Transplantation of young adult Angiostrongylus cantonensis into the rat pulmonary vessels and its application to the assessment of acquired resistance. International Journal for Parasitology9: 97–103. A simple procedure for the transplantation of young adult Angiostrongylus cantonensis into the rat pulmonary vessels was described and its potential usefulness was discussed. Acquired resistance to A. cantonensis was conferred on rats by infection with 30 third-stage larvae or by a similar infection followed by treatment with thiabendazole during 14–19 days postinfection. Nearly comparable levels of resistance to reinfection were also observed in rats that were transplanted with 30 young adult worms, collected from the brain surface of donor animals at 21 and 22 days postinfection. The time-course development of reaginic, hemagglutinating and precipitating antibodies differed in these groups of rats although there appeared to be no positive correlation between the appearance of these antibodies and acquired resistance.     

The passive transfer of protective immunity against Angiostrongylus cantonensis with immune lymph node cells from different lymphoid tissues in rats

-Yong W. K. and Dobson C. 1982. The passive transfer of proctective immunity against Angiostrongylus cantonensis with immune lymph node cells from different lymphoid tissues in rats. International Journal for Parasitology12: 423–425. Lymph node cells from the posterior cervical and mesenteric lymph nodes of immune rats passively protected syngeneic recipient rats against Angiostrongylus cantonensis better than cells from the spleen, thymic and inguinal lymph nodes either as reduced worms burdens and/or stunted growth. No antibody was detected in the sera of recipient rats after transfer of the cells and before infection which suggested that the protection was cell- rather than antibody-mediated.     

The role of the spleen in protective immunity against Angiostrongylus cantonensis in rats: Splenectomy and passive spleen cell transfers

Splenectonuzed rats infected with Angiostrongylus cantonensis had more and longer worms and lower anti-A. cantonensis antibody titres compared to intact and sham operated rats. Splenectomy, however, had no effect on the capacity of rats to mount a cell-mediated immune response, as assessed by the in vitro uptake of tritiated thymidine by peripheral blood lymphocytes following stimulation by PHA and adult worm antigen. Adoptive protection and antibody production against A. cantonensis could be transferred with immune spleen cells. This protection was strongly dose and time dependent. Protection was adoptively conferred with 3 × 10⁹ spleen cells transferred 7 days before infection.     

Peripheral blood white cell responses during Angiostrongylus cantonensis infection in rats

Yono W. K. and Dobson C. 1984. Peripheral blood white cell responses during Angiostrongylus cantonensis infections in rats. Interntional Journal for Parasitology14: 207–211. Changes in white blood cell (WBC) populations and their proliferative responses to phytohaemagglutinin (PHA) and parasite antigens in vitro were studied in rats given one to three concurrent infections with Angiostrongylus cantonensis. WBC counts were elevated following infection; these changes were augmented following each successive reinfection. The WBC response could be partitioned into variations in the numbers of four major cell types. There was a loss of lymphocytes from the circulation after infection or reinfection followed by an increase in circulating lymphocytes when the parasite migrated to the lungs and matured. An eosinophilia was observed in all rats immediately after infection which was enhanced successively after each reinfection. The monocyte populations increased in a similar, but less obvious manner, to the eosinophil leucocytes. Neutrophil leucocytes increased after infection, but the numbers declined after reinfection. All rats given one to three infections showed a neutrophilia late in the experiment. A reversal in the neutrophil leucocyte-lymphocyte ratio was observed after each infection. A peak response in the proliferation of peripheral blood lymphocytes in vitro to PHA preceded and exceeded that stimulated by A. cantonensis antigen. These responses were interpreted as the dissemination of uncommitted thymus-dependent lymphocytes involved in the induction of antigen sensitized memory cells released following the protective immune reaction. The degree of lymphocyte responsiveness to mitogens correlated with the numbers of these cells circulating at each time interval. The relationships between in vitro lymphocyte responses and protective immunity in the rat against A. cantonensis are discussed.     

Immunodiagnosis of parasitic zoonoses: purification of Toxocara canis antigens by affinity chromatography

A method of affinity chromatography developed for the purification of species-specific antigens from Toxocara canis adult worms is described. Immunochemical analyses by polyacrylamide gel electrophoresis, immunoelectrophoresis and immunodiffusion showed that ‘pure’ antigen contained fewer but more specific proteins than ‘crude’ antigen. Purified antigens and parasite sections from four parasite species (Toxocara canis, Dirofilaria immitis, Angiostrongylus cantonensis and Ascaris lumbricoides) were used in immunofluorescence tests to measure serum antibody levels in animals with natural or experimental T. canis infections and people with zoonotic toxocariasis. ‘Pure’ antigen showed higher specificity and sensitivity than ‘crude’ antigen in serological testing.     

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.     

Hematological and histopathological changes in Rattus norvegicus (Wistar) experimentally infected by Angiostrongylus cantonensis (Chen, 1935)

Eosinophilic meningitis caused by Angiostrongylus cantonensis is a zoonosis endemic to Southeast Asia and the Pacific islands. It is considered an emerging disease because it has been expanding both geographically and in terms of the range of hosts. In South America, the first cases were reported in Brazil and were attributed to eating infected snails. In this study, 70 adult females of Rattus norvegicus (Wistar) were used to evaluate hematology, blood gases, cardiac markers and lung histopathology changes caused by this parasite. Of them, 60 were individually infected by orogastric gavage with 100 L₃ larvae and 10 uninfected animals formed the control group. The results obtained demonstrate that infection caused by A. cantonensis in R. norvegicus promotes significant hematological changes induced in the vertebrate host, manifested mainly in the form of regenerative anemia, thrombocytopenia and eosinophilia. Additionally, histopathological changes in the lung parenchyma demonstrated in rodents reveal the occurrence of areas of necrosis and extensive fibrosis, being directly related to the development of cellular hypoxia and enzyme cardiac changes. This study can contribute to a better understanding of the relationship between A. cantonensis and R. norvegicus.     

Angiostrongylus cantonensis: preparation of a specific antigen using immunoadsorbent columns.

Antigenic components which reacted specifically with sera of rats infected with Angiostrongylus cantonensis were prepared from adult worm extract by the use of immunoadsorbent columns. Components common to rat serum were removed from a whole worm extract by passing it through a column of cyanogen bromide-activated Sepharose coupled to rabbit antirat immunoglobulin. Consequently, components common to Ascaris suum were similarly removed using Sepharose linked to immunoglobulin to A. suum. Finally, the sample was applied to a column of Sepharose coupled with immunoglobulin from A. cantonensis infected rats and eluted with glycine-hydrochloric acid buffer. Intradermal skin tests using this preparation gave sensitive and specific reactions.     

First record of a nematode Metastrongyloidea (Aelurostrongylus abstrusus larvae) in Achatina (Lissachatina) fulica (Mollusca, Achatinidae) in Brazil

Achatina (Lissachatina) fulica was introduced in Brazil in the 1980s for commercial purposes (“escargot” farming) and nowadays, mainly by human activity, it is widespread in at least 23 out of 26 Brazilian states and Brasília, including the Amazonian region and natural reserves, where besides a general nuisance for people it is a pest and also a public health concern, since it is one of the natural intermediate host of Angiostrongylus cantonensis, ethiological agent of the meningoencephalitis in Asia. As Brazil is experiencing the explosive phase of the invasion, the Laboratório de Malacologia do Instituto Oswaldo Cruz/Fiocruz has been receiving samples of these molluscs for identification and search for Angiostrongylus cantonensis and Angiostrongylus costaricensis larvae. While examining samples of A. fulica different nematode larvae were obtained, including Aelurostrongylus, whose different species are parasites of felids, dogs, primates, and badger. Morphological and morphometric analyses presented herein indicated the species Aelurostrongylus abstrusus, as well as the occurrence of other nematode larvae (Strongyluris-like) found in the interior of the pallial cavity of A. fulica. This is the first report in Brazil of the development of A. abstrusus infective larvae in A. fulica evidencing the veterinary importance of this mollusc in the transmission of A. abstrusus to domestic cats. Since the spread of A. fulica is pointed out in the literature as one of the main causative spread of the meningoencephalitis caused by A. cantonensis the authors emphasize the need of sanitary vigilance of snails and rats from vulnerable areas for A. cantonensis introduction as the port side areas.     

Identification and characterisation of microRNAs in young adults of Angiostrongylus cantonensis via a deep-sequencing approach

Angiostrongylus cantonensis is an important causative agent of eosinophilic meningitis and eosinophilic meningoencephalitis in humans. MicroRNAs (miRNAs) are small non-coding RNAs that participate in a wide range of biological processes. This study employed a deep-sequencing approach to study miRNAs from young adults of A. cantonensis. Based on 16,880,456 high-quality reads, 252 conserved mature miRNAs including 10 antisense miRNAs that belonging to 90 families, together with 10 antisense miRNAs were identified and characterised. Among these sequences, 53 miRNAs from 25 families displayed 50 or more reads. The conserved miRNA families were divided into four groups according to their phylogenetic distribution and a total of nine families without any members showing homology to other nematodes or adult worms were identified. Stem-loop real-time polymerase chain reaction analysis of aca-miR-1-1 and aca-miR-71-1 demonstrated that their level of expression increased dramatically from infective larvae to young adults and then decreased in adult worms, with the male worms exhibiting significantly higher levels of expression than female worms. These findings provide information related to the regulation of gene expression during the growth, development and pathogenesis of young adults of A. cantonensis.     

Detection of Angiostrongylus cantonensis in the Blood and Peripheral Tissues of Wild Hawaiian Rats (Rattus rattus) by a Quantitative PCR (qPCR) Assay

The nematode Angiostrongylus cantonensis is a rat lungworm, a zoonotic pathogen that causes human eosinophilic meningitis and ocular angiostrongyliasis characteristic of rat lungworm (RLW) disease. Definitive diagnosis is made by finding and identifying A. cantonensis larvae in the cerebral spinal fluid or by using a custom immunological or molecular test. This study was conducted to determine if genomic DNA from A. cantonensis is detectable by qPCR in the blood or tissues of experimentally infected rats. F1 offspring from wild rats were subjected to experimental infection with RLW larvae isolated from slugs, then blood or tissue samples were collected over multiple time points. Blood samples were collected from 21 rats throughout the course of two trials (15 rats in Trial I, and 6 rats in Trial II). In addition to a control group, each trial had two treatment groups: the rats in the low dose (LD) group were infected by approximately 10 larvae and the rats in the high dose (HD) group were infected with approximately 50 larvae. In Trial I, parasite DNA was detected in cardiac bleed samples from five of five LD rats and five of five HD rats at six weeks post-infection (PI), and three of five LD rats and five of five HD rats from tail tissue. In Trial II, parasite DNA was detected in peripheral blood samples from one of two HD rats at 53 minutes PI, one of two LD rats at 1.5 hours PI, one of two HD rats at 18 hours PI, one of two LD rats at five weeks PI and two of two at six weeks PI, and two of two HD rats at weeks five and six PI. These data demonstrate that parasite DNA can be detected in peripheral blood at various time points throughout RLW infection in rats.     

Development-Specific Differences in the Proteomics of Angiostrongylus cantonensis

Angiostrongyliasis is an emerging communicable disease. Several different hosts are required to complete the life cycle of Angiostrongylus cantonensis. However, we lack a complete understanding of variability of proteins across different developmental stages and their contribution to parasite survival and progression. In this study, we extracted soluble proteins from various stages of the A. cantonensis life cycle [female adults, male adults, the fifth-stage female larvae (FL5), the fifth-stage male larvae (ML5) and third-stage larvae (L3)], separated those proteins using two-dimensional difference gel electrophoresis (2D-DIGE) at pH 4–7, and analyzed the gel images using DeCyder 7.0 software. This proteomic analysis produced a total of 183 different dominant protein spots. Thirty-seven protein spots were found to have high confidence scores (>95%) by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Comparative proteomic analyses revealed that 29 spots represented cytoskeleton-associated proteins and functional proteins. Eight spots were unnamed proteins. Twelve protein spots that were matched to the EST of different-stage larvae of A. cantonensis were identified. Two genes and the internal control 18s were chosen for quantitative real-time PCR (qPCR) and the qPCR results were consistent with those of the DIGE studies. These findings will provide a new basis for understanding the characteristics of growth and development of A. cantonensis and the host–parasite relationship. They may also assist searches for candidate proteins suitable for use in diagnostic assays and as drug targets for the control of eosinophilic meningitis caused by A. cantonensis.     

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.     

Molecular differentiation and phylogenetic relationships of three Angiostrongylus species and Angiostrongylus cantonensis geographical isolates based on a 66-kDa protein gene of A. cantonensis (Nematoda: Angiostrongylidae)

The phylogenetic relationships and molecular differentiation of three species of angiostrongylid nematodes (Angiostrongylus cantonensis, Angiostrongylus costaricensis and Angiostrongylus malaysiensis) were studied using the AC primers for a 66-kDa protein gene of A. cantonensis. The AC primers successfully amplified the genomic DNA of these angiostrongylid nematodes. No amplification was detected for the DNA of Ascaris lumbricoides, Ascaris suum, Anisakis simplex, Gnathostoma spinigerum, Toxocara canis, and Trichinella spiralis. The maximum-parsimony (MP) consensus tree and the maximum-likelihood (ML) tree both showed that the Angiostrongylus taxa could be divided into two major clades - Clade 1 (A. costaricensis) and Clade 2 (A. cantonensis and A. malaysiensis) with a full support bootstrap value. A. costaricensis is the most distant taxon. A. cantonensis is a sister group to A. malaysiensis; these two taxa (species) are clearly separated. There is no clear distinction between the A. cantonensis samples from four different geographical localities (Thailand, China, Japan and Hawaii); only some of the samples are grouped ranging from no support or low support to moderate support of bootstrap values. The published nucleotide sequences of A. cantonensis adult-specific native 66 kDa protein mRNA, clone L5-400 from Taiwan (U17585) appear to be very distant from the A. cantonensis samples from Thailand, China, Japan and Hawaii, with the uncorrected p-distance values ranging from 26.87% to 29.92%.     

Alien parasitic infections in the endangered Ryukyu long-furred rat (Diplothrix legata) on Amami-Oshima Island,Japan

There is limited knowledge about parasites of the endangered Ryukyu long-furred rat, Diplothrix legata (Murinae, Rodentia) endemic to Okinawa, Tokunoshima, and Amami-Oshima Islands in Japan. In the present study, postmortem histopathological examination of an individual found on Amami-Oshima Island revealed a mixed helminth infection of Calodium hepaticum, Hydatigera taeniaeformis, and Angiostrongylus cantonensis. These helminths are considered non-native to Amami-Oshima Island and are maintained by invasive mammals, such as non-native rats and outdoor cats. This observation presents a new host record for C. hepaticum and H. taeniaeformis and the first record of A. cantonensis in Ryukyu long-furred rat on Amami-Oshima Island.     

Angiostrongylus cantonensis: Rejection of pulmonary arterial transfers of lung stage worms

Experimental transfer of the lung stage worms of Angiostrongylus cantonensis was performed between permissive hosts (rats) and between permissive (rat) and nonpermissive hosts (guinea pigs and rabbits). These worms from rats were rejected when implanted into nonpermissive hosts. Unexpectedly, similar worms did not survive well even in permissive hosts; the majority of recipient rats did not have first-stage larvae (L₁) in their stools and, even when positive for L₁, the number of the larvae shed was few. These findings contrast with the successful pulmonary arterial transfer of younger, intracranial-stage worms. It was shown that differences in rat strain between donor and recipient had no significant effect on the subsequent worm survival in recipient hosts. The alteration of maintaining conditions of the intrapulmonary worms, prior to transfer, in terms of temperature, media, and maintaining period, also showed no profound effect on the subsequent worm survival. The kinetics of precipitating and reaginic antibody levels in rats implanted with the intrapulmonary worms were analogous to those in rats with intracranial-stage worms. The findings indicate that some qualitative differences may exist between the worms obtained from two different sites.     

Design,synthesis and evaluation of phthalide alkyl tertiary amine derivatives as promising acetylcholinesterase inhibitors with high potency and selectivity against Alzheimer's disease

A series of phthalide alkyl tertiary amine derivatives were designed, synthesized and evaluated as potential multi-target agents against Alzheimer’s disease (AD). The results indicated that almost all the compounds displayed significant AChE inhibitory and selective activities. Besides, most of the derivatives exhibited increased self-induced Aβ_1-42 aggregation inhibitory activity compared to the lead compound dl-NBP, and some compounds also exerted good antioxidant activity. Specifically, compound I-8 showed the highest inhibitory potency toward AChE (IC₅₀ = 2.66 nM), which was significantly better than Donepezil (IC₅₀ = 26.4 nM). Moreover, molecular docking studies revealed that compound I-8 could bind to both the catalytic active site and peripheral anionic site of AChE. Furthermore, compound I-8 displayed excellent BBB permeability in vitro. Importantly, the step-down passive avoidance test indicated that I-8 significantly reversed scopolamine-induced memory deficit in mice. Collectively, these results suggested that I-8 might be a potent and selective AChE inhibitor for further anti-AD drug development.     

Steroidal alkaloids from Holarrhena antidysenterica as acetylcholinesterase inhibitors and the investigation for structure-activity relationships

AimsInhibition of acetylcholinesterase (AChE) is still considered as a strategy for the treatment of neurological disorders such as Alzheimer's disease (AD). Many plant derived alkaloids (such as huperzine A, galanthamine and rivastigmine) are known for their AChE inhibitory activity. The aim of the present work was to isolate and identify new AChE inhibitors from Holarrhen antidysenterica.Main methodsThese compounds were tested for AChE inhibiting activity by the Ellman's method in 96-well microplates. In addition, molecular modeling was performed to explore the binding mode of inhibitors 1–5 at the active site of AChE, and the preliminary structure–activity relationships (SARs) were discussed.Key findingsIn the course of searching for AChE inhibitors from herb medicines, the total alkaloidal extract from the seeds of H. antidysenterica was found having potent AChE inhibitory activity with an IC₅₀ value of 6.1 μg/mL. Further bioactivity-guided chromatographic fractionation afforded five steroidal alkaloids, conessine 1, isoconessimine 2, conessimin 3, conarrhimin 4 and conimin 5. All the isolated compounds, except for 2, showed strong AChE inhibiting activity with IC₅₀ values ranging from 4 to 28 μM. The most active inhibitor is compound 3 with an IC₅₀ value of 4 μM. The mode of AChE inhibition by 3 was reversible and non-competitive.SignificanceThe results suggest that these alkaloids could be potential candidates for further development of new drugs against AD.     

The development of 2-acetylphenol-donepezil hybrids as multifunctional agents for the treatment of Alzheimer’s disease

A series of 2-acetylphenol-donepezil hybrids was designed and synthesized based on multi-target-directed ligands strategy. The biological activities were evaluated by AChE/BChE inhibition and MAO-A/MAO-B inhibition. The results revealed that the tertiary amines and methylene chain length significantly affected the eeAChE inhibitory potency, in particular, compound TM-14 showed the best eeAChE inhibitory activity with IC₅₀ value of 2.9 μM, in addition, both kinetic analysis of AChE inhibition and docking study displayed that TM-14 could simultaneously bind to the catalytic active site and peripheral anionic site of AChE. Moreover, compound TM-14 was a selective metal chelator and could form 1:1 TM-14-Cu²⁺ complex. The structure-active-relationship also indicated that the O-alkylamine fragment remarkably decreased hMAO-B inhibitory activity, compound TM-2 exhibited potent hMAO-B inhibitory activity (IC₅₀ = 6.8 μM), which was supported by the molecular docking study. More interestingly, compounds TM-14 and TM-2 could cross the blood-brain barrier in vitro. Therefore, the structure-active-relationship of 2-acetylphenol-donepezil hybrids could encourage the development of multifunction agents with selective AChE inhibition or selective MAO-B inhibition for the treatment of Alzheimer’s disease.     

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.