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.     

Angiostrongylus cantonensis: apoptosis of inflammatory cells induced by treatment with mebendazole or/and interleukin 12 in mice

Angiostrongylus cantonensis is the major cause of human eosinophilic meningoencephalitis. ICR mice were infected orally with 35 infective larvae and sacrificed at 4-14 days, 25 days or 32 days post infection (dpi) for pathological and immunocytochemical examinations. In the non-treated group, no apoptosis signal was found in the meninges or parenchyma of the brains (4-14 dpi). Only a few apoptotic cells were noticed at 25 dpi (3%) and 32 dpi (10%). In the groups, the animals were given a single dose of mebendazole (20 mg/kg, per os at various times) or injections of interleukin 12 (IL-12) (10 ng/daily, intraperitoneally), all the animals were sacrificed at 14 dpi; the number of apoptotic cells was increased (17-21%). In the group that received a single dose of mebendazole (4 dpi) in combination with IL-12 injections (4-13 dpi), mild meningitis was observed, and most of the infiltrated inflammatory cells were in the apoptotic program (55%). Taken together, apoptosis of the inflammatory cells (most were eosinophils) could be induced when the infected mice were treated with mebendazole or/and IL-12.     

Apoptosis in meningoencephalitis of Angiostrongylus cantonensis-infected mice

A hallmark of eosinophilic meningoencephalitis is infiltration of leukocytes into brain parenchyma and subarachnoid space infected by Angiostrongylus cantonensis. Apoptosis, a process that eliminates useless cells and counterbalances tissue homeostasis, is important for homeostasis of the immune system. In this study, we investigated the characteristics of cell death induced in BABL/c mice infected with A. cantonensis. We observed increased expression of the apoptotic proteins, caspase-3, caspase-8, caspase-9, and cytochrome c, and decreased expression of anti-apoptotic proteins, B-cell leukemia 2 and inhibitor of apoptosis protein 1. On immunohistochemistry, apoptotic proteins were localized within the leukocytes infiltrate. A terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling assay to detect DNA fragmentation confirmed these observations. The infiltration of leukocytes present in the brain parenchyma and subarachnoid space in vivo may also express these apoptotic regulatory molecules, which demonstrates the capacity of these cells to undergo apoptosis.     

Ectopic migration of an adult heartworm in a dog with dirofilariasis (case report)

A 3-yr-old female mongrel dog was referred to the Veterinary Teaching Hospital of Chungnam National University in the Republic of Korea. An adult heartworm, Dirofilaria immitis, was found in the abdominal cavity of the dog during spaying. Dirofilariasis in this dog was also diagnosed by modified Knott's test, ELISA test, and PCR analysis. The present case is the first report on the migration of an adult dog heartworm to the abdominal cavity of a dog in the Republic of Korea.     

Development of Lateral Flow Immunoassay for Antigen Detection in Human Angiostrongylus cantonensis Infection

Angiostrongyliasis is difficult to be diagnosed for the reason that no ideal method can be used. Serologic tests require specific equipment and are not always available in poverty-stricken zone and are time-consuming. A lateral flow immunoassay (LFIA) may be useful for angiostrongyliasis control. We established a LFIA for the diagnosis of angiostrongyliasis based on 2 monoclonal antibodies (mAbs) against antigens of Angiostrongylus cantonensis adults. The sensitivity and specificity were 91.1% and 100% in LFIA, while those of commercial ELISA kit was 97.8% and 86.3%, respectively. Youden index was 0.91 in LFIA and 0.84 in commercial ELISA kit. LFIA showed detection limit of 1 ng/ml of A. cantonensis ES antigens. This LFIA was simple, rapid, highly sensitive and specific, which opened an alternative approach for the diagnosis of human angiostrongyliasis.     

Angiostrongylus cantonensis: experimental study on the susceptibility of apple snails, Pomacea canaliculata compared to Pila polita

Six groups (15 snails/group) of Pomacea canaliculata and Pila polita were infected orally with 0 (control), 200, 400, 800, 1600 and 3200 first-stage Angiostrongylus cantonensis larvae (L1). The respective mean+/-SD third stage larvae (L3) worm recovery 1-month post-infection (p.i.) for P. canaliculata was 0, 1.4+/-5.42 (0.7%), 0.13+/-0.35 (0.03%), 0.07+/-0.26 (0.009%), 0.07+/-0.26 (0.004%), 0, and for P. polita 0, 64.33+/-21.38 (32.25%), 115.36+/-36.82 (28.93%), 265.33+/-90.01 (33.27%), 471.33+/-92.98 (29.60%) and 849.00+/-243.23 (26.61%). The susceptibility of A. cantonensis in P. polita was dose-dependent (p<0.001). In the three groups (nine snails/group) of P. polita given 500 L1, we studied the distribution of L3 in the internal organs (i.e., foot, head+esophagus, kidney, albumin gland, mantle, intestine, digestive gland) and found the highest density after 1, 2 and 3 months p.i. in the mantle at 29.37%, 31.09% and 37.45%. The infection rate in P. canaliculata was too low to study distribution rates.     

Effects of catechins on vascular tone in rat thoracic aorta with endothelium

The effects of eight catechin derivatives on vascular tone in rat thoracic aorta were examined. Catechin derivatives (10 microM) potentiated the contractile response to phenylephrine in endothelium-intact arteries. The potentiations produced by EGCg and EGC were almost absent in endothelium-denuded arteries and abolished by N(G)-nitro-L-arginine methyl ester, an inhibitor of nitric oxide synthesis. The catechin derivatives also inhibited endothelium-dependent relaxation in response to acetylcholine. The order of catechin derivatives ranked in terms of both increasing vascular reactivity and impairing endothelium-dependent relaxation was similar; (-)-gallocatechin (GC) >or= (-)-epigallocatechin (EGC) >or= (-)-gallocatechin gallate (GCg) >or= (-)-epigallocatechin gallate (EGCg) >or= (-)-catechin (C) >or= (-)-epicatechin (EC) >or= (-)-catechin gallate (Cg) >or= (-)-epicatechin gallate (ECg). In addition, EGC inhibited the endothelium-independent relaxation evoked by both sodium nitroprusside and NOC-7, a spontanous NO releaser, but EGCg inhibited only that by NOC-7. These findings indicate that catechin derivatives produce a potentiation of the contractile response and an inhibition of the vasorelaxant response, probably through inactivation of endothelium-derived nitric oxide (NO), and that the hydroxyl on C-5 of the B ring together with the stereoscopic structure between the C-3 group and the B ring of flavanols was of importance in mediating the above effects and that the substitution of a gallate group of C-3 attenuated the effects, probably due to a decreased response to solube guanylate cyclase in vascular smooth muscle cells.     

Spontaneous Splenic Rupture in a Vivax Malaria Case Treated with Transcatheter Coil Embolization of the Splenic Artery

An enlarged spleen is considered one of the most common signs of malaria, and splenic rupture rarely occurs as an important life-threatening complication. Splenectomy has been recommended as the treatment of choice for hemodynamically unstable patients. However, a very limited number of splenic rupture patients have been treated with transcatheter coil embolization. Here we report a 38-year-old Korean vivax malaria patient with ruptured spleen who was treated successfully by embolization of the splenic artery. The present study showed that angiographic embolization of the splenic artery may be an appropriate option to avoid perioperative harmful effects of splenectomy in malaria patients.     

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.     

有氧运动训练影响大鼠胸主动脉环内皮依赖性舒张功能机制的研究

目的:探讨有氧运动对大鼠胸主动脉内皮依赖性舒张功能影响的机制。方法:12只SD大鼠随机分为有氧运动和对照两组(n=6),经过8周每天1 h的游泳训练后(每周5天),测定比较2组间大鼠胸主动脉舒张功能的改变。结果:有氧运动组一氧化氮(NO)和前列环素PGI2途径胸主动脉舒张功能的Rmax值较对照组明显提高(P<0.05)。结论:有氧运动对大鼠胸主动脉舒张功能的有益影响,主要是由NO和PGI2途径介导的。     

A Case of Ocular Angiostrongyliasis with Molecular Identification of the Species in Vietnam

A 23-year-old female residing in a village of Cao Bang Province, North Vietnam, visited the Hospital of Hanoi Medical University in July 2013. She felt dim eyes and a bulge-sticking pain in her left eye for some days before visiting the hospital. In the hospital, a clinical examination, an eye endoscopy, and an operation were carried out. A nematode specimen was collected from the eye of this patient. The body of this worm was thin and long and measured 22.0×0.3 mm. It was morphologically suggested as an immature female worm of Angiostrongylus cantonensis. By a molecular method using 18S rRNA gene, this nematode was confirmed as A. cantonensis. This is the first molecular study for identification of A. cantonensis in Vietnam.     

An innovative numerical approach to resolve the pulse wave velocity in a healthy thoracic aorta model

Aortic dissection and atherosclerosis are highly fatal diseases. The development of both diseases is closely associated with highly complex haemodynamics. Thus, in predicting the onset of cardiac disease, it is desirable to obtain a detailed understanding of the flowfield characteristics in the human cardiovascular circulatory system. Accordingly, in this study, a numerical model of a normal human thoracic aorta is constructed using the geometry information obtained from a phase-contrast magnetic resonance imaging (PC-MRI) technique. The interaction between the blood flow and the vessel wall dynamics is then investigated using a coupled fluid–structure interaction (FSI) analysis. The simulations focus specifically on the flowfield characteristics and pulse wave velocity (PWV) of the blood flow. Instead of using a conventional PC-MRI method to measure PWV, we present an innovative application of using the FSI approach to numerically resolve PWV for the assessment of wall compliance in a thoracic aorta model. The estimated PWV for a normal thoracic aorta agrees well with the results obtained via PC-MRI measurement. In addition, simulations which consider the FSI effect yield a lower predicted value of the wall shear stress at certain locations in the cardiac cycle than models which assume a rigid vessel wall. Consequently, the model provides a suitable basis for the future development of more sophisticated methods capable of performing the computer-aided analysis of aortic blood flows.     

Elevation of plasminogen activators in cerebrospinal fluid of mice with eosinophilic meningitis caused by Angiostrongylus cantonensis

A hallmark of parasitic meningitis is the infiltration of eosinophils into the subarachnoid space. Infection with Angiostrongylus cantonensis in mice induced proteinase activity in parallel with the pathological changes of eosinophilic meningitis. Zymogram analysis demonstrated that 70 and 55 kDa proteinases from cerebrospinal fluid (CSF) were active against the casein/plasminogen substrate. The proteinase activities were clearly inhibited by phenylmethanesulphonyl fluoride but not by ethylenediamine tetraacetic acid, 1,10-phenanthroline or leupeptin. Western blotting confirmed these enzymes to be tissue-type plasminogen activator and urokinase-type plasminogen activator, respectively. High activities of tissue-type plasminogen activator and urokinase-type plasminogen activator were detected in the CSF of mice with eosinophilic meningitis, and correlated positively with CSF eosinophil numbers and total protein, respectively. Immunohistochemistry demonstrated that tissue-type plasminogen activator and urokinase-type plasminogen activator localised in the endothelial cells of blood vessels, in blood clots and in infiltrated leukocytes. These results suggest that tissue-type plasminogen activator and urokinase-type plasminogen activator may be play a role in the pathogenesis of eosinophilic meningitis of angiostrongyliasis.     

Association of matrix metalloproteinase-9 and Purkinje cell degeneration in mouse cerebellum caused by Angiostrongylus cantonensis

Angiostrongylosis is a neurological disorder caused by invasion of the central nervous system by developing larvae of Angiostrongylus cantonensis. Purkinje cells in infected mouse cerebellums are small and irregular with degenerative atrophy or partial loss. Ultrastructural changes in degenerative cells included enlarged vacuolar structures and swollen mitochondria within the cytoplasm. The matrix metalloproteinase-9 mRNA which is low in normal cerebellums was expressed in A. cantonensis-infected mice cerebellum prior to Purkinje cell degeneration. Matrix metalloproteinase-9 protein level and enzyme activity increased when the Purkinje cells appeared degenerated. Using immunohistochemistry, matrix metalloproteinase-9 was localised within degenerative Purkinje cells. In addition, when the specific matrix metalloproteinase inhibitor, GM6001, was added, matrix metalloproteinase-9 enzyme activity was reduced by 41.6%. The numbers of degenerative Purkinje cells increased significantly upon establishment of infection but subsided upon inhibition. These results suggested that the expression of matrix metalloproteinase-9 may be associated with degeneration of Purkinje cells in mouse cerebellum infected by A. cantonensis.     

Fruit flies learn to avoid odours associated with virulent infection

While learning to avoid toxic food is common in mammals and occurs in some insects, learning to avoid cues associated with infectious pathogens has received little attention. We demonstrate that Drosophila melanogaster show olfactory learning in response to infection with their virulent intestinal pathogen Pseudomonas entomophila. This pathogen was not aversive to taste when added to food. Nonetheless, flies exposed for 3 h to food laced with P. entomophila, and scented with an odorant, became subsequently less likely to choose this odorant than flies exposed to pathogen-laced food scented with another odorant. No such effect occurred after an otherwise identical treatment with an avirulent mutant of P. entomophila, indicating that the response is mediated by pathogen virulence. These results demonstrate that a virulent pathogen infection can act as an aversive unconditioned stimulus which flies can associate with food odours, and thus become less attracted to pathogen-contaminated food.     

Numerical simulation of non-Newtonian blood flow dynamics in human thoracic aorta

Three non-Newtonian blood viscosity models plus the Newtonian one are analysed for a patient-specific thoracic aorta anatomical model under steady-state flow conditions via wall shear stress (WSS) distribution, non-Newtonian importance factors, blood viscosity and shear rate. All blood viscosity models yield a consistent WSS distribution pattern. The WSS magnitude, however, is influenced by the model used. WSS is found to be the lowest in the vicinity of the three arch branches and along the distal walls of the branches themselves. In this region, the local non-Newtonian importance factor and the blood viscosity are elevated, and the shear rate is low. The present study revealed that the Newtonian assumption is a good approximation at mid-and-high flow velocities, as the greater the blood flow, the higher the shear rate near the arterial wall. Furthermore, the capabilities of the applied non-Newtonian models appeared at low-flow velocities. It is concluded that, while the non-Newtonian power-law model approximates the blood viscosity and WSS calculations in a more satisfactory way than the other non-Newtonian models at low shear rates, a cautious approach is given in the use of this blood viscosity model. Finally, some preliminary transient results are presented.     

Angiostrongylus cantonensis: induction of urokinase-type PA and degradation of type IV collagen in rat lung granulomatous fibrosis

Granuloma formation and subsequent fibrosis around Angiostrongylus cantonensis larvae in the lungs were induced experimentally in Sprague-Dawley strain rats. Casein zymogram analysis demonstrated that urokinase-type plasminogen activator (uPA) activity was increased during lung inflammation and fibrosis. Granulomatous fibrosis, type IV collagen degradation and activation of uPA occur simultaneously. Furthermore, the present study demonstrated that collagen avidly binds uPA. Immunohistochemical observations showed localization of uPA within the infiltrating leucocytes. We propose that uPA may participate in A. cantonensis-induced granulomatous fibrosis.