Experimental hybridization of Angiostrongylus mackerrasae, Bhaibulaya, 1968 and Angiostrongylus cantonensis (Chen, 1935)

Angiostrongylus mackerrasae and A. cantonensis are closely related and their status as distinct species was tested by experimental hybridization. Hybridization between A. mackerrasae and A. cantonensis was observed and the morphology of the F₁ hybrids was found to be intermediate between that of the parental species which suggests strongly that A. mackerrasae and A. cantonensis are both valid species. However, male F₁ hybrids were sterile, whereas females were fertile; no evidence of parthenogenesis or other form of asexual reproduction were observed in either species. It is suggested that A. mackerrasae and A. cantonensis may cross breed in nature.     

Detection of anti-oxidant enzymatic activities and purification of glutathione transferases from Angiostrongylus cantonensis

There are several anti-oxidant enzyme families that play pivotal roles in facilitating the survival of parasites. Glutathione transferases (GSTs) are members of the anti-oxidant family that can detoxify a broad range of exogenous or endogenous compounds including reactive oxidative species. GSTs have been studied as vaccine candidates, immunodiagnostic markers and as treatment targets. Helminths of the genus Angiostrongylus live inside arteries of vertebrates and two main species are associated with accidental human infections: Angiostrongylus costaricensis adult worms live inside the mesenteric arteries and larvae of Angiostrongylus cantonensis become trapped in the central nervous system vasculature. Since the interactions between angiostrongylid nematodes and their vertebrate hosts are poorly understood, this study characterized the anti-oxidant enzymatic activities of A. cantonensis from female worms by collecting excreted and secreted (ES) and total extract (TE) molecules. Catalase (CAT) and superoxide dismutase (SOD) activities were found both in the ES and TE while glutathione peroxidase (GPX) and GST were found only in the TE. GSTs were purified by glutathione agarose affinity column (AcGST) and the pool of eluted GSTs was analyzed by mass spectrometry (LC-MS/MS) and de novo sequencing (Masslynx software). Sequences from two peptides (AcGSTpep1 and AcGSTpep2) present high identity to the N-terminal and C-terminal from sigma class GSTs of nematodes. It is known that these GST enzymes are associated with host immune regulation. Furthermore, understanding the role of parasite-derived anti-oxidant molecules is important in understanding host-parasite interactions.     

Eosinophils in the cerebrospinal fluid of mice infected with Angiostrongylus cantonensis are resistant to apoptosis

Interleukin-5 (IL-5) transgenic mice were used to assess the immunological features of CSF eosinophils from mice infected with Angiostrongylus cantonensis. CSF eosinophils were hypodense by day 14 post infection (p.i.). CSF eosinophils survived longer in vitro than peritoneal eosinophils collected from cadmium sulphate (CdSO₄) -treated normal IL-5 transgenic mice. Apoptosis was measured by Annexin V binding and the presence of a distinct laddering pattern of DNA fragmentation on agarose electrophoresis. Regardless of the presence or absence of Actinomycin D, CSF eosinophils collected from IL-5 transgenic mice from days 15–36 p.i. exhibited less apoptosis than peritoneal eosinophils collected from uninfected IL-5 transgenic mice. CSF eosinophils collected from A. cantonensis infected C57BL/6 mice at days 15–34 p.i. showed elongation of survival time and less apoptosis during in vitro cultivation. Reduced apoptosis was noted only in CSF eosinophils, but not in peritoneal eosinophils recovered from the same infected IL-5 transgenic mice. CPP32/Caspase 3 activity of cultured peritoneal eosinophils from both infected and uninfected IL-5 transgenic mice was higher than that of cultured CSF eosinophils. Stimulation with A23187 readily induced apoptosis of peritoneal eosinophils, but not CSF eosinophils or peritoneal eosinophils cultured with mouse recombinant IL-5. The latter cells were morphologically identical to hypodense eosinophils. RT-PCR analysis indicated that bcl-2 and bcl-x_L mRNA expression was higher in CSF eosinophils compared with peritoneal eosinophils and this expression in the latter cells was upregulated after culture with mouse recombinant IL-5. These results suggest that CSF eosinophils, shifting to hypodense status through an accumulation from peripheral blood, are resistant to apoptosis. These changes may explain the long-lasting, helminthotoxic and neurotoxic actions of CSF eosinophils in A. cantonensis infection.     

The effect of cyclosporin A on Angiostrongylus cantonensis infection and eosinophilia in mice

Male BALB/c mice were infected with Angiostrongylus cantonensis and at various times p.i. treated with cyclosporin A (CsA) either for 5 days continuously or intermittently, or for 12–16 days on alternate days. They were monitored for peripheral blood eosinophilia and at necropsy examined for CSF and bone marrow eosinophilia, and worm recovery. CsA treatment provoked a transient inhibition of peripheral blood eosinophilia in all groups examined, followed by rebounding eosinophilia. There was no significant difference in bone marrow and CSF eosinophilia between CsA- and vehicle-treated groups. Mice treated with CsA on alternate days yielded lower intracranial worm recovery with small-sized worms at days 7, 21 and 30 p.i. than vehicle-treated groups did. No significant reduction in worm recovery was noted in mice treated for 5 days either continuously or intermittently, although worms, especially female ones, harvested from groups treated with CsA from days — 1 to 3 or from days 13 to 17 were significantly smaller than those from vehicle-treated groups. CsA-treatment suppressed blastogenic responses of spleen cells against Con A or worm antigen at days 7 and 21 p.i. In vitro treatment of the 3rd stage larvae with CsA did not adversely influence survival of A. cantonensis in mice. These data indicate that CsA exerts antiparasitic effects against A. cantonensis in mice.     

In vivo evaluation of the efficacy of albendazole sulfoxide and albendazole sulfoxide loaded solid lipid nanoparticles against hydatid cyst

Cystic echinococcosis (CE) is caused by the larval stage of Echinococcus granulosus, which in this disease the metacestode develop in visceral organs especially liver and lungs. The disease is present worldwide and affects humans as well as herbivores including cattle, sheep, camels, horses and others. Benzimidazole carbamate derivatives, such as mebendazole and albendazole, are currently used for chemotherapeutic treatment of CE in inoperable patients and have to be applied in high doses for extended periods of time, and therefore adverse side effects are frequently observed. This study was designed to evaluate and compare the in vivo effects of 0.5 mg/kg, BID, albendazole sulfoxide (ricobendazole) and two different therapeutic regimens of 0.5 mg/kg BID and 2 mg/kg every 48 h of albendazole sulfoxide loaded solid lipid nanoparticles. Albendazole sulfoxide loaded solid lipid nanoparticles was prepared by solvent diffusion–evaporation method. Fifty Balb/c mice were infected by intraperitoneal injection of protoscoleces and 8 months post infection, the infected mice were treated for 15 days with the above mentioned regimens. They were then euthanized and the size and weight of the cysts as well as their ultrastructural changes were investigated. Although the cysts showed reduced size and weight in the treated animals but these reductions were not statistically significant. The cysts in the animals which received albendazole sulfoxide loaded SLN every 48 h showed more ultrastructural modification. However, these ultrastructural changes should be supported by further biochemical and molecular studies before introducing it as an efficient therapeutic regimen for treatment of human and animal hydatid disease.     

Simultaneous liquid chromatography-tandem mass spectrometric determination of albendazole sulfoxide and albendazole sulfone in plasma

This paper describes a simple, fast, sensitive and reliable method for the simultaneous determination of albendazole sulfoxide (ASOX) and albendazole sulfone (ASON), the two most important metabolites of the drug albendazole (ABZ), in plasma samples using liquid chromatography and tandem mass spectrometry. After liquid-liquid extraction with dichloromethane, the two albendazole metabolites and the internal standard phenacetin were resolved in a CN column using the mobile phase methanol-water (4:6, v/v) acidified with 1% acetic acid. Detection by electrospray mass spectrometry was carried out in the positive ion mode. The method was linear up to 2500 and 250 ng/ml for ASOX and ASON, respectively, with mean recoveries of more than 85%. The precision and accuracy data, based on within- and between-day variations over 5 days, were lower than 15%. The quantitation limits of 0.5 and 5.0 ng/ml for ASON and ASOX are low enough for the method to be suitable for pharmacokinetic studies. Pharmacokinetic data obtained with the proposed method following oral administration of ABZ to a patient with neurocysticercosis are also reported.     

Trace analysis of albendazole and its sulphoxide and sulphone metabolites in milk by liquid chromatography

Analytical methodology for determination of albendazole and its sulphoxide and sulphone metabolites in milk at levels down to 2–5 ng/ml has been developed. Extraction was carried out with ethyl acetate under alkaline conditions, and extracts were analyzed on a silica-based C₁₈ column in the presence of positively-charged pairing ions. Accuracy data showed overall recoveries ranged from 78.4% to 100%, whereas precision data, based on within- and between-day variation, suggested overall precision values better than 4.9%. The method was successfully applied to determine residues in milk of a dairy cow orally given albendazole.     

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%.     

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.     

Secondary and primary murine alveolar echinococcosis: combined albendazole/nitazoxanide chemotherapy exhibits profound anti-parasitic activity

In this study, the efficacies of chemotherapy employing nitazoxanide (NTZ), albendazole (ABZ), and a NTZ/ABZ-combination against alveolar echinococcosis (AE) were investigated in an experimental murine model. Following secondary infection, meaning i.p. injection of 20 Echinococcus multilocularis metacestodes, the drugs were administered by intragastric inoculation on a daily bases for a period of 5 weeks. Treatment was started either immediately on the day of infection, or at 2 months p.i., respectively. Application of the NTZ/ABZ-combination starting at 2 months p.i. was proven to be most effective in terms of reducing parasite weight (from 4.42+/-1.03 to 1+/-0.05 g; P=0.01). Inspection of treated parasites by transmission electron microscopy showed that ABZ- and NTZ-treated metacestode tissues, respectively, were heterogeneous in that both largely intact parasites as well as severely altered metacestodes could be observed. NTZ/ABZ-combination treatment induced the most severe ultrastructural alterations, including massive reduction in length and number of microtriches, severely damaged tegumental architecture, and progressive loss of viability of the germinal layer, associated with encapsulation by host connective tissue. A comparative pharmacokinetic study in mice revealed that the application of ABZ and NTZ in combination resulted in a two- to four-fold increase of albendazole sulfoxide serum levels for the period of 4-8 h following drug uptake compared to application of ABZ alone. In a third experiment, mice were orally infected with E. multilocularis eggs, and treated with NTZ starting at 2 months p.i. This resulted in a significantly lower lesion number in treated versus untreated mice (P=0.01). This investigation indicates the potential value for NTZ and/or a combined ABZ/NTZ chemotherapy against AE.     

Zataria multiflora would attenuate the hepatotoxicity of long-term albendazole treatment in mice with cystic echinococcosis

Hepatic injury is the major limitation of long-term albendazole administration in patients with cystic echinococcosis (CE), which could give rise to cessation of treatment. The objective of the present study was to evaluate the protective effects of Zataria multiflora aromatic water (AW) against the hepatic injury induced by long-term albendazole treatment in mice with CE. Fifty healthy BALB/c female mice were infected intraperitoneally by injection of 1500 protoscoleces per animal. Five months after infection, the infected animals were divided into five treatment groups including Z. multiflora (40 ml/l in drinking water for 90 days), albendazole (200 mg/kg/day for 90 days), Z. multiflora + albendazole 200 (40 ml/l Z. multiflora and 200 mg/kg/day albendazole for 90 days), Z. multiflora + albendazole100 (40 ml/l Z. multiflora and 100 mg/kg/day albendazole for 90 days), and untreated (control) group. At the end of the treatment period, anesthesia was performed and blood samples were collected directly from the heart prior to euthanasia. Liver variables and oxidative stress markers were measured in the blood serum samples. A decrease in serum liver enzyme activity in the both Z. multiflora + albendazole groups was observed when compared to control, Z. multiflora and albendazole groups; however, the results for Z. multiflora + albendazole 100 were significant (p < 0.007) and superior compared to those for Z. multiflora + albendazole 200. No significant differences for oxidative stress markers were observed between the different groups. The results of the present study revealed that a combined therapy with Z. multiflora AW and albendazole is effective against hepatic injury induced by CE and/or long term albendazole administration in mice with cystic echinococcosis.     

Lecithin in synthetic larval diet for Aedes aegypti improves larval and adult performance

A chemically defined synthetic rearing medium was used to compare larval growth of Aedes aegypti with or without crude animal lecithin or synthetic dipalmitoyl lecithin. Pupal weights, adult female life spans and oviposition histories of pupae and adults derived from synthetic diet rearings and from crude culture (liver powder) rearings were also compared. Both lecithins improved larval growth rate; optimal concentrations reduced time to pupation by 2 and 1 days for animal and synthetic lecithins respectively, and animal lecithin was better tolerated by the mosquito larvae at the higher concentrations tested. Addition of crude animal lecithin to the basal synthetic rearing medium had little effect on the weights of male and female pupae, but it increased adult female life spans and improved weekly egg production. In no case did mosquitoes from axenic rearings in synthetic media deviate greatly from limits specified by earlier workers for field-derived mosquitoes.

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Résumé Les développements larvaires d'Aedes aegypti ont été comparés sur des substrats alimentaires avec ou sans lecithine animale brute ou DL-a-dipalmitoyle lecithine synthétique.Les comparaisons ont porté aussi sur les poids nymphaux, la longé-vité et la fécondité de femelles élevées sur régime synthétique ou à partir de poudre de foie.Les deux types de lecithines améliorent le développement larvaire; les concentrations optimales des deux régimes (lecithine animale et lecithine synthétique) avancent respectivement la pupaison de 2 et 1 jour; les lecithines animale et synthétique sont tolérées par les larves respectivement jusqu'aux concentrations de 0,008 et 0,006%.L'addition de lecithine animale brute au régime contenant de la lecithine synthétique a eu peu d'effets sur les poids des nymphes mâles et femelles mais a augmenté la longévité et la fécondité hebdomadaire des femelles.Les performances des moustiques élevés sur substrat aseptique synthétique n'ont jamais été très éloignées des limites indiquées antérieurement par les travaux sur moustiques provenant de la nature.

     

In vivo effect of single oral dose of artemether against early juvenile stages of Schistosoma mansoni Egyptian strain

The current treatment and control of schistosomiasis, rely on a single drug, praziquantel, although, it has minor activity against juvenile stages of the parasite. Studies have shown that artemether (ART) exhibits effects against juveniles of Schistosoma mansoni Liberian and Puerto Rican strains, Schistosoma japonicum and Schistosoma haematobium. Aiming to assess the in vivo activity of single oral dose of ART against early juvenile stages of S. mansoni Egyptian strain, this study was established. Mice were treated with ART (400 mg/kg) at two time points evenly spaced over the period of larval development (7 and 21 days post-infection; pi), and a third treatment point (day 49 pi) was included to elucidate when susceptibility decreases. Administration of ART on day 7 pi reduced the total worm burden by 85.94%. The greatest reductions were seen when treatment was given on day 21 pi, with total and female worm burden reductions of 91.52% and 90.57%, respectively, and cessation of oviposition. Similar dose given on day 49 pi reduced total worm burden by 55.17% and female worm burden by 66.51%. Moreover, it induced significant reduction in the tissue egg load and significant alterations in the oogram pattern with decreased immature eggs and increased dead eggs. Antipathological activities were evident in significant reductions in granulomata count and diameter. In conclusion, ART exhibits major in vivo schistosomicidal effects against the early larval migratory stages of S. mansoni Egyptian strain, mainly the 21-day old schistosomula, hence preventing disease progression and morbidity.     

Development of an injectable formulation of albendazole and in vivo evaluation of its efficacy against Echinococcus multilocularis metacestode

The loading of poly (D,L-lactide) nanoparticles with ABZ has led us to evaluate the potential of this new colloidal drug delivery system against E. multilocularis, using a murine model of hepatic alveolar echinococcosis. ABZ-loaded nanoparticles had a monodisperse size distribution between 100 and 150 nm. The efficiency of drug loading to nanoparticles was over 97%. In vitro, at an ABZ concentration of 1.0 μg ml⁻¹, the formulation had no toxicity for peritoneal macrophages harvested from uninfected mice, In vivo, the ABZ-loaded nanoparticles exhibited no signs of toxicity at any of the doses tested. Intravenous injections of 6 mg kg⁻¹ of bound ABZ to infected mice had an equivalent antiparasitic effect on the metacestode growth to that of a treatment with 1500 mg kg⁻¹ of orally administered free ABZ. The parasite hepatic superficial size as well as the peritoneal metastatic burden was significantly reduced by these 2 courses of treatment, as compared to those of untreated mice. Our results should encourage further study in order to explain the absence of dose-dependent efficacy of ABZ-loaded nanoparticles demonstrated in the present study.     

Effects of testosterone on spatial learning and memory in adult male rats

A male advantage over females for spatial tasks has been well documented in both humans and rodents, but it remains unclear how the activational effects of testosterone influence spatial ability in males. In a series of experiments, we tested how injections of testosterone influenced the spatial working and reference memory of castrated male rats. In the eight-arm radial maze, testosterone injections (0.500 mg/rat) reduced the number of working memory errors during the early blocks of testing but had no effect on the number of reference memory errors relative to the castrated control group. In a reference memory version of the Morris water maze, injections of a wide range of testosterone doses (0.0625-1.000 mg/rat) reduced path lengths to the hidden platform, indicative of improved spatial learning. This improved learning was independent of testosterone dose, with all treatment groups showing better performance than the castrated control males. Furthermore, this effect was only observed when rats were given testosterone injections starting 7 days prior to water maze testing and not when injections were given only on the testing days. We also observed that certain doses of testosterone (0.250 and 1.000 mg/rat) increased perseverative behavior in a reversal-learning task. Finally, testosterone did not have a clear effect on spatial working memory in the Morris water maze, although intermediate doses seemed to optimize performance. Overall, the results indicate that testosterone can have positive activational effects on spatial learning and memory, but the duration of testosterone replacement and the nature of the spatial task modify these effects.     

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.     

Protective effect of cannabidiol against cadmium hepatotoxicity in rats

The protective effect of cannabidiol, the non-psychoactive component of Cannabis sativa, against liver toxicity induced by a single dose of cadmium chloride (6.5 mg kg^?1 i.p.) was investigated in rats. Cannabidiol treatment (5 mg kg^?1/day, i.p.) was applied for five days starting three days before cadmium administration. Cannabidiol significantly reduced serum alanine aminotransferase, and suppressed hepatic lipid peroxidation, prevented the depletion of reduced glutathione and nitric oxide, and catalase activity, and attenuated the elevation of cadmium level in the liver tissue resulted from cadmium administration. Histopathological examination showed that cadmium-induced liver tissue injury was ameliorated by cannabidiol treatment. Immunohistochemical analysis revealed that cannabidiol significantly decreased the cadmium-induced expression of tumor necrosis factor-α, cyclooxygenase-2, nuclear factor-κB, caspase-3, and caspase-9, and increased the expression of endothelial nitric oxide synthase in liver tissue. It was concluded that cannabidiol may represent a potential option to protect the liver tissue from the detrimental effects of cadmium toxicity.     

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.