Proteomic analysis of Ascaridia galli. Identification of immunoreactive proteins in naturally and experimentally infected hens.

Ascaridia galli, intestinal parasite of domestic fowl, is responsible of economic losses in avian exploitations. However, molecular mechanisms that govern avian ascaridiasis remain largely unknown. The aim of the present work was to identify proteins of A. galli recognized by the immune system of naturally and experimentally infected hens, using two-dimensional electrophoresis (2-DE) and mass spectrometry (MS). Sixteen immunoreactive proteins of A. galli were identified. These proteins are mainly related to different metabolic processes, cell motility and binding activities. The timing evolution of this recognition pattern was studied using serum samples from experimentally infected hens, allowing us to observe an early recognition of many of these antigens. Many of them were isoforms from lipid and plasminogen-binding proteins. Moreover, plasminogen-binding activity has been related in other parasites with the facilitation of intra-organic migration, which represents an important fact in avian ascaridiasis. This work represents the first proteomic study of A. galli and could contribute to explain some aspects of parasite/host relationships of avian ascaridiasis.

Effect of selenium and Ascaridia galli infection on antioxidant biomarkers in broiler chickens: a mathematical model for parasite reduction and host growth.

The activity of selenium-dependent glutathione peroxidase (GPX), liver concentration of vitamin E, and plasma and liver selenium levels were used for estimation of the antioxidant status of broiler chickens infected with Ascaridia galli. These biomarkers were recorded in an experiment covering 70 days p.i. At the same time the establishment rate of A. galli in chicken intestines, gain in the host body weight and chicken survival were studied. Broiler chickens (Cobb hybrids) were infected with 1450 embryonated A. galli eggs and treated with Sel-plex. A mathematical model was applied to determine the rate of nematode reduction and the relative rate of gain of host body weight, which are essential kinetic parameters of parasite-host interaction. The activity of GPX increased with both elevated selenium and reduced infection levels. The concentrations of selenium and vitamin E, and the GPX activity in the infected chickens demonstrated a similar pattern of change with time after day 30 p.i. The supplementation of the broilers with dietary selenium in the form of Sel-plex improved their antioxidant status. Increases by 29% in vitamin E concentration, 15% in GPX activity, and 22% in liver selenium concentration, respectively, were recorded in the infected and treated, compared to infected and untreated broilers.

Sphingomyelin synthesis in helminths: a minireview.

Sphingolipids are a diverse and ubiquitous group of lipids. They are widely distributed in parasites and a number of novel forms have been described. Sphingolipid synthesis has been investigated in the malarial parasite, cestodes, digeneans and nematodes. Although there are differences in detail, the synthetic pathways involved are similar to those found in mammals.

The role of cAMP in the actions of the peptide AF3 in the parasitic nematodes Ascaris suum and Ascaridia galli.

AF3 (AVPGVLRFamide) is an endogenous RFamide-like peptide isolated from the parasitic nematode Ascaris suum. It has a potent and long lasting excitatory effect in A. suum and Ascaridia galli. This is mediated by a mechanism independent of the nicotinic-like acetylcholine (ACh) receptor, which mediates excitatory transmission at the neuromuscular junction of both nematodes. In addition, AF3 has been found to sensitise A. suum muscle to the contractile effect of ACh. In this study, the involvement of the second messenger cAMP in mediating the action of AF3 on the somatic musculature of A. suum and A. galli has been investigated. Two approaches have been used; the effects of drugs which raise intracellular cAMP levels on the contractile responses to AF3 have been examined and biochemical assays have been used to measure the effects of AF3 on cAMP levels. AF3 contractions were inhibited in A. suum by 10 microM forskolin (by 22% of control; P < 0.05; n = 9) and by 500 microM isobutylmethylxanthine (IBMX, by 27% of control; P < 0.001; n = 6). AF3 decreased cAMP concentrations in A. suum somatic muscle (basal, 1721 +/- 134 pmol mg-1 protein; with 1 microM AF3, 1148 +/- 133 pmol mg-1 protein; P < 0.05, n = 5). AF3 (1 microM) also reduced the 10 microM forskolin induced potentiation of cAMP concentrations in A. suum (forskolin 3242 +/- 471 pmol mg-1 protein; forskolin and AF3, 1524 +/- 143 pmol mg-1 protein; P < 0.001, n = 6) and A. galli (forskolin 291 +/- 32 pmol mg-1 protein, forskolin +AF3, 185 +/- 12 pmol mg-1 protein; P < 0.005, n = 5). These data suggest that in both nematodes the contractile effect of AF3 is, at least in part, regulated by cAMP.

Vasoactive intestinal polypeptide-like and peptide histidine isoleucine-like proteins excreted/secreted by Nippostrongylus brasiliensis, Nematodirus battus and Ascaridia galli.

Vasoactive intestinal polypeptide (VIP)-like protein was detected by dot blot analysis in the excretions/secretions (E/S) of Nematodirus battus and Ascaridia galli and was confirmed in the E/S of Nippostrongylus brasiliensis. ELISA analysis showed that N. brasiliensis E/S contained the highest proportion of VIP-like protein (28.04 pmoles/mg of total E/S protein) and A. galli E/S contained the lowest (10.89 pmoles/mg of total E/S protein). Peptide histidine isoleucine (PHI)-like protein was detected by dot blot analysis in the E/S products of N. brasiliensis, N. battus and A. galli. ELISA analysis suggested that A. galli E/S contained the highest proportion of PHI (20.77 nmoles/mg of total E/S protein) and N. battus E/S contained the lowest (0.67 nmoles/mg of total E/S protein). The possible significance of VIP-like and PHI-like substances in the E/S of gastrointestinal nematodes is discussed.

A novel membrane glycoprotein involved in ascidian hemocyte aggregation and phagocytosis.

Invertebrate hemocytes undergo several cellular defense reactions. To clarify the molecular mechanisms for cellular recognition between hemocytes and also between hemocytes and foreign materials, we established hybridoma clones producing monoclonal antibodies that inhibit hemocyte aggregation (i.e. a cellular reaction between hemocytes) in the solitary ascidian, Halocynthia roretzi. The antibody, A74, also inhibited phagocytosis of foreign materials by H. roretzi hemocytes. Immunocytochemistry of H. roretzi hemocytes using A74 antibody revealed the localization of the A74 antigen on the surface of hemocytes. The A74 antigen, which is referred to as A74 protein, was purified from a hemocyte membrane preparation by three chromatographies on phenyl-Sepharose, A74 antibody-immobilized Sepharose and Mono Q. The A74 protein was a glycoprotein with a molecular mass of 160 kDa; N-glycosidase or neuraminidase treatment resulted in a reduction of its molecular mass. The N-terminal amino acid sequence of A74 protein showed little similarity to other known proteins. Thus, the A74 protein is a novel membrane protein that plays an important role in ascidian cellular defense reactions.

Sphingomyelin synthesis in Ascaridia galli.

Adult Ascaridia galli incorporate label from [U-14C] serine into various intermediates of sphingomyelin synthesis (ketosphinganine, sphinganine, sphingosine, ceramide and sphingomyelin). From the results it is concluded that A. galli possesses the five enzymes involved in sphingomyelin synthesis, namely: serine palmitoyltransferase, 3-ketosphinganine reductase, flavoprotein sphinganine reductase, sphingosine acyltransferase and ceramide choline phosphotransferase.

Polyamine metabolism in some helminth parasites.

Polyamine levels of some helminth parasites were analyzed by reverse phase HPLC of benzoyl derivatives. Setaria cervi, Acanthocheilonema viteae, Hymenolepis nana, H. diminuta, and Ascaridia galli contained higher levels of spermine than spermidine while in Ancylostoma ceylanicum and Nippostrongylus brasiliensis the spermidine levels were higher than spermine; putrescine was either absent or present in minor quantities. The enzymes of polyamine biosynthesis viz., ornithine decarboxylase, S-adenosyl methionine (SAM)-decarboxylase, and arginine decarboxylase were present in very low to negligible amounts in all the parasites examined. A. ceylanicum exhibited high activity of ornithine amino transferase (OAT) and catalyzed appreciable decarboxylation of ornithine. The ornithine decarboxylating activity of A. ceylanicum was localized in the particulate fraction containing mitochondria, not inhibited by alpha-difluoromethyl ornithine, the specific inhibitor of ornithine decarboxylase (ODC), but inhibited in the presence of glutamate, suggesting the involvement of mitochondrial OAT rather than a true ODC in ornithine decarboxylation in this parasite. Significant activity of polyamine oxidase was also detected in helminth parasites. The absence of polyamine biosynthesizing enzymes in helminth parasites suggests their dependence on hosts for uptake and interconversion of polyamines, providing a potential target for chemotherapy.

Effect of cambendazole and haloxon on the carbohydrate metabolism of Ascaridia galli and Heterakis gallinae (Nematoda).

Cambendazole 10(-4) M significantly reduced the level of glycogen in both the parasites. Lactic acid level was found enhanced. Oxygen consumption was suppressed by 63 and 94% in A. galli and H. gallinae, respectively, by 10(-4) M cambendazole. Haloxon did not cause any significant change in glycogen and lactic acid level in either parasite, but reduced oxygen consumption by 23 and 31% in Ascaridia galli and Heterakis gallinae, respectively.

Ascaridia galli: effect of some anthelmintics on amino acid uptake and macromolecular synthesis in vitro.

L-(U-14C) aspartic acid, L-(U-14C) alanine and L-(U-14C) leucine uptake by Ascaridia galli was found to be a non-linear function of time and limiting substrate concentration. The uptake was rapid initially but achieved steady state thereafter, possibly owing to the saturation of transport loci. Linear transformations of substrate saturation kinetics by Lineweaver-Burk plots of L-(U-14C) aspartic acid, L-(U-14C) alanine and L-(U-14C) leucine gave Kt values of 4.76, 3.03 and 2.0 mM and Jmax of 5.0, 3.57 and 2.08 m moles/100 mg dry weight/2 min, respectively. D1-tetramisole and 1-tetramisole (levamisole) inhibited the uptake of amino acids. The uptaken amino acids were readily metabolized into different tissue fractions. D1-tetramisole and levamisole significantly inhibited the incorporation of the three amino acids into the nematode's total protein, RNA and lipid fractions in an in vitro incubation system.

Lipid metabolism and low molecular weight solute uptake in Ascaridia galli.

Adult Ascaridia galli, an intestinal nematode parasite of fowl, reveals a large variety of complex lipids such as phospholipids containing choline, ethanolamine, inositol, serine and glycerol. Lysophospholipid species, vinyl ether phospholipid (plasmalogen), neutral acylglycerols, cholesterol and non-esterified fatty acids are also present. Sugar-containing lipids, such as cerebrosides, sulphatides and gangliosides are abundantly present. Female parasites contain more lipids, particularly acylglycerols and phospholipids. Acylglycerols, phosphatidyl choline, phosphatidyl ethanolamine and glycolipids incorporate a large amount of radiolabelled precursor substrate in A. galli. The presence of important enzymes of lipid biosynthesis like glucose-6-phosphate dehydrogenase, malate dehydrogenase and hydroxymethyl glutaryl-CoA reductase as well as an enzyme of lipid ester hydrolysis, triacylglycerol lipase is detected in the parasite. These enzymes show subcellular distribution patterns and Michaelis-Menten kinetic characteristics comparable with that from rat liver homogenate. Studies on the uptake of labelled precursor molecules for lipid biosynthesis, glucose, acetate and palmitate show that the parasites can take up the isotopes readily in a time-dependent manner, showing substrate saturation kinetics, dependence upon Na ions, and can be inhibited by the presence of the bile salts sodium cholate and sodium deoxycholate. The substrate affinity constant (Kt) and maximum apparent velocity of glucose uptake in A. galli were found to be 9.09 mM and 26.67 mM per 100 mg tissue dry weight per min at 37 degrees C.

The O2-dependence of respiration and H2O2 production in the parasitic nematode Ascaridia galli.

1. Respiration in the parasitic nematode worm Ascaridia galli was inhibited at O2 concentrations in excess of 255 microM, and an apparent Km,O2 of 174 microM was determined. 2. Mitochondria-enriched fractions isolated from the tissues of A. galli have much lower apparent Km,O2 values (approx. 5 microM). They produce H2O2 in the energized state; higher rates of H2O2 production were observed in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone. 3. Antimycin A inhibited respiration in muscle tissue mitochondria by 10%, but had no effect on respiration in gut + reproductive tissue mitochondria; the major portion of respiration in both types of mitochondria could be attributed to an alternative electron-transport pathway. 4. o-Hydroxydiphenyl, an inhibitor of alternative electron-transport pathways, inhibits respiration by 98% and completely inhibits the production of H2O2 in gut-plus-reproductive-tissue mitochondria; respiration and H2O2 production in muscle tissue mitochondria were inhibited by 90 and 86% respectively. 5. Another inhibitor of alternative electron transport, salicylhydroxamic acid, had the same effect as o-hydroxydiphenyl on H2O2 production and respiration in gut-plus-reproductive-tissue mitochondria. However, its effect on muscle tissue mitochondria was complex; a low concentration (0.35 mM) stimulated H2O2 production, whereas 3 mM inhibited respiration by 87% and prevented H2O2 production completely. 6. The similarities between the apparent Km,O2 values for H2O2 production and respiration in muscle mitochondria and in gut-plus-reproductive-tissue mitochondria suggests that the site of H2O2 production on the alternative electron-transport chain is cytochrome 'o'. 7. These results are discussed in relation to potential O2 toxicity in A. galli.

The effect of piperazine adipate and parbendazole on the carbohydrate metabolism of Ascaridia galli and Heterakis gallinae.

Carbohydrate metabolism of Ascardia galli and Heterakis gallinae was studied after their exposure in vitro to 10(-2) to 10(-5) M piperazine adipate and parbendazole for 10 to 60 min. Following treatment with 10(-2) M piperazine adipate O2 uptake was reduced by 87% in A. galli and 70% in H. gallinae, while parbendazole (10(-2) M) reduced O2 uptake by 70% and 86%, respectively. Glycogen contents were reduced significantly by 10(-2) M piperazine adipate in both the worms, while lactic acid level was enhanced. Parbendazole, however, did not cause any significant change in glycogen contents and lactic acid level in either parasite.

[Cadaverine and its possible role in parasite and host interrelations in ascaridiasis]. / Kadaverin i ego vozmozhnaia rol'vo vzaimootnosheniiakh parazita i khoziaina pri askaridioze.

A comparative study of the formation process of cadaverine in tissues of ascarides, intestine and liver of hen was conducted. Data are given on the activity, pH-optimum of lysine decarboxylase obtained from tissues of helminth and its host. The question on a toxic role of cadaverine during helminthiasis is considered. It has been concluded that the accumulation of cadaverine in the host's intestine can break the permeability of the intestine walls and favour the penetration of toxins of helminths into the host's organism.

Nematoda: aerobic respiratory pathways of adult parasitic species.

Aerobic respiratory pathways have been compared in adult parasitic nematodes, including Trichostrongylus colubriformis, Nippostrongylus brasiliensis, Ostertagia ostertagi, Cooperia oncophora, Haemonchus contortus, Oesophagostomum venulosum, Chabertia ovina, Dictyocaulus filaria, Dictyocaulus viviparus, and Ascaridia galli. Respiration was measured in both whole worm or tissue homogenates and isolated mitochondrial fractions, and delineated into the mammalian type or alternative respiratory pathways on the basis of their inhibition by antimycin A. The alternative, antimycin A-insensitive respiratory pathway was of comparable activity in all parasitic nematodes studied, irrespective of the body diameter or habitat of the worm. The mammalian-type, antimycin A-sensitive respiratory pathway showed variations; the extent of this pathway correlated with both the body diameter and habitat of the worm, being greater in thinner worms and those worms whose habitat is supposedly more aerobic.

Nippostrongylus brasiliensis and Ascaridia galli: mitochondrial respiration in free-living and parasitic stages.

Aerobic respiratory pathways have been delineated and respiratory efficiency has been assessed in mitochondria isolated from embryonated eggs, infective larvae, and adult Nippostrongylus brasiliensis and Ascaridia galli. Mitochondrial respiration in free-living stages of N. brasiliensis is mediated mainly by a mammalian-like antimycin A- and cyanide-sensitive pathway; specific respiratory activity is high and oxidative phosphorylation efficient. In mitochondria of adult N. brasiliensis, antimycin A- and cyanide-sensitive respiration is decreased relative to respiration though an alternative pathway, and specific respiratory activity and mitochondrial efficiency are lower. Respiration in mitochondria from embryonated eggs and tissues of adult A. galli is comparable, and apparently mediated by an antimycin A- and cyanide-insensitive alternative respiratory pathway; no evidence for the presence of a mammalian-like respiratory pathway in embryonated eggs of A. galli was found. The results of this study are compared to mitochondrial respiration in eggs, larvae, and adult body wall muscle of Ascaris suum.