Change of subunit composition of mitochondrial complex II (succinate-ubiquinone reductase/quinol-fumarate reductase) in Ascaris suum during the migration in the experimental host.

The mitochondrial metabolic pathway of the parasitic nematode Ascaris suum changes dramatically during its life cycle, to adapt to changes in the environmental oxygen concentration. We previously showed that A. suum mitochondria express stage-specific isoforms of complex II (succinate-ubiquinone reductase: SQR/quinol-fumarate reductase: QFR). The flavoprotein (Fp) and small subunit of cytochrome b (CybS) in adult complex II differ from those of infective third stage larval (L3) complex II. However, there is no difference in the iron-sulfur cluster (Ip) or the large subunit of cytochrome b (CybL) between adult and L3 isoforms of complex II. In the present study, to clarify the changes that occur in the respiratory chain of A. suum larvae during their migration in the host, we examined enzymatic activity, quinone content and complex II subunit composition in mitochondria of lung stage L3 (LL3) A. suum larvae. LL3 mitochondria showed higher QFR activity ( approximately 160 nmol/min/mg) than mitochondria of A. suum at other stages (L3: approximately 80 nmol/min/mg; adult: approximately 70 nmol/min/mg). Ubiquinone content in LL3 mitochondria was more abundant than rhodoquinone ( approximately 1.8 nmol/mg versus approximately 0.9 nmol/mg). Interestingly, the results of two-dimensional bule-native/sodium dodecyl sulfate polyacrylamide gel electrophoresis analyses showed that LL3 mitochondria contained larval Fp (Fp(L)) and adult Fp (Fp(A)) at a ratio of 1:0.56, and that most LL3 CybS subunits were of the adult form (CybS(A)). This clearly indicates that the rearrangement of complex II begins with a change in the isoform of the anchor CybS subunit, followed by a similar change in the Fp subunit.

A comparison of the susceptibility of growing mukota and large white pigs to infection with Ascaris suum.

The influence of A. suum infection on the haematology, liver-related serum enzymes, blood urea and live weight gain in Mukota and Large White (LW) weaner pigs was compared. Six pigs of each genotype were infected with a single dose of 4000 A. suum eggs per pig and another six were not. The pigs were kept for 100 days. Blood was collected daily for the first 7 days and also after 100 days. In the infected pigs, there was an increase (p<0.05) in alanine aminotransferase (ALT) activity in the LW but not in the Mukota pigs. Although the alkaline phosphatase (ALP) activity rose (p<0.05) in both infected and non-infected LW pigs from day 1 to day 3, the activity in the non-infected LW pigs then decreased, while that of the infected LW pigs remained elevated. The infected LW pigs had higher (p<0.05) levels of ALT, ALP and aspartate aminotransferase than their non-infected counterparts. Non-infected LW pigs tended to have higher (p<0.05) haematological parameters, daily weight gain and urea concentrations than infected LW pigs, but these differences were not significant. These preliminary findings suggest that more A. suum larvae reached the livers in the LW than in the Mukota pigs and that the latter may be more resistant to A. suum infection.

Diagnosis of parasitic zoonoses by immunoenzymatic assays--analysis of cross-reactivity among the excretory/secretory antigens of Fasciola hepatica, Toxocara canis, and Ascaris suum.

Several parasitic infections such fasciolosis, toxocariosis or ascariosis are important zoonoses. During the infection with Fasciola hepatica, Toxocara canis and Ascaris suum, an important intraorganic phase in their hosts takes place, releasing antigens responsible for a humoral immune response, which enables the diagnosis of that parasitosis. A study to identify the existence of cross-reactivity among the excretory/ secretory antigens of F. hepatica, T. canis and A. suum was developed. One group of Sprague-Dawley rats was infected with 20 metacercariae of F. hepatica and another group remained uninfected as control. By means of an Indirect-ELISA, the rat humoral immune response (IgG and IgM) against the excretory/secretory antigens of F. hepatica was analysed and measured for cross reactivity with T. canis and A. suum. IgM cross-reaction was mainly observed in the first 10 weeks post-infection. IgG cross-reaction was observed throughout the study, and was maximal at the 2-3 weeks and 3-6 weeks post-infection, which corresponds to the intraorganic migratory phase of these parasites. The western-blot showed that the rat IgG recognised three proteins of 190, 160 and 33 kDa in the antigens from F. hepatica, T. canis and A. suum. The existence of cross-reactivity among these antigens seems to demonstrate also the presence of structural similarities, such as tegumental proteins. These results should be consider when immunoassay probes are used in the diagnosis of parasitic infections.

Characterization of a putative nitric oxide synthase in the neuromuscular system of the parasitic nematode, Ascaris suum.

In this paper we report on the biochemical presence of nitric oxide synthase (NOS)-like activity in Ascaris suum tissue and examine the pharmacological effect of NO donors on A. suum muscle strip preparation. NOS activity was determined by monitoring the formation of [3H]citrulline from [3H]L-arginine and NO formation via the oxyhaemoglobin assay. Neuromuscular tissue from A. suum which stained positively for NADPH diaphorase, contained NOS activity. Neither NOS activity nor NADPH diaphorase staining was detected in intestinal tissue. The absence of Ca2+, NADPH and other co-factors normally required for mammalian neuronal NOS activity only partially reduced the formation of both citrulline and NO by A. suum neuromuscular homogenate. The results of the biochemical assays indicate the presence of an enzyme capable of producing NO and citrulline, but with a different profile from that of rat neuronal NOS. We also present preliminary evidence for the action of NO (NO donors) in the neuromuscular system of A. suum.

The effect of Ascaris trypsin inhibitor on proteolytic activity of lungs and kidneys of guinea pig with larval ascariosis and after ascarid homogenate administration.

Trypsin inhibitor, isolated from Ascaris suum tegument reduces in vitro proteolytic activity (pH 7) of lungs and kidneys post-nuclear fractions of guinea pigs with larval ascariosis, pigs after subcutaneous ascarid homogenate administration as well as the activity in control groups. The comparative analysis of effect curves of trypsin inhibitor on the organ proteases revealed stronger reduction of the activity of post-nuclear lung proteases in ascariosis and of kidneys proteases after the injection of Ascaris homogenate than the inhibition of the fractions activity from respective control groups.

Stimulation of leukocyte lysophospholipase activity by noninfectious agents.

Mouse peritoneal leukocyte lysophospholipase (LPL) activity was studied to determine whether or not noninfectious agents cause increased enzyme activity and whether neutrophils have LPL activity. In the first study, mice infected with Ascaris suum, a known inducer of LPL activity, were given intraperitoneal injections of proteose peptone, thioglycolate, bovine albumin, paraffin, glycogen, or A. suum whole worm extract (WWE). Cell populations collected from mice injected with A. suum WWE, proteose peptone, thioglycolate, or bovine albumin contained increased numbers of neutrophils and eosinophils. These cell populations had increased LPL activity when treated, in vitro, with either A. suum WWE, zymosan-activated complement, or with the agent they were induced with. However, the LPL activity of the different cell populations did not respond to all treatments in the same way. In a second study, A. suum-infected or noninfected mice were given intraperitoneal injections of paraffin, thioglycolate, glycogen, or A. suum WWE. Enriched cell populations containing either lymphocytes or macrophages, from infected or noninfected mice, did not have increased LPL activity following in vitro stimulation with A. suum WWE, zymosan-activated complement, or with the agent they were induced with. Enriched neutrophil populations from infected or noninfected mice had increased LPL activity following in vitro treatment with A. suum WWE or zymosan-activated complement. Results demonstrate that the LPL activity of peritoneal leukocytes can be induced by noninfectious agents and that neutrophils have increased LPL activity following in vitro stimulation.

[Trypsin and alpha-amylase activity in the pancreas of guinea pigs. IV. Comparison of the effects of administration of the mixtures of volatile organic acids, invasive ova of Ascaris suum and their homogenates]. / Aktywnosc trypsyny i alfa-amylazy w trzustce swinek morskich. IV. Porównanie wplywu podawania mieszaniny lotnych kwasów organicznych, inwazyjnych jaj Ascaris suum i ich homogenatów.

The activity of alpha-amylase in the experimental groups of guinea pigs was lower than in control animals. The decreased level of activity of trypsin was noted (p less than 0.05) also in infected animals, and in treatment with organic acids. In the group of guinea pigs, which was administrated homogenized eggs of parasite, the activity of enzyme was a little higher than in control animals. The infection of animals was associated with increase of relative weight of lungs. The guinea pigs treatment with organic acids had also a higher weight of spleen.

[Effects of administration of vitamin A, B 2 and B 15 to guinea pigs during the larval stage of ascariasis on alpha-amylase activity in the lungs and kidneys]. / Wplyw witamin A, B2 i B15 podawanych swinkom morskim w czasie glistnicy larwalnej na aktywnosc alfa-amylazy w plucach i nerkach.

The guinea pigs were administrated vit. A (400 i.u.), vit. B2 (1 mg) or vit. B15 (5 mg). On the 9-th day of the experiment part of them was infected with 5000 invasive eggs of Ascaris suum. The invasion lasted 6 days and was controlled by lungs and kidney weight, and number of larvae in the lungs. The activity of amylase was determined by saccharogenic method in both organs. In the lungs of infected animals the activity of alpha-amylase was about 3 times lower than in the control. The infection of guinea pigs which were given vitamins did not cause change of enzyme's activity. In the kidney directive tendency was the same, but the differences were smaller. The infection resulted in an increase of relative mass of lungs. This index and number of larvae was considerable smaller in guinea pigs with vitamins A and B15 administration. Any testing agent did not cause change of relative weight of kidney.

[Trypsin and alpha-amylase activity in the pancreas of guinea pigs. V. Effects of administration of vitamin A and B 2 during the larval stage of ascariasis]. / Aktywnosc trypsyny i alfa-amylazy w trzustce swinek morskich. V. Wplyw podawania witamin A i B2 w czasie askarydozy larwalnej.

The infection of guinea pigs with Ascaris suum larvae resulted in decrease of the activities of trypsin and alpha-amylase, and in increase of lipase activity in extracts from their pancreas. The activity of alpha-amylase, lipase and the relative weight of lungs of infected animals which were given vitamin A, did not differ from control animals. The activity of trypsin from pancreas these animals was higher than that measured in only infected guinea pigs but it was lower than in control animals. Application of vitamin B2 and the infection of guinea pigs with A. suum did not lead to the synonymous results.

Effect of dietary zinc on larval burdens, tissue eosinophil numbers, and lysophospholipase activity of Ascaris suum infected mice.

The effect of dietary zinc on larval burdens, tissue eosinophil numbers, and lysophospholipase (LPL) activity in the lungs and livers of BALB/c mice infected with Ascaris suum was evaluated. A suum larval numbers were increased in both liver and lungs in low zinc groups during primary and secondary infection as assessed at days 2 and 7 after egg administration. In the same groups, LPL activity and eosinophil numbers were reduced at both time points and in both tissues, with the exception of lung eosinophils in nonimmunized mice, since these animals did not develop an eosinophil response during primary infection.

[Trypsin and alpha-amylase activities in the pancreas of guinea pigs. II. Effect of per os and intra-intestinal infection with invasive ova of Ascaris suum]. / Aktywnosc trypsyny i alfa-amylazy w trzustce swinek morskich. II. Wplyw zarazenia per os i dojelitowo inwazyjnymi jajami ascaris suum.

The guinea pigs were infected orally or intraintestinally (after laparotomy) with 5000 invasive eggs of Ascaris suum. On the 7th day after infection the invasion was controlled by lung's weight, degree of congestion, and number of larvae in the lungs. The activity of amylase was determined according to Fennel method, trypsin to Anson method. The activities of both enzymes in pancreas of orally infected animals (after laparotomy or not) were lower, and relative weight of lungs of these animals was higher than in control animals. In pancreas of guinea pig with invasive material introduced into its, the activities of amylase and trypsin were higher than in control animals, but only the activity of trypsin differed significantly.

[Trypsin and alpha-amylase activities in the pancreas of guinea pigs. III. Effect of homogenates of invasive ova of Ascaris suum and organic acids C2-C6 on the activity of enzymes in pancreatic extracts]. / Aktywnosc trypsyny i alfa-amylazy w trzustce swinek morskich. III. Wplyw homogenatów z inwazyjnych jaj ascaris suum oraz mieszaniny kwasów organicznych C2-C6 na aktywnosc enzymów w wyciagach z trzustki.

The studies were carried out on pancreas extracts from guinea pigs free of parasite invasion. The activity of trypsin was determined according to the method of Anson, and amylase of Fennel method. The measurements of activity were done at the presence of homogenized invasive eggs of Ascaris suum, and the mixture of volatile organic acids C2-C6 (at concentrations of 30.3 and 0.3 mM/l). In samples with homogenized invasive eggs of A. suum the amylase activity remained unchanged while the activity of trypsin was considerably higher (p less than 0.01). The introduction of the organic acids at conc. 30 mM/l into the samples was the reason of lower amylase activity. No influence of these acids on trypsin activity in pancreas extracts was observed.

Alterations in Ascaris suum larval burdens, eosinophil numbers, and lysophospholipase activity associated with low levels of dietary iron.

The effect of various amounts of dietary iron on the immune response was investigated using BALB/cAnNCr/BR mice infected with Ascaris suum. Changes in numbers of larvae, numbers of eosinophils, and levels of lysophospholipase (LPL) activity in lung or liver tissues were analyzed from nonimmune and immunized mice at 2 and 7 days postinfection (PI). Various iron diets did not influence the numbers of tissue larvae, eosinophils, or the LPL activity in lungs or livers of nonimmunized mice at various times after infection. Lung and liver LPL activity was reduced in immunized mice without significant changes in larval numbers at 2 days PI. At 7 days PI, lung and liver LPL activity, eosinophil numbers, and numbers of larvae were increased in immunized mice receiving low iron diets. Results confirm that low iron diets affect the host response to A. suum.

[Changes in the serum levels of lactate dehydrogenase, gamma- glutamyltransferase, glucose and alpha amino nitrogen in guinea pigs with experimental ascariasis]. / Zmiany poziomu dehydrogenazy mleczanowej, gammaglutamylotranspeptydazy, glukozy i azotu alfa-aminowego w surowicy swinek morskich w przebiegu doswiadczalnej glistnicy.

The studies were carried out on 90 male guinea pigs of which 75 were infected per os with a dose of ca. 5000 invasive eggs of Ascaris lumbricoides suis. At the 1st, 2nd, 4th, 7th and 20th day of infection blood was taken from experimental and control animals. The activity of LDH and its liver fraction (spectrophotometric method), GGTP (circle test method) was determined. The level of glucose (orthotoluidin method) and alpha amino nitrogen (ninhydrin method was determined. In the course of larval ascariasis an increase in the values of the studied parameters was noted, especially at the 4th and 7th day of infection. The changes in the activity of the studied enzymes evidence disturbances of the structure and function of the liver of infected animals. The increased level of glucose and alpha amino nitrogen is probably associated with a disturbance of basic energy metabolism of the host organism.

[Alpha-amylase activity in serum and some organs of guinea pigs with larval ascariasis]. / Aktywnosc alfa-amylazy w surowicy i niektórych narzadach swinek morskich podczas askarydozy larwalnej.

The studies were carried out on 40 guinea pig males weighting about 230 g. The experimental animals were infected with 7000 invasive eggs of Ascaris suum. On the 3-rd, 7-th and 10-th day after infection the activity of alpha-amylase was estimated according to Caraway's method in serum, liver, pancreas, lungs, kidneys and spleen. The infection of guinea pigs results in increased activity of enzyme in serum and spleen, and decreased in pancreas, liver, kidneys and lungs. On the 3-rd day after infection the changes in amylase activity were the most intense.

[Trypsin and alpha-amylase activity in the pancreas of guinea pigs. I. The influence of infective dose and duration of infection by Ascaris suum Goeze, 1782]. / Aktywnosc trypsyny i alfa-amylazy w trzustce swinek morskich. I. Wplyw wielkosci dawki zarazeniowej i czasu trwania inwazji Ascaris suum Goeze, 1782.

The studies were carried out on guinea pig males weighting about 280 g. The animals were infected with 5000 or 20,000 invasive eggs of Ascaris suum. On the 3-rd, 7-th, 10-th and 14-th day of invasion the animals were dissected. Lungs were weighted, and the number of Ascaris larvae was estimated using Baermann method. The activity of alpha-amylase (Fennel's method) and trypsin (Anson's method) were examined in pancreas homogenate. The infection of guinea pigs with A. suum larvae resulted in a decrease of amylase and trypsin activity in pancreas. The activities were the lowest at the 3-rd day of invasion for amylase and at the 7-th day for trypsin. The results obtained for the animals infected 5 or 20,000 invasive eggs A. suum did not differ statically.