A time-lapse study of interactions between Echinostoma paraensei intramolluscan larval stages and adherent hemocytes from Biomphalaria glabrata and Helix aspersa

In vitro interactions between intramolluscan stages (sporocyst, daughter rediae, and metacercariae) of the trematode parasite Echinostoma paraensei and adherent hemocytes from the gastropods Biomphalaria glabrata (intermediate host) and Helix aspersa (non-host) were visualized by time-lapse videomicroscopy. Hemocytes of either species not exposed to E. paraensei displayed extensive mobility and activity of cellular extensions. Image analysis disclosed no significant change in the total surface area occupied by hemocytes in a selected field of view over 2 hr. Echinostoma paraensei exerted life stage-specific effects on the behavior of B. glabrata hemocytes; the cells moved away from sporocysts and daughter rediae but not encysted metacercariae. In the presence of sporocysts, hemocytes rounded up, whereas hemocytes adjacent to rediae assumed a stringy, beady appearance. Hemocytes close to the parasite were affected more rapidly than more distant cells. In 2 hr, a hemocyte-free "halo" formed around the parasite larvae, significantly reducing the hemocyte-occupied surface area (to 43% by sporocysts and to 70% by rediae). The changes induced by sporocysts and rediae are similar to those noted in both in vivo and in vitro studies of the B. glabrata-E. paraensei model system and are interpreted as manifestations of parasite-mediated interference with host hemocyte function. Helix aspersa (non-host) hemocytes were not affected, suggesting that E. paraensei-mediated effects on hemocytes exhibit a degree of specificity.     

Tissue reactions induced by Schistosoma mansoni in Biomphalaria glabrata

In Biomphalaria glabrata with a strong natural resistance, Schistosoma mansoni sporocysts are rapidly encapsulated by granulocytes and killed, mainly by the strong phagocytic activity of the cells. Irradiated Echinostoma paraensei sporocysts seem able to suppress the function of the granulocytes. Tissue reactions in snails with self-cure demonstrate: involvement of two types of cells, granulocytes and hyalinocyte-like cells; formation of amoeba-fibrous capsules; limited tendency of granulocytes to become attracted to the parasites; a slow process of parasite destruction; and a possible involvement of humoral factors. It seems that there is partial suppression of the granulocyte function in smails with self-cure.     

Ilyanassa obsoleta (Gastropoda) as a host for trematodes in Delaware estuaries

Extensive data characterizing larval trematode communities in marine gastropods do not exist for many systems. The purpose here is to report on the infections encountered in Delaware Ilyanassa obsoleta and to compare this trematode community with certain others that have been well studied. Over 15 yr, 11,774 I. obsoleta from 9 different estuarine habitats were examined. Trematode parasitism, sex, and size were determined for each snail. In the total collection, 9 trematode species were encountered; 51.04% of snails were infected with 1 or more species. The sexes were equally susceptible to being parasitized. Smaller snails were less likely to be infected than larger ones. Juvenile snails can be infected, but few have been colonized because of the short time they have been exposed to infective stages. Other studies of trematodes in this snail have revealed few multispecies infections. In contrast, they were strikingly common in this study (12.57% of all snails). Himasthla quissetensis seldom infects the same snail with Lepocreadium setiferoides or Austrobilharzia variglandis, but other species combinations can coexist. The important element in this trematode community seems to be time. The observed frequencies of different infracommunities (single and multispecies combinations) in samples of snails are considered to result from a variety of causes. These include commonness of the trematode species in the system, where and how the sample was collected, definitive host behavior, the low probability of a snail becoming infected, the long life of the snail and its infections, the vagility of the snail, and the incompatibility of certain species in terms of infecting the same snail.     

Pulmonary hemosiderosis

The cercarial emergence rhythms of Schistosoma mansoni were analyzed for mixed infection of the snail Biomphalaria glabrata with two chronobiological strains, one with an early shedding pattern and the other with a late shedding pattern. Under these conditions, each parasite strain kept its own circadian cercarial emergence pattern. When the chronobiological variants were of the same species, no interference between the trematode larvae occurred during the cercarial emergence process. These results confirm that within the host-parasite system the parasite component is directly responsible for the cercarial emergence pattern.     

Trematode hemoglobins show exceptionally high oxygen affinity

Ligand binding studies were made with hemoglobin (Hb) isolated from trematode species Gastrothylax crumenifer (Gc), Paramphistomum epiclitum (Pe), Explanatum explanatum (Ee), parasitic worms of water buffalo Bubalus bubalis, and Isoparorchis hypselobagri (Ih) parasitic in the catfish Wallago attu. The kinetics of oxygen and carbon monoxide binding show very fast association rates. Whereas oxygen can be displaced on a millisecond time scale from human Hb at 25 degrees C, the dissociation of oxygen from trematode Hb may require a few seconds to over 20 s (for Hb Pe). Carbon monoxide dissociation is faster, however, than for other monomeric hemoglobins or myoglobins. Trematode hemoglobins also show a reduced rate of autoxidation; the oxy form is not readily oxidized by potassium ferricyanide, indicating that only the deoxy form reacts rapidly with this oxidizing agent. Unlike most vertebrate Hbs, the trematodes have a tyrosine residue at position E7 instead of the usual distal histidine. As for Hb Ascaris, which also displays a high oxygen affinity, the trematodes have a tyrosine in position B10; two H-bonds to the oxygen molecule are thought to be responsible for the very high oxygen affinity. The trematode hemoglobins display a combination of high association rates and very low dissociation rates, resulting in some of the highest oxygen affinities ever observed.     

Evolution of ITS1 rDNA in the Digenea (Platyhelminthes: trematoda): 3' end sequence conservation and its phylogenetic utility

A comparison of ribosomal internal transcribed spacer 1 (ITS1) elements of digenetic trematodes (Platyhelminthes) including unidentified digeneans isolated from Cyathura carinata (Crustacea: Isopoda) revealed DNA sequence similarities at more than half of the spacer at its 3' end. Primary sequence similarity was shown to be associated with secondary structure conservation, which suggested that similarity is due to identity by descent and not chance. Using an analysis of apomorphies, the sequence data were shown to produce a distinct phylogenetic signal. This was confirmed by the consistency of results of different tree reconstruction methods such as distance approaches, maximum parsimony, and maximum likelihood. Morphological evidence additionally supported the phylogenetic tree based on ITS1 data and the inferred phylogenetic position of the unidentified digeneans of C. carinata met the expectations from known trematode life-cycle patterns. Although ribosomal ITS1 elements are generally believed to be too variable for phylogenetic analysis above the species or genus level, the overall consistency of the results of this study strongly suggests that this is not the case in digenetic trematodes. Here, 3' end ITS1 sequence data seem to provide a valuable tool for elucidating phylogenetic relationships of a broad range of phylogenetically distinct taxa.     

Reservoir competence of Alphitobius diaperinus (Coleoptera:Tenebrionidae) for Escherichia coli (Eubacteriales:Enterobacteriaceae)

Larval and adult lesser mealworms, Alphitobius diaperinus (Panzer), were found to harbor a Congo red-binding strain of Escherichia coli (Migula) Castellani & Chalmers both on the external surface of their body and internally for 12 d. Thereafter, E. coli was not detected, even though the beetles were exposed continuously to a food source inoculated with the bacteria. Lesser mealworm larvae and adults discharge E. coli bacteria in their feces for up to 6 and 10 d, respectively. However, bacteria were no longer detected in their feces after larvae underwent a single molt to the next larval stage. This indicated there was no transstadial transmission of this strain of E. coli. Consumed infected larvae were found to cause more 1-d-old chicks to have positive cloacal swabs for Congo red-binding E. coli than consumed infected adults. The data indicated that the lesser mealworm may play a role in the direct transmission of E. coli and contribute to the spread of this bacteria in broiler production systems. This may be achieved by beetles being directly consumed by chickens or indirectly by spread of the bacteria throughout the broiler house by lesser mealworm feces.     

Fas/APO-1 (CD95) expression in myelodysplastic syndromes

Increased apoptosis of myeloid precursors appears to contribute to the pathophysiology of cytopenias in myelodysplastic syndromes (MDS). Fas /APO-1(CD95) is a cell surface protein inducing an apoptotic signal after its binding to Fas ligand or to a functional anti-Fas antibody. Here we studied Fas expression by immunocytochemistry on marrow slides from 30 cases of MDS. Increased Fas expression in erythroblasts and/or immature granulocytes, compared to controls, was seen in 12 (40%) of the cases. In addition, in 16 of the 18 cases with > or = 5% marrow blasts, a variable proportion of blasts expressed Fas. Increased apoptosis was found by morphological analysis and/or TUNEL technique in marrow cells from 8 of the 26 cases analyzed (31%) The ability of Fas antigen to trigger apoptosis was studied after addition of a functional anti Fas antibody in 5 of the patients with Fas overexpression. Addition of this antibody, however, only lead to mild increase of apoptosis in immature granulocytes (but not other myeloid cells) in 2 of the 5 cases. Thus, increased Fas expression is seen in myeloid and/or blast cells in the majority of MDS cases. However, the relationship between this finding and increased apoptosis in MDS still remains to be established.     

Mitochondrial ND1 gene sequences used to identify echinostome isolates from Australia and New Zealand

Echinostomes were collected in Australia and New Zealand as cercariae, metacercariae or adults. Using DNA sequences from the mitochondrial ND1 gene Echinostoma revolutum and Echinostoma paraensei were discovered in Australia. The presence of a further five, as yet unidentified, echinostome species was inferred in Northern Australia and a further isolate, closely allied to E. revolutum, occurs in New Zealand. ND1 sequences of species within the genus diverge from each other by 9.6-30.8%. Sequence divergence levels among strains within a single species are 0-3.6%. The phylogenetic tree produced from the Australasian isolates, in addition to species described previously, identifies the 37-collar-spine species as a well supported monophyletic group. The five unidentified Australian species cluster away from the 37-collar-spine group. These unidentified species appear to divide further into > 37-collar-spine and < 37-collar-spine clusters. Three strains of E. revolutum, collected as metacercariae from snails, were identified from two ponds located 6 km apart. Two of these strains may be cycling through a planorbid snail, Glyptophysa sp., as first intermediate host; however, this hypothesis could not be confirmed as specimens could not be obtained to match sequences between larvae and adults.     

Clinical study in vertiginuous patients suspected of having neurovascular compression syndrome of the eighth cranial nerve

The in vitro activity of root-tuber-peel extract of Flemingia vestita, an indigenous plant consumed by the natives in Northeast India, was tested against helminth parasites. Live parasites (nematode: Ascaris suum from pigs, A. lumbricoides from humans, Ascaridia galli and Heterakis gallinarum from domestic fowl; cestode: Raillietina echinobothrida from domestic fowl; trematode: Paramphistomum sp. from cattle) were collected in 0.9 % physiological buffered saline (PBS) and maintained at 37 +/- 1 degrees C. In vitro treatment of the parasites with the crude extract (50 mg/ml) in PBS revealed complete immobilization of the trematode and cestode in about 43 and 20 min, respectively. However, the cuticle-covered nematodes did not show any change in physical activity and remained viable even after a long period of exposure to the extract. Exposure of R. echinobothrida to genistein (0.5 mg/ml), an active principle isolated from the root-tuber peel, caused spontaneous loss of movement (paralysis) in 4.5 h, which was slower than the time required for praziquantel, the reference flukicide and cestodicide. The treated parasites showed structural alteration in their tegumental architecture. This study suggests the vermifugal activity of this plant extract against trematodes and cestodes.     

Methodology of penile isotope imaging. Evaluation]

Increased mortality in a flock of non-breeding mute swans (Cygnus olor) on a Scottish loch was investigated. Postmortem examinations were carried out on eight adult and six immature swans. The commonest cause of death, found in eight birds, was lead poisoning associated with the ingestion of large lead fishing weights. Heavy parasitic burdens were found in five immature birds, involving combinations of the gizzard worm Amidostomum species, the thornyheaded worms Polymorphus minutus and Profilicollis anatis, and the tracheal trematode Orchipedum tracheicola. Other parasites of lesser significance were the biting louse Trinoton anserinum, the tapeworm Wardoides nyrocae, the hairworm Capillaria species and the intestinal trematode Echinoparyphium recurvatum. Eight of the 14 swans carried trematodes of the family Schistosomatidae, which may be involved in human cercarial dermatitis or 'swimmers' itch'. It is suggested that the increased mortality arose through a combination of increased numbers of swans on the loch, and a fall in the water level of the loch which exposed the birds to previously inaccessible lead fishing weights and to the intermediate hosts of a range of internal parasites.     

Immunological investigations in vaginal mycoses

In 42 women with chronically recurrent and 20 women with acute Candida albicans vulvovaginitis, as well as 14 women with Candida glabrata vaginitis, the following investigations were carried out: determination of protein content and secretory immunoglobulin A (sIgA) in the cervicovaginal secretion by a self-modified ELISA technique; determination of immunocells and cellbound IgA in the cervicovaginal secretion by immunofluorescence and nephelometric analysis of IgA in the serum. The results were compared with those of 77 pre-menopausal non-pregnant women with or without intake of anti-ovulants, 17 healthy pregnant women and four hysterectomised pre-menopausal women. Due to inflammation, women with acute and chronically recurrent Candida albicans vulvovaginitis had a higher protein content in the cervicovaginal secretion than healthy women. However, the content of secretory IgA was not increased but even slightly decreased in chronic cases. The number of macrophages and granulocytes in the vaginal content was not increased compared with healthy patients. In only a few cases was IgA detected on yeast cells and in the cervicovaginal secretion by fluorescence microscopy. In chronically-relapsing vaginal candidosis, the frequency of the serotype B of C. albicans was strikingly high. Women with Candida glabrata vaginitis showed lower values of secretory sIgA in the vaginal secretion compared with healthy patients as well as women with vaginitis caused by C. albicans. However, like healthy women, they had normal protein values in the cervicovaginal secretion and also lower values of IgA in the serum compared with women of C. albicans vulvovaginitis patients. Macrophages and granulocytes were demonstrable in the cervicovaginal secretion just as in healthy persons. Women with C. glabrata vaginitis showed a more conspicuous, although not a significantly more frequent, binding of IgA to budding cells demonstrated by fluorescence microscopy than women with C. albicans.     

Computerized spirometers (analysis of problem solving)

It has been experimentally found that larval and pulmonary paragonimiasis types are caused by the trematode Paragonimus westermani ichunensis. The development of either paragonimiasis in nontypical definitive hosts depends on the stage of larval development when they penetrate into the mammal. Only the P. westermani ichunensis trematode is likely to develop with the participation of Juga mollusks on the territory of the Primorye.     

Distribution and movement of infective-stage larvae of Dipetalonema viteae (Filarioidea) in the vector tick, Ornithodoros tartakowskyi (Argasidae)

To determine the behavior of Dipetalonema viteae in its tick vector, Ornithodoros tartakowskyi, the ticks were fed on jirds at successive intervals of 30 to 35 days after a single infective blood meal, and the number of larvae passing from the tick during each bite was determined by recovery of: 1) adult worms from the jirds' tissues; 2) larvae from skin snips taken at the feeding site immediately after the bite; and 3) larvae from serum and tissue after artificial feeding through a skin-membrane. All methods gave similar results. Ticks harboring few larvae released most of them (82%) during the first bite, and required only 2 bites to transmit all. Ticks with moderate or heavy infections required 3 or 4 bites to transfer all larvae. Factors which may explain this are: 1) relatively short duration of the bite of heavily infected ticks due to irritation and damage to the muscles of mouthparts and pharynx by the larvae; 2) resistance of the anterior alimentary tract to penetration by the larvae; and 3) retarding effects of crowding on development and migration of larvae. Aging of infection in the tick apparently did not influence the rate of transfer of larvae. Infection adversely affected the feeding and retarded the molting of young nymphs, but with the loss of larvae in successive bites the ability to suck blood was regained.     

Increased concentration of circulating acid glycosaminoglycans in chronic lymphocytic leukaemia and essential thrombocythaemia

The phagocytic cell function is an important factor of resistance to infection in fish. Although it is known that several nutritional factors affect phagocyte function and that vitamins C and E modulate the vertebrate immune system, there are no data on the direct effect of these vitamins or on their possible synergistic effects on the fish phagocyte functions. We report the in vitro effect of vitamin C and/or E on the functions and ultrastructure of gilthead seabream (Sparus aurata L.) head-kidney phagocytic leukocytes. Head-kidney leukocytes were incubated with different concentrations of vitamins C and/or E for 48 h and then their migration, respiratory burst, phagocytosis and ultrastructure were studied. Leukocyte migration and phagocytosis increased when vitamin C or E were added, while the respiratory burst was synergistically enhanced when both vitamins were used together. Noticeable ultrastructural changes not previously reported were induced by these vitamins in gilthead seabream phagocytes (acidophilic granulocytes and macrophages). These results indicated that vitamins C and E added in vitro modulate the main functions of gilthead seabream phagocytes and induced ultrastructural changes characteristic of cell activation on such cells.     

Host choice by larval parasites: a study of Biomphalaria glabrata snails and Schistosoma mansoni miracidia related to host size

Within snail/trematode associations the age/size of the host at infection has consequences with regard to miracidial infection success, further intramolluscan parasite development and reproduction, and the host response, mainly in terms of growth and reproductive effort. Taking into account these differences, we were interested in determining whether miracidia could discriminate and make a choice between snails of different sizes. Using the Schistosoma mansoni/Biomphalaria glabrata system, we compared data on the snail infection rate and the mother sporocyst abundance among three size classes of snails (juvenile, subadult, and adult) exposed separately or together to the parasite larvae. When exposed individually, juvenile snails (3-5 mm) had significantly higher prevalence and abundance values than did subadult snails, followed by adult snails. In contrast, when snails of the three size classes were exposed together in heterogeneous size groups the prevalence and abundance values were always significantly higher for subadult snails of the 7- to 9-mm class than for juvenile and adult snails. A host choice experiment confirmed that significantly more miracidia were attracted by subadult snails, suggesting that the parasite has been selected for specific locating and recognition mechanisms increasing the infection rate of subadult snails when the latter have been exposed in a heterogeneous size group. Selective forces that may be responsible for such a preferential infectivity of the parasite vis-à-vis particular host age/size class are discussed in relation to host resources and host responses.     

High-pressure washing treatments to remove obscure mealybug (Homoptera: Pseudococcidae) and lightbrown apple moth (Lepidoptera: Tortricidae) from harvested apples

Mixed life stages of obscure mealybug, Pseudococcus viburni (Signoret), and late 1st-instar or early 2nd-instar lightbrown apple moth, Epiphyas postvittana (Walker), on 'Royal Gala' apples (Malus domestica Borkhausen) were exposed to standard packhouse processing with and without addition of high-pressure apple washer treatments. Insect removal and mortality were assessed. After standard packhouse processing approximately 60% of P. viburni remained on their host apples. The 2 high-pressure apple washer treatments (500 and 800 psi at 2.0 rods/s) were equally effective and significantly reduced the number of P. viburni on apples compared with the packhouse control. High-pressure apple washer removal by location decreased in the following order: calyx cavity outside the sepals > cheek approximately stem cavity > or = calyx beneath sepals. About half of the E. postvittana larvae infesting apples was removed by standard packhouse processing. Removal rates were similar for all locations on open-calyxed apples. However, no removal occurred from the calyx beneath the sepals if the apple calyx was closed. All 4 high-pressure apple washer treatments tested (500 and 800 psi at 1.0 and 2.0 rods/s) halved the number of larvae on the apple exterior relative to the packhouse control. The pattern of removal for larvae on open-calyxed apples was calyx outside sepals approximately stem cavity > calyx beneath the sepals approximately cheek. A similar pattern was evident for larvae on closed-calyxed apples, except insects beneath the sepals evaded removal. The persistence of insects on the apple cheek reflects the high proportion of larvae inside tunnels in this location compared with other apple locations. Removal of internally positioned insects was much lower than that of externally positioned insects.     

Granulocytic protein p25 is a DNA-binding subunit of protein M(r) = 50,000: subcellular localization, cell and species specificity

We have previously reported the presence and isolation of the novel protein M(r) = 25,000 (p25) from human granulocytes. In this study, the protein p25 was characterized by its: (a) ability to bind DNA, (b) subunit association, (c) partial protein sequencing, (d) subcellular localization, (e) cellular and species specificity and (f) stability in the presence of released granulocytic proteinases. For the detection of p25 in various extracts, fractions and types of human or animal hematopoietic cells, SDS-PAGE/Western blotting and immunohistochemical staining were used. The protein p25 was subjected to N-terminal amino acid sequence analysis. Protein p25-DNA interactions were monitored using Southwestern blotting. Selective inhibition of granulocytic proteinases was performed. Granulocytic protein p25 was found to be a product of oxidative cleavage of disulfide bridges in the p50 dimer. It was shown that neither protein p50 nor the p25 subunit is a degradation product of a protein of higher molecular weight. The N-terminal amino acid sequence of p25 was: RLNYNKPHAA. Binding capacity for double stranded DNA without significant sequence specificity was revealed and nuclear localization of some fraction of p50 dimer was established. The data concerning the cell and species specificity demonstrated that the protein is expressed only in normal human granulocytes. In summary, protein p25 originates from splitting of the p50 dimer. This subunit shows no identity with proteins already sequenced. DNA-binding of p25 is not sequence specific. It is concluded that the protein p50 is localized in the nuclei and cytoplasmic granules of mature human polymorphonuclear leukocytes or granulocytes of species high on the evolutionary tree. The functions of this protein remain to be determined.     

Parenteral infection in mice with Baylisascaris transfuga (Nematoda: Ascaridae) infective eggs

In order to investigate the ability of infective larvae of the nematode Baylisascaris transfuga (Rudolphi, 1819) Sprent, 1968 to hatch from the egg-shells and then to migrate in tissues, parenteral infections of mice with embryonated eggs were performed. Two groups of outbred albino mice were infected with approximately 3500 B. transfuga infective eggs subcutaneously (s.c.) or intraperitoneally (i.p.). B. transfuga larvae in the i.p. group rapidly hatched and migrated to the intestine, liver, lungs, brain and carcass. Subcutaneous inoculation of eggs was followed also by migration of hatched larvae in the examined organs. In the s.c. mice, extensive encapsulating reactions involving the subcutaneous tissues and carcass, and containing large numbers of hatched eggs and free motile larvae, were found at the sites of inoculation. Some differences in the migratory behaviour were observed between the two groups. It is shown that B. transfuga infective larvae are able to hatch and migrate in tissues of mice, and tend to settle and/or to be trapped in the intestinal wall and muscles, even after parenteral inoculations of embryonated eggs. These results could provide basic data for further investigations on the migratory pathways of B. transfuga larvae or to perform immunological and therapeutical studies.