Persistent infection of Myxobolus cerebralis, the causative agent of salmonid whirling disease, in Tubifex tubifex.

The objective of this study was to quantify and determine the periodicity in the release of the triactinomyxon (TAM) stage of Myxobolus cerebralis, the causative agent of salmonid whirling disease, by its aquatic oligochaete host Tubifex tubifex. For this, 24 individual T. tubifex (infected as a group at 15 C) were examined daily for the release of M. cerebralis TAMs, and the number of waterborne TAMs released by each worm was quantified. The duration of the infection in these worms was also monitored using a polymerase chain reaction (PCR) diagnostic test. TAMs were first released 74 days postexposure (PE) and continued to be released until 132 days PE. During this period, each worm released on average, 1.5 x 10(3) waterborne TAMs 12 times; however, no pattern or periodicity was noted. The results of the PCR diagnostic tests conducted at 5, 7, 9, and 15 mo PE were positive, and the persistent infection was confirmed at 606 days PE (approximately 20 mo) when the remaining worms began releasing TAMs again. Similar results were observed in naturally infected T. tubifex, indicating that these worms remain infected for the duration of their natural lifespan and are capable of shedding viable TAMs, in temporally separate periods. These findings open the possibility of a seasonal periodicity in TAM release by T. tubifex.

Recombinant human interleukin-1-mediated killing of Schistosoma mansoni primary sporocysts in Biomphalaria glabrata.

Previous work has indicated that injection of recombinant-human interleukin (rhIL)-1beta in Schistosoma mansoni-infected M-line Biomphalaria glabrata resulted in a significant reduction in the number of cercariae shed. The purpose of the present work was to determine if primary sporocysts were killed following rhIL-1beta injection in susceptible snails and, if so, to determine if killing was the direct result of hemocyte activity. Counting of primary sporocysts indicated a 50% reduction in the number surviving at 3 days PE in snails from 2 susceptible strains following injection. Histological analysis indicated that killing occurred with little-to-no observable hemocyte/parasite contact, whereas short-term culture of primary sporocysts with cell-free plasma (hemolymph) from injected snails rapidly initiated killing in vitro. Because levels of a snail IL-1-like molecule (SnaIL-1) drop significantly following schistosome exposure in M-line snails, because resistant snails maintain higher SnaIL-1 levels following infection, and because rhIL-1beta upregulates hemocyte cytotoxic mechanisms, these data support the contention that SnaIL-1 plays a role in determining resistance in B. glabrata. These data also indicate that schistosome death may be separated from parasite encapsulation by hemocytes and that an as yet unidentified humoral killing mechanism/factor may exist in B. glabrata. Lastly, these data further support the hypothesis that cytokine-like molecules are important, functionally conserved immunodefense mediators in both vertebrates and invertebrates.

Prevalence and transmission of cercariae causing schistosome dermatitis in Flathead Lake, Montana.

Numerous studies have been conducted on swimmer's itch, but very few have been in Montana and none on Flathead Lake, the largest, natural freshwater lake in the western United States. We conducted a study to determine the prevalence of hosts transmitting cercariae causing swimmer's itch in this lake. Hosts for this life cycle were determined by direct observation of fresh waterfowl fecal material for the presence of miracidia, and snails for the presence of cercariae. Swimmer's itch-producing cercariae were verified directly by placing various species on the arms of human volunteers and waiting for a reaction. Results of the study were further substantiated using a controlled experiment in which snails were individually infected with miracidia from the suspected waterfowl host and then checked for infection after a 6-wk incubation period. Our findings show that the common merganser (Mergus merganser) and the snail Stagnicola elrodi were natural hosts of the swimmer's itch parasite (Trichobiharzia ocellata) with prevalences of 84% and 2.0%, respectively. To our knowledge this is the first documented case of S. elrodi transmitting the swimmer's itch cercariae.

Hemocytes of schistosome-resistant and -susceptible Biomphalaria glabrata recognize different antigens on the surface of Schistosoma mansoni sporocysts.

A cytoadherence assay was used to determine whether antibodies to plasma and hemocyte components of Schistosoma mansoni-susceptible (M-line) and -resistant (10-R2, 13-16-R1) strains of Biomphalaria glabrata affected attachment of hemocytes to chemically fixed schistosome sporocysts differentially. Experiments used purified, intact IgG and purified Fab fragments of each antibody. Indirect fluorescent antibody tests confirmed that the intact purified IgG to plasma and hemocytes from the 3 strains of snails bound to sporocysts. There was no qualitative difference in fluorescence among any of the antibodies to snail components, although the various antibodies affected adherence of hemocytes to the parasite differentially. Adherence assays revealed that antibodies to plasma components inhibited binding of hemocytes from the snail strain to which the antibody was generated. Additionally, antibody to plasma from resistant strains of B. glabrata inhibited binding of hemocytes from the homologous and heterologous resistant strains but not hemocytes from susceptible snails. Antibody to hemocytes from M-line snails did not inhibit binding of hemocytes from any of the snail strains. Since results of assays using intact IgG correlated well with assays using their Fab fragment counterparts, it was concluded that hemocytes from the 3 snail strains utilize specific antigen-binding sites on sporocysts. Results of these assays also indicate that, with regard to the mechanism of hemocyte binding to sporocysts, M-line and 13-16-R1 snails are more dissimilar than M-line and 10-R2 or 10-R2 and 13-16-R1 B. glabrata.

Schistosoma mansoni: interleukin-1 increases phagocytosis and superoxide production by hemocytes and decreases output of cercariae in schistosome-susceptible Biomphalaria glabrata.

Decreases in the number of Schistosoma mansoni cercariae released from susceptible M-line Biomphalaria glabrata were detected following injection with the recombinant human cytokine, interleukin-1. No differences in either the time post-exposure at which shedding began or the percentage of snails shedding cercariae were detected between interleukin-1 injected, heat-inactivated interleukin-1 injected, or sham injected controls. However, sham injected and heat-inactivated interleukin-1 injected snails maintained significantly higher (approximately three-fold) levels of cercarial production compared to interleukin-1 injected snails over 8 weeks of cercarial shedding. Injection of interleukin-1 into schistosome-susceptible (M-line) and resistant (13-16-R1) strains of B. glabrata increased hemocyte phagocytosis of target particles and phagocytosis stimulated O2- production in both snail strains at 24 hr postexposure to the parasite. Resistant 13-16-R1 snails maintained, on average, 2.4 times the number of O2- producing phagocytic cells than did M-line susceptible snails, indicating that the incomplete abrogation of cercarial shedding in M-line snails may be due to an inadequate number of activated circulating effector cells in these snails. These data strongly support the contention that the evolutionarily conserved cytokine, interleukin-1, or a molecule in snail plasma with interleukin-1-like immunospecificity, biological activity, and function plays a significant role in the maintenance of susceptibility or resistance to S. mansoni infection in B. glabrata. Finally, these data also supply evidence for the evolutionary conservation of the function and role of interleukin-1, O2-, and antioxidant defense mechanisms in this host-parasite relationship.

Interleukin 1 activity in haemolymph from strains of the snail Biomphalaria glabrata varying in susceptibility to the human blood fluke, Schistosoma mansoni: presence, differential expression, and biological function.

Cytokines control many of the steps in the complex pathways of immune and inflammatory responses in mammals. Recent reports also indicate that some invertebrates may produce cytokines such as interleukin 1 (IL-1). Certain strains of the snail, Biomphalaria glabrata (intermediate host for the human blood fluke, Schistosoma mansoni), possess a soluble plasma factor that stimulates the haemocyte-mediated killing of larval schistosomes, making them resistant to infection. In this study, we have sought to determine whether these snails possessed IL-1 in their plasma, and whether this cytokine was associated with resistance of B. glabrata to S. mansoni. Plasma from susceptible (M-line) and resistant (10-R2, 13-16-R1) strains of B. glabrata that had been unexposed or exposed to S. mansoni were tested for the presence of IL-1-like activity. Experiments employing both a bioassay and an immunoassay indicated that an IL-1-like molecule was present, in varying quantities, among the snail strains. Further, plasma IL-1 levels were significantly affected by exposure to S. mansoni, with levels dropping in M-line and 10-R2 snails, but increasing in the 13-16-R1 strain. However, both resistant strains maintained significantly higher IL-1 levels than M-line snails. Recombinant, human IL-1 (rhIL-1) was shown to prime haemocytes from both resistant snail strains for superoxide production, but had no effect on haemocytes from susceptible B. glabrata. Moreover, the addition of an IL-1 receptor antagonist protein (IRAP) eliminated this priming effect. Priming with rhIL-1 and/or IRAP had no effect on phagocytosis rates in any of the snail strains tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of hemocyte components from Schistosoma mansoni (Trematoda)-susceptible and resistant Biomphalaria glabrata (Gastropoda) that cross-react with larval schistosome surface proteins.

1. Cross-reactive antigens between larval Schistosoma mansoni and hemocytes from schistosome-resistant and susceptible strains of Biomphalaria glabrata were identified and compared using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting and surface radioiodination. 2. Four sporocyst (larval S. mansoni) surface antigens (27, 39, 40 and 70 kDa) reacted with antibodies to hemocytes from resistant snails whereas only one surface antigen (70 kDa) was recognized by antibodies to hemocytes from susceptible B. glabrata. 3. The wider distribution and greater frequency of cross-reactive antigens between resistant hemocytes and sporocysts suggests that such cross-reactivity may be involved in the hemocyte-mediated killing of larval schistosomes that occurs in this host-parasite system.

Identification, comparison and partial characterization of glycoproteins in the hemolymph of Schistosoma mansoni (Trematoda)-susceptible and resistant Biomphalaria glabrata (Gastropoda).

1. Glycoproteins in the plasma (cell-free hemolymph) of Schistosoma mansoni-resistant and susceptible strains of Biomphalaria glabrata were identified, characterized and compared using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroblotting and a series of peroxidase-labeled lectins with different sugar specificities. 2. Schistosome-resistant snails possessed at least three glycoproteins (70, 116, 205 kDa) that were not present in plasma from susceptible snails. 3. Sporocysts (larval S. mansoni) preincubated in plasma from schistosome-resistant or susceptible B. glabrata adsorbed certain glycoproteins to their surface and some altered the binding of lectin probes to the parasite. 4. This study indicates that glycoproteins in the plasma of B. glabrata are associated with S. mansoni-susceptibility and resistance.

Differential binding of hemolymph proteins from schistosome-resistant and -susceptible Biomphalaria glabrata to Schistosoma mansoni sporocysts.

Humoral factors have been associated with resistance of Biomphalaria glabrata to infection by Schistosoma mansoni. The goal of this study was to determine which serum (cell-free hemolymph) proteins bind to the surface of S. mansoni sporocysts. For this, 125I-labeled serum from schistosome-resistant (10-R2) and -susceptible (M-line) B. glabrata was incubated with sporocysts, washed, and then subjected to SDS-PAGE and autoradiography. Other samples examined included radiolabeled 10-R2 and M-line serum, sporocysts incubated with unlabeled serum followed by incubation with radiolabeled serum, and radiolabeled sporocysts. Results indicated that many polypeptides in the serum from both strains of B. glabrata were radiolabeled. Dominating both profiles were bands in the 90-210-kDa range. However, some differences between the serum of the 2 snail strains were observed with M-line serum having several radiolabeled polypeptides in the 31-40- and 66-85-kDa range that were absent in serum from 10-R2 B. glabrata. When sporocysts were incubated with radiolabeled serum, 3 polypeptides (116, 180, 210 kDa) from both snail strains bound to the surface of the parasite. Further, a 55-kDa polypeptide bound to sporocysts incubated with 10-R2 serum but did not bind to those parasites incubated with M-line serum. Preincubation of sporocysts with unlabeled serum prior to incubation with radiolabeled serum significantly inhibited the uptake of radiolabeled proteins. This differential binding of serum polypeptides from different strains of B. glabrata may be important in determining resistance or susceptibility of the snail to larval schistosome infection.

Structural analysis of hemolymph proteins from Schistosoma mansoni (Trematoda)-susceptible and resistant Biomphalaria glabrata (Gastropoda).

1. Five different molecular weight polypeptides from serum (cell-free hemolymph) of Schistosoma mansoni-resistant and susceptible strains of Biomphalaria glabrata, were examined by two-dimensional 125I-peptide mapping and high performance liquid chromatography (HPLC). 2. Peptide mapping indicated that all five radiolabeled polypeptides within and between the two snail strains had similar migration patterns when cleaved with pepsin or alpha-chymotrypsin, thus revealing a shared structural homology. All peptides chosen for analysis appeared to be structurally similar to the 160 kDa hemoglobin molecule. 3. Separations of the radiolabeled enzyme digests by HPLC confirmed results seen in the mapping experiments since all chromatograms had similar elution patterns. 4. Minor differences in the peptide maps and chromatograms within and between snail strains may be due to quantitative differences in the amount of protein present and/or variations in the primary amino acid sequences of the proteins chosen for analysis.

Comparison of haemolymph proteins from Schistosoma mansoni (Trematoda)-susceptible and resistant Biomphalaria glabrata (Gastropoda).

1. Whole and haemoglobin (Hb)-depleted serum fractions from Schistosoma mansoni-resistant (10-R2) and schistosome-susceptible (PR albino, M-line) strains of Biomphalaria glabrata were studied using sodium dodecyl sulphate-polyacrylamide gel electrophoresis, immunoblotting and protein assays. 2. Whole serum of the PR albino strain had more total protein (11.4 +/- 1.9 mg protein/ml) than the 10-R2 strain (7.8 +/- 3.0 mg protein/ml). 3. There are specific differences in the polypeptide profiles of the Hb-depleted fractions between the 10-R2 and PR albino strains of B. glabrata. 4. Antibodies against whole 10-R2 or PR albino serum reacted similarly when incubated with whole or Hb-depleted snail sera although differences in staining intensities were observed. 5. Antibodies against snail Hb reacted with many proteins in whole sera and Hb-depleted fractions from both B. glabrata strains indicating that they may be Hb-associated polypeptides. 6. Polypeptides in whole or Hb-depleted snail serum are modified by reduction indicating the presence of disulphide bonds.

Analysis of the interacting components between larval Schistosoma mansoni and schistosome-susceptible and resistant Biomphalaria glabrata.

Cell-free hemolymph (serum) and hemocytes from Schistosoma mansoni-susceptible (PR albino M-line) and resistant (10-R2) strains of Biomphalaria glabrata were compared by SDS-PAGE, immunoblotting and radioiodination. Whole serum of both snail strains is dominated by hemoglobin (Hb) (MW = 160 Kd). SDS-PAGE. of Hb-depleted serum indicated that the 10-R2 strain has dominant polypeptides in the 50 to 30 Kd range whereas PR albino snails have few low MW proteins. Antibodies raised to whole PR albino and 10-R2 serum, and the 160 Kd (Hb) band reacted similarly in immunoblot assays. Analysis of hemocytes revealed that 10-R2 snails have a surface-exposed protein at about 80 Kd which is not present on PR albino hemocytes. An examination of primary cultured sporocysts indicated the presence of four major surface proteins (40, 50, 55, 70 Kd) and two minor surface-exposed polypeptides (92, 170 Kd). Antibodies raised against live, intact sporocysts reacted almost exclusively with sporocyst-surface proteins when tested by immunoblotting.

Schistosoma mansoni: passive transfer of resistance by serum in the vector snail, Biomphalaria glabrata.

Passive transfer of natural resistance to Schistosoma mansoni (PR-1 strain) has been successfully accomplished in the snail intermediate host, Biomphalaria glabrata (PR albino, M-line strain). Injection of serum (cell-free hemolymph) from a naturally schistosome-resistant strain of B. glabrata (10-R2) into PR albino snails induced a complete protection from a primary infection with the parasite in 29 of 48 snails (60.4%). In comparison, inoculation of homologous PR albino serum or heterologous proteins (fetal calf serum) had no effect. Moreover, this protection could be induced 24 hr prior to, or 24 hr after, exposure to the parasite, although heating of 10-R2 serum to 70 C for 30 min destroyed its protective ability. When in vitro transformed sporocysts were preincubated in 10-R2 or PR albino serum and then were injected into susceptible snails, a high level of infection (88.5 and 83.3%, respectively) was produced in both groups. Thus, the 10-R2 serum factor does not appear to be mediating specific parasite recognition by host hemocytes. Alternatively, our results suggest that 10-R2 serum possesses a heat-labile factor which specifically activate B. glabrata hemocytes to encapsulate and destroy sporocysts whereas PR albino serum lacks this factor.

Lysosomal enzyme activities in susceptible and refractory strains of Biomphalaria glabrata during the course of infection with Schistosoma mansoni.

The distribution and abundance of the lysosomal enzyme markers, acid phosphatase (AP), peroxidase (PO), and nonspecific esterase (NE), within circulating blood cells (hemocytes) were examined in a schistosome-susceptible (PR albino M-line) and a resistant (10-R2) strain of Biomphalaria glabrata during the course of infection with Schistosoma mansoni. The dynamics of serum (cell-free hemolymph) AP activities and total hemocyte numbers in infected snails also were investigated. Hemocyte subpopulations, as determined by these enzyme markers, responded differently to parasite infection between snail strains. Generally, the hemocyte subpopulations within PR albino snails remained largely unchanged, whereas the same subpopulations in 10-R2 snails fluctuated considerably. The distribution of AP in the hemocytes of 10-R2 snails decreased by 1 hr postexposure (PE) to the parasite and remained low through 12 hr before increasing to control values at 24 hr and 2 wk PE. In comparison, PO activity increased by 1 hr PE and peaked at 12 hr before dropping to 0 hr values by 2 wk PE. The NE activity exhibited still another pattern with the percentage of NE-positive cells decreasing from 0 to 12 hr PE followed by a recovery to 0-hr values by 24 hr. The abundance of these hemocyte enzymes followed a similar pattern to that of their distribution, although some differences were observed. Serum AP values varied little in PR albino snails except for a significant increase at 2 wk PE, indicating a possible response to tissue damage resulting from migrating daughter sporocysts.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of antigenically distinct hemocyte subpopulations in Biomphalaria glabrata (Gastropoda) using monoclonal antibodies to surface membrane markers.

Five monoclonal antibodies (mABs) against surface antigens on circulating, glass-adherent hemocytes of the snail, Biomphalaria glabrata, were produced by somatic cell hybridization methods. Two mABs (IID2.6-Bg and IID4.8-Bg) are pan-hemocytic, reacting uniformly with epitopes shared by all adherent hemocytes. Determinants recognized by these mABs also are present in soluble form and appear to be associated with a hemoglobin-depleted ultracentrifuged fraction of snail hemolymph. Hybridoma-derived mABs IIC6.8-Bg and VB10.3-Bg recognize hemocyte surface epitopes expressed by only 50-60% of the adherent cell population. These mABs also are reactive with soluble hemolymph antigens but apparently recognize determinants which are different from the IID2.6-Bg and IID4.8-Bg reactive sites. Another antigenically distinct hemocyte subpopulation is recognized by mAB IID7.1-Bg. Epitopes that are reactive with this mAB differ from the previously described determinants by their asymmetrical distribution on the surface of positive cells and the absence of soluble antigenic components in hemolymph. Furthermore, unlike the other mABs, the prevalence of hemocytes staining with IID7.1-Bg antibodies differed between two strains of B. glabrata. Results of this study clearly demonstrate that circulating B. glabrata hemocytes, consisting of a single, predominant population of adherent cells, is composed of several distinct antigenic subpopulations based on the specific binding of anti-hemocyte mAB probes. Our successful application of hybridoma techniques to the study of molluscan hemocyte surface antigens underscores further the great potential usefulness of this method in analysing the molecular basis of hemocyte reactivity.