The 10 kDa protein of Taenia solium metacestodes shows genus specific antigenicity

Genus specific antigenicity of the 10 kDa protein in cyst fluid (CF) of Taenia solium metacestodes was demonstrated by comparative immunoblot analysis. When CFs from taeniid metacestodes of T. saginata, T. solium, T. taeniaeformis and T. crassiceps were probed with specific monoclonal antibody (mAb) raised against 150 kDa protein of T. solium metacestodes, specific antibody reactions were observed in 7 and 10 kDa proteins of T. solium and in 7/8 kDa of T. saginata, T. taeniaeformis and T. crassiceps. The mAb did not react with any protein in hydatid fluid of Echinococcus granulosus and E. multilocularis. This result revealed that the 10 kDa peptide of T. solium metacestodes and its equivalent proteins of different Taenia metacestodes are genus specific antigens that are shared among different Taenia species.     

Seasonal prevalence of Taenia taeniaeformis: relationship to age, sex, reproduction and abundance of an intermediate host (Peromyscus maniculatus).

Livers from 4,501 deer mice (Peromyscus maniculatus) collected from a weedy habitat in northeastern California during 48 consecutive monthly samplings were examined microscopically for Taenia taeniaeformis larva. Although there were pronounced seasonal fluctuations in host density, there were no significant annual or season-related differences in cestode intensities in adult deer mice. There were no significant differences in prevalences associated with sex of the host, nor were there significant changes in level of reproduction noted between infected and non-infected hosts. There were, however, significant differences in prevalences between young (1.2%) and adult (4.2%) hosts. Plausible mechanisms for this age-related difference in prevalence rates include (1) differential susceptibility due to the activity pattern of adult mice and/or (2) passive immunity in neonates as a result of colostrum- and/or transplacentally-transferred immunoglobulins and (3) capture of subadult animals before they had completed the period of highest susceptibility to T. taeniaeformis. Density of larvae per mouse liver was determined during a 21 mo consecutive period. The intensity of T. taeniaeformis larvae was not significantly different between the sexes of the adult mice. The larval stage showed an overdispersion pattern within the adult population. These results suggest that determinations of T. taeniaeformis abundances can be accurately made, at least in this P. maniculatus population, at any time of the year provided adjustment is made for the relative age structure of the host population.     

Comparison of biochemical and immunochemical properties of myosin II in taeniid parasites

Type II myosins are highly conserved proteins, though differences have been observed among organisms, mainly in the filamentous region. Myosin isoforms have been identified in Taenia solium, a helminth parasite of public health importance in many developing countries. These isoforms are probably associated with the physiological requirements of each developmental stage of the parasite. In this paper we extend the characterization of myosin to several other Taenia species. Type II myosins were purified from the larvae (cysticerci) of Taenia solium, T. taeniaeformis and T. crassiceps and the adult stages of T. solium, T. taeniaeformis and T. saginata. Rabbit polyclonal antibodies against some of these myosins were specific at high dilutions but cross-reacted at low dilutions. ATPase activity was evaluated and kinetic values were calculated for each myosin. Homologous actin-myosin interactions increased both the affinity of myosin for ATP and the hydrolysis rate. The results indicate immunological and biochemical differences among taeniid myosins. This variability suggests that different isoforms are found not only in different taeniid species but also at different developmental stages. Further characterization of myosin isoforms should include determination of their amino acid composition.     

Proteomic characterisation of Echinococcus granulosus hydatid cyst fluid from sheep, cattle and humans

The hydatid cyst fluid (HCF) of Echinococcus granulosus is a complex biological mixture containing a wide range of proteins of both parasite and host origin. Using a combination of in- and off-gel protein fractionation techniques and tandem mass spectrometry 130 HCF proteins were identified from fertile cysts of sheep and human origin and infertile cysts from cattle. Forty-eight proteins were of parasite origin including Antigen 5 and Antigen B--the most abundant parasite proteins, thioredoxin, low-density lipoprotein receptors, cyclophilin and ferritin. Across the three host species the identified HCF proteins were broadly similar although, based on spectral counts, three proteins, including an antigen B isoform, were more abundant in sheep HCF compared with the fluids of cattle and human origin. Eighty-two host proteins were identified in HCF from the three species. Host plasma proteins were the most abundant, although approximately thirty of the host proteins that were identified are not considered constituents of plasma. The identification of parasite heat shock proteins and annexin A13 exclusively in infertile cysts, along with an increased spectral count for cathepsin B, supports the hypothesis of increased cellular stress and apoptosis as the cause of their infertility.     

Incorporation of calcium into the soft tissues and calcareous corpuscles of larval Taenia taeniaeformis.

Larval Taenia taeniaeformis in vivo accumulates 45Ca2+ in soft tissues and calcareous corpuscles. Radioactivity was demonstrable in the corpuscles six months after a single dose of 45Ca2+ was administered to the host by means of a stomach tube. Ca2+ also was taken up by isolated larvae. Accumulation in vitro was more rapid then in vivo and was correlated with the external Ca2+ concentration. Temperature variation, oxygen availability, and metabolic inhibitors had little effect on the Ca2+ uptake, indicating that active transport of Ca2+ is unlikely in this parasite. Variations in the external Pi concentrations had no effect on Ca2+ accumulation or on its distribution. Addition of 5% CO2 increased the uptake of Ca2+ by the calcareous corpuscles under anaerobic conditions. Radioactivity from NaH14CO3 also was accumulated in soft tissues and corpuscles of T. taeniaeformis. Assuming that the 14C taken up by the corpuscles was in the form of 14CO3(2-), the ratio of Ca2+ to CO3(2-) accumulation in the corpuscles approximates the ratio of these constituents in dolomite: CaMg(CO3)2.     

Comparative cytotoxicity of secondary hydatid cysts, protoscoleces, and in vitro developed microcysts of Echinococcus granulosus.

Infection with the metacestode of Echinococcus granulosus is characterized by a concomitant immunity. Survival of established and developing hydatid cysts in the intermediate host implies a mechanism to modulate its immunological reactions. In order to investigate this mechanism, secondary hydatid cysts were isolated from intraperitoneally infected laboratory white mice (strain NMRI) 12 months p.i. A number of hydatid cysts were freed from the surrounding host adventitial tissue. Monolayer cultures of non-stimulated peritoneal macrophages of NMRI mice were prepared and incubated in the presence of the hydatid cysts. By means of a trypan blue exclusion test and by measuring the incorporation of tritium labelled uridine, it was found that the presence of hydatid cysts reduced the viability of the macrophages in vitro. Toxic substances are probably secreted since the medium of cultured hydatid cysts also displayed cytotoxic activity. Hydatid cysts with adventitia, as well as culture medium of those cysts, were less toxic. When toxins, partially purified from hydatid cyst fluid, were previously incubated on a collagen coated surface, a reduced level of toxicity was found, suggesting that collagen of the host adventitia may play a role in controlling the liberation of toxins by the hydatid cyst. Virtually no toxicity was exerted by protoscoleces or by the medium of cultured protoscoleces, in contrast to in vitro vesiculated protoscoleces (so called microcysts). The results reveal a novel feature of hydatid cysts that may play a role in the survival of the parasite in the immunized host.     

Studies on the mechanism of long term survival of Taenia taeniaeformis in rats.

An attempt was made to determine if blocking antibody is involved in protecting cysticerci of Taenia taeniaeformis against a host immune response. Immunoflourescence microscopy confirmed that host antibody is presnet on the parasite surface within the capsule. To test if the larvae can still survive after such a coat of blocking antibody is removed, the larvae were trysinised and then implanted into recipients. The results indicate that blocking antibody could be involved in the survival of 1 year old established larvae. Untrypsinised larvae were normal 14 days after implantation into control or immunised rats. Trypsinised larvae implanted in control rats were alive but showed on intense cell adherence on their surface. On the other hand, trypsinised larvae implanted into immunised rats were dead and completely encapsulated. However, experiments with 1 month old larvae were inconclusive.     

Lytic effects of normal serum on isolated postonchospheral and metacestode stages of Taenia taeniaeformis

Postonchospheral stages of Taenia taeniaeformis liberated from rat livers by enzymatic digestion at 1 to 10 days postinfection (DPI) and metacestodes dissected from infected livers at 22, 34, and 69 DPI were exposed in vitro to immune rat serum (IRS) and to normal serum from rats (NRS), human beings (NHS), or guinea pigs (NGS). The onset of rapid and destructive tegumental changes in all organisms exposed to any of the sera was demonstrated to be complement-dependent because the reaction was: (a) inhibited by treatment of serum at 56 C for 30 min; (b) inhibited by prior incubation of serum with zymosan or with complement-fixing, soluble products derived from larvae of T. taeniaeformis maintained in vitro (IVP); and (c) abolished by the addition of EDTA. Lytic effects occurred on exposure to agammaglobulinemic sheep serum, and lysis in the presence of IRS and NRS was shown to result in consumption of available hemolytic complement. Surface changes consisted of vesiculation in the microvillar or microthrix layers followed by sloughing of the tegument, eventually leading to collapse of the cystic bladder and cessation of flame cell activity, or, in the case of early postonchospheral forms, complete disintegration of the organism. When IVP was added to NHS, reduction of hemolytic complement activity was associated with the electrophoretic conversion of C3, and Factor B, but there was little or no consumption of C1. The observations support the hypothesis that complement-mediated effector mechanisms must be counteracted to ensure survival of parasites in vivo, and that the capacity for release of soluble nonspecific complement-fixing factors by taeniid larvae may have an important role to play in this process.     

Biochemical profiles of hydatid cyst fluids of Echinococcus granulosus of human and animal origin in Libya.

A comparative study on the biochemical parameters in hydatid cyst fluids of sheep, goats, camels, cattle and human cystic forms of Echinococcus granulosus has been made in Libya. Quantitative variations in the levels of sodium, potassium, calcium, cholesterol, glucose, urea, creatinine and gamma glutamyl transpeptidase (gamma GT) were found in the cystic fluids of different host origins although these differences were statistically insignificant compared with the hydatid fluids of sheep. However, the concentration of triglycerides and proteins were significantly elevated in the cyst fluids of sheep compared with the other fluids studied. Similarities in the biochemical composition of different hydatid cyst fluids suggest the existence of sheep strains of E. granulosus in human and other domestic animal intermediate hosts in Libya.     

Uptake and secretion of host proteins by Taenia crassiceps metacestodes

Although the presence of intact host proteins in the cyst fluid of cyclophyllidean metacestodes has been well documented, the underlying reason for protein uptake is poorly understood. To investigate this discrepancy, both the cyst fluid (CF) and excreted/secreted (E/S) proteins were collected in vitro from Taenia crassiceps metacestodes 16 wk postinfection in Balb/cJ female mice. The CF and E/S were subsequently immunoblotted using rabbit anti-mouse whole serum antibodies as a probe. The results show that whole host proteins were not only internalized by metacestodes but also secreted as well. The predominant secreted host protein was 66 kDa and was confirmed to be mouse serum albumin. The amount of secreted albumin decreased daily, whereas the concentration of albumin in the cyst fluid remained consistent. This suggests that the secretion of albumin is a coordinated function rather than a random event. It is probable that albumin cycling may be an evolved mechanism providing multiple benefits for the larvae, including osmoregulation and protection from innate immune responses.     

Taenia taeniaeformis: immunoprecipitation analysis of the protein antigens of oncospheres and larvae

Biosynthetically or exogenously labeled proteins and immunoprecipitated protein antigens of established 28-day-old larvae of Taenia taeniaeformis were compared with proteins and antigens of infective oncospheres using single and two-dimensional gel electrophoresis. Immunoprecipitation was carried out using sera from infected mice and mouse antisera raised to larvae or oncospheres, and emphasis was placed on identifying antigens common to both oncospheres and larvae. Two major larval antigens of Mr 40,000 and 200,000, designated Tt40 and Tt200, are common to somatic larval preparations and oncospheres. Additionally, two major oncosphere antigens of Mr 55,000 and 60,000, designated Tt55 and Tt60, are also present in larval excretory and secretory (i.e., ES or exoantigen) products. Information obtained from these immunoprecipitation analyses will facilitate isolation and production of common as well as stage-specific protein antigens in the development of defined-antigen vaccines in this model system of cysticercosis.     

Echinococcus granulosus: development of resistance to albendazole in an animal model

Gerbils with well developed peritoneal cysts of Echinococcus granulosus were randomized to albendazole 50 mg/kg/day or untreated control. Treated animals had less disease at post mortem after 3 months of treatment. Cysts were then taken from both albendazole-treated and control animals and cultured in vitro either with or without albendazole sulphoxide (Alb Sx) 500 micrograms/L for 14 days. Viability of cysts was then established by implantation of whole cysts into gerbils. Whilst naive cysts were affected by Alb Sx (only 2 cysts developed/gerbil) cysts from animals treated with albendazole were not sensitive to further therapy (6.4 cysts/gerbil).     

Infrapopulation dynamics of a wild strain of Taenia crassiceps (WFU) (Cestoda: Taeniidae) in BALB/cJ mice

Taenia crassiceps cysticerci form large infrapopulations that persist in the tissues of their rodent hosts. Early infrapopulation growth appears inhibited and is followed by rapid increases that appear not to be controlled by the host immune response. This investigation was undertaken to examine the infrapopulation growth dynamics of a normally developing strain (WFU) of T. crassiceps during a 60-day primary intraperitoneal (i.p.) infection. Three, 6, 9, 14, 28, and 60 days after i.p. inoculation of 5 cysticerci, mice were killed, and the numbers of larvae, developmental stage, and buds per larva were recorded. Larval infrapopulation abundance increased exponentially beginning on day 6 postinoculation (PI), indicating an initial lag in reproduction. A stage-structured exponential growth model, assuming no mortality, fits the larval infrapopulation dynamics in terms of the numbers of larvae in reproductive and nonreproductive stages, indicating that cysticerci evade or suppress (or both) host immune mechanisms that are parasite restrictive after the first week of infection.     

Studies on immune responses to larval cestodes in mice. Increased susceptibility of certain mouse strains and hypothymic mice to Taenia taeniaeformis and analysis of passive transfer of resistance with serum.

Various inbred strains of mice vary markedly in their susceptibility to the larvae of the cestode, Taenia taeniaeformis. Males are generally more susceptible than females and the most susceptible common inbred mouse strains are those which are deficient in C5 and/or C4 components of complement. However, no genetic evidence is yet available to implicate loci controlling complement levels in susceptibility/resistance, and multiple genetic factors appear to be operative. Hypothymic, nu/nu ("nude") mice of the relatively resistant mouse strain, BALB/c, are highly susceptible in that cystic larvae in the liver develop in large numbers and more rapidly than in intact BALB/c.nu/+litter-mates. Cyclophosphamide pretreatment also increases the susceptibility of relatively resistant strains of mice in terms of both the number and size of liver cysts. Hypothymic and intact mice can be protected, absolutely, by an injection of serum from infected intact mice, provided the serum is given to recipient mice close to the time of oral egg administration. The protective activity of immune serum is absorbed totally by staphylococcal protein A-Sepharose columns and can be abolished by treatment of recipients with cobra venom factor. Cyst fluid from established larvae facilitates the activity of subhaemolytic amounts of guinea pig complement in a standard direct PFC assay. The data suggest that complement-fixing antibodies are responsible for inhibition of establishing larvae in mice and that one method of protection for established cystic larvae involves the alteration of host complement activity within the cyst.     

Taenia crassiceps: experimental model of intraocular cysticercosis

An experimental model of Taenia crassiceps intraocular cysticercosis was developed in rabbits. The objectives of this study were to analyze the pathophysiology of this parasitic infection and to evaluate the humoral immune response. Cysticerci, inoculated in the anterior chamber of the eye, were able to grow; no inflammatory changes in the eye or anticysticercus antibodies in serum or in aqueous humor were detected during the 12-day period. In contrast, rabbits that had previously been either infected intraperitoneally with living T. crassiceps cysts or immunized intramuscularly with T. crassiceps antigenic extract developed an intense inflammatory reaction in the eye and high levels of antibodies were detected in serum and aqueous humor even before the intraocular inoculation of parasites. Furthermore, intraocular cysticerci showed minimal growth and some were eliminated. These findings support the concept that the eye is an immunologically privileged site in the nonimmunized host and the importance of the immune response in the elimination of this parasitic infection.     

The growth of the metacestodes of Taenia crassiceps in white mice.

The effect of the sex of the intermediate host on the growth and development of the metacestodes of Taenia crassiceps has been investigated. Three different strains of mice were used, the C R S, L A C A and C F L P strains and two strains of metacestode, the Toi and the E R S strains. The results show that matacestodes of T. crassiceps grow and multiply more rapidly in female than in male mice, although both sexes are equally susceptible to the infection. The origin of the parasite, i.e., from a rat or mouse host, affects the parasite growth.     

Septate-like junctions between cells of the immune system in vivo.

Septate-like junctions between lymphocytes, lymphocytes and macrophages, and macrophages were observed in cellular capsules surrounding larvae of the tapeworm Taenia crassiceps after intraperitoneal inoculation into immunized mice. The morphology of the junctions found in vivo is similar to that reported for septate-like junctions between cells of the immune system in vitro. These findings support the possibility that septate-like junctions may be involved in functional interactions between cells of the immune system both in vivo and in vitro.     

Parasitism of Lacanobia oleracea (lepidoptera) by the ectoparasitic wasp, Eulophus pennicornis, disrupts the cytoskeleton of host haemocytes and suppresses encapsulation in vivo

Parasitism of Lacanobia oleracea larvae by the ectoparasitic wasp Eulophus pennicornis suppressed host haemocyte-mediated encapsulation of Sephadex DEAE A-25 beads in vivo. Beads dissected out of parasitized larvae had fewer haemocytes associated with them. Moreover, those haemocytes that were associated with the beads tended to retain a rounded configuration and rarely flattened. Similar results were obtained using in vitro encapsulation assays. SDS PAGE indicated that for parasitized and PBS injected larvae, there were some differences in the plasma proteins that bound to Sephadex DEAE A-25 beads, suggesting that parasitism-mediated changes to host plasma proteins might contribute to the differences in the encapsulation response occurring in these larvae. However, in vitro encapsulation assays using beads that had been pre-incubated in plasma from parasitized and unparasitized larvae, demonstrated that major differences in the extent of encapsulation did not occur. These results, plus in vitro haemocyte attachment and spreading assays, suggest that parasitism-mediated suppression of encapsulation is primarily due to reductions in the ability of host haemocytes to attach to (i.e., recognize) and flatten over non-self surfaces and other haemocytes. This proposal is corroborated by staining of actin in the haemocyte cytoskeleton by FITC-labelled phalloidin, which indicated that parasitism disrupts the formation of stress fibers and focal adhesions in plasmatocytes. By contrast, experimental injection of adult female wasp venom into unparasitized L. oleracea larvae had no significant effect on in vivo encapsulation responses or the haemocyte cytoskeleton. Arch. Insect Biochem. Physiol. 49:108-124, 2002. Published 2002 Wiley-Liss, Inc.     

Molecular mechanisms involved in the differential effects of sex steroids on the reproduction and infectivity of Taenia crassiceps

The in vitro exposure of Taenia crassiceps cysticerci to 17-beta estradiol (E2) and progesterone (P4) stimulated their reproduction and infectivity. Testosterone (T4) and dihydrotestosterone (DHT) inhibited their reproduction and reduced their motility and infectivity. E2 and P4 increased, whereas T4 and DHT reduced, the expression of parasite c-fos and c-jun and DNA synthesis. In vitro exposure of cysticerci to sex steroids before their inoculation into recipient noninfected mice resulted in large parasite loads when pretreated with E2 and P4 and in smaller loads when pretreated with T4 and DHT To determine the possible molecular mechanisms by which sex steroids affect T. crassiceps, sex steroid receptors were amplified. Taenia crassiceps expressed estrogen receptors (both alpha and beta isoforms) and androgen receptors but no P4 receptors. These results demonstrate that sex steroids act directly on parasite reproduction by binding to a classic and specific sex steroid receptor on the parasite. The differential response of cysticerci to sex steroids may also be involved in their ability to grow faster in the murine female or feminized male host. This is the first report of direct sex steroid effects on the parasite possibly through sex steroid receptors in the cysticerci.     

Diversity of the helminth community of the Pampean grassland mouse (Akodon azarae) on poultry farms in central Argentina

This paper describes the helminth community of the Pampean grassland mouse (Akodon azarae) inhabiting poultry farms in central Argentina. Winter diversity (season of high rodent abundance) has been compared to spring diversity (season of low rodent abundance). Species richness was seven in both seasons: five nematodes (Syphacia carlitosi, Stilestrongylus spp., Trichuris laevitestis, Pterygodermatites (Paucipectines) azarai and Protospirura numidica criceticola) and two cestodes (adults of Cyclophyllidea and Taenia taeniaeformis hepatic cysts). No difference in richness was detected between host sexes in each season or among host age classes. However, the helminth community showed 67% similarity between winter and spring, with diversity being significantly higher in spring (H = 0.873) than in winter (H = 0.546; P < 0.0005). This could be attributed to different factors, such as host abundance, host diet or environmental factors, that affect the transmission of each species differently. On the other hand, Stilestrongylus spp. and S. carlitosi showed higher dominance and intensity in both seasons compared to their cohabiting species, P. (P.) azarai and T. laevitestis, respectively. The lower values of the latter two species may be related to a crowding effect due to their large body sizes. This is the first report of cestodes in A. azarae. The finding of T. taeniaeformis strobilocerci could be important in the epidemiology of parasitosis in domestic animals of the farms.