Abstract: Cucumber vein yellowing virus (CVYV) (genus Ipomovirus, family Potyviridae) and Cucurbit yellow stunting disorder virus (CYSDV) (genus Crinivirus, family Closteroviridae) are included in the EPPO A2 Action List of pathogens and have become a limiting factor of cucumber production in the southeast of Spain. Due to the importance of these two pathogens and the need to have robust tools that can be used in risk assessment procedures to predict future epidemics, a real-time RT-PCR method based on TaqMan® chemistry has been developed. This method is fast, sensitive, specific and easy to automate in plant health laboratories.
Abstract: Symptom expression and levels of the ipomovirus Cucumber vein yellowing virus (CVYV) and the crinivirus Cucurbit yellow stunting disorder virus (CYSDV) were compared in greenhouse cucumbers in single and mixed infections. Results were contrasted with those obtained for plants infected with the potyvirus Zucchini yellow mosaic virus(ZYMV) in single and in mixed infections with either CVYV or CYSDV. Cucumber showed leaf symptoms of each co-infecting virus, except for the combination of CYSDV with ZYMV, where the typical CYSDV-like symptoms of interveinal leaf yellowing were inconspicuous or absent. The progression of CVYV as quanti¿ed by real-time RT-PCR was similar in plants with single infections and in mixed infection with CYSDV between 15 and 60 days post-inoculation (dpi). However, CYSDV was detected at signi¿cantly enhanced levels in plants when co-infected with CVYV but not when co-infected with ZYMV. In the latter case, ZYMV levels were reduced when compared with single infections. During mixed infections of ZYMV and CVYV, the titre levels of the ipomovirus were signi¿cantly lower when compared with single infections. Cucumber had reduced plant height, internode length, dry weight and fruit yield, positively correlated with the titre levels of CVYV and not of CYSDV during mixed infections. It is concluded that co-infections with CVYV enhance the titre of CYSDV, which could have epidemiological signficance.
Abstract: Bean yellow disorder virus (BnYDV) was recently identified as the first crinivirus (family Closteroviridae) that infects members of the family Leguminosae. It was first observed during the autumn of 2003, causing heavy losses in French bean (Phaseolus vulgaris L.) grown commercially in Spain. The virus is transmitted by the
sweetpotato whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae) Q-biotype, and disease symptoms resemble nutritional disorders consisting of interveinal mottling and yellowing in leaves, combined with stiffness or brittleness, and are typically produced on the middle to lower parts of the plant. Transmission experiments showed that 50% and 100% of B. tabaci adults acquired the virus after a feeding period of 3 and 7 h, respectively.Viruliferous whiteflies infected 66% and 100% of P. vulgaris plants after a feeding period of 12 and
24 h, respectively. The transmission efficiency of single whiteflies was 37% and persistence of BnYDV in the vector lasted up to 2 weeks with a half-life of 9 days. BnYDV was transmitted to P. vulgaris, Pisum sativum L., Lens culinaris Medik., and Vicia faba L., but not to Vigna unguiculata L., Glycine max (L.) Merr., Cicer arietum L., and to crop species belonging to families of the Solanaceae and Cucurbitaceae. No virus was detected in field
samples collected from 30 different species from Boraginaceae, Asteraceae, Geraniaceae, Lamiaceae, Leguminosae, Malvaceae, Scrophulariaceae, Thymelaeaceae andVerbenaceae. The restricted host range and efficient management of crops regarding whitefly infestation may be key elements in the control of BnYDV.
Abstract: Zucchini squash is host to Cucurbit yellow stunting disorder virus (CYSDV), a member of the genus Crinivirus, and Cucumber vein yellowing virus (CVYV), a member of the genus Ipomovirus, both transmitted by the whitefly Bemisia tabaci. Field observations suggest the appearance of new symptoms observed on leaves of zucchini squash crops when both viruses were present. When infected during controlled experiments with CYSDV only, zucchini plants showed no obvious symptoms and the virus titer decreased between 15 and 45 days postinoculation (dpi), after which it was no longer detected. CVYV caused inconspicuous symptoms restricted to vein clearing on some of the apical leaves and the virus accumulated progressively between 15 and 60 dpi. Similar accumulations of virus followed single inoculations with the potyvirus Zucchini yellow mosaic virus (ZYMV) and plants showed severe stunting, leaf deformation, and mosaic yellowing. However, in mixed infections with CYSDV and CVYV, intermediate leaves showed chlorotic mottling which evolved later to rolling, brittleness, and complete yellowing of the leaf lamina, with exception of the veins. No consistent alteration of CVYV accumulation was detected but the amounts of CYSDV increased ≈100-fold and remained detectable at 60 dpi. Such synergistic effects on the titer of the crinivirus and symptom expression were not observed when co-infected with ZYMV.
Abstract: Epidemics of whitefly-transmitted Tomato chlorosis virus, Tomato yellow leaf curl Sardinia virus and Tomato yellow leaf curl virus have been present in the south east of Spain since the 1990s. A survey was performed in 40 greenhouses and nethouses during 2003 to establish the relationship between the disease incidence and the quality of greenhouse or nethouse coverings, providing a physical protection of crops against whiteflies. For tomato chlorosis virus disease (ToCD), the incidence correlated with the type of greenhouse cover and was most reduced under higher quality covers. Control of tomato yellow leaf curl disease (TYLCD) was achieved only for crops grown in the highest quality greenhouses. TYLCD incidence in tolerant tomatoes remained below 100% within the 5 months of sampling, despite the disease progress rate at the initial stage of the cultivation being higher than that of ToCD, which did reach 100% incidence in many greenhouses. Linear regression analysis showed that the development of ToCD and TYLCD in most of the greenhouses was best described by the monomolecular model and the Gompertz model, respectively. Tomato infectious chlorosis virus was not detected in parallel surveys carried out during this study, although it has been described previously in the area studied.
Abstract: The population structure and genetic diversity of Cucumber vein yellowing virus (CVYV) from Spain were estimated by analyses of partial nucleotide sequences of the P1-proteinase (P1-Pro), P3 protein (P3), and the coat protein (CP) coding regions. Analysis of 56 CVYV Spanish field isolates collected from 2001 to 2005 showed low genetic diversity (0.0026, 0.0013, and 0.0012 for the P1-Pro, P3, and CP regions, respectively). The ratio between nonsynonymous and synonymous substitutions was among the lowest found in a plant virus, indicating a strong negative selective pressure in the regions analyzed. Nonsynonymous nucleotide substitutions were only found within the P1-Pro regions, although these do not appear to have been selected with time. The results support the hypothesis that the Spanish CVYV population could derive from a single origin of recent introduction.
Abstract: Reverse transcription followed by real-time PCR assays based on TaqMan chemistry have been developed for the detection and quantification of Cucumber vein yellowing virus (CVYV) and Cucurbit yellow stunting disorder virus (CYSDV) in individual adults of the whitefly vector Bemisia tabaci. The method includes an internal control for the detection of a gene from B. tabaci to compensate for variations in extraction efficiency. The assays designed were used to estimate proportions of viruliferous whiteflies collected from commercial greenhouse-grown crops in Spain. In a significant number of whiteflies, both viruses were detected and their amounts were estimated. The assays could be used to assist risk assessment of CVYV and CYSDV which constitute limiting factors in cucurbit crops. They are also suited to investigating the epidemiology and plant-virus-vector relationships in these diseases.
Abstract: Amaranthus leaf mottle virus (AmLMV) was classified as a member of the genus Potyvirus on the basis of its particle morphology, serology, and biological properties (Casetta et al., 1986). Based on these properties, an Amaranthus viridis-infecting virus isolated in Spain, causing mottle and leaf blistering as well as reduced growth has been identified as AmLMV. The 3' terminal genomic region of this and a reference isolate from Italy has been sequenced and reveals a 95% nucleotide identity between the two isolates. The sequenced part comprises the coat protein with 281 amino acids and 315 nucleotides of the 3' untranslated region (UTR) preceding a polyadenylated tail. Pairwise comparisons and phylogenetic analysis of the nucleotide and deduced amino acid sequences of the CP and 3' UTR of the cloned cDNAs with those of other potyviruses shows that AmLMV is a distinct potyvirus closely related to Potato virus Y.
Abstract: Cucurbit yellow stunting disorder virus (CYSDV) has been present in greenhouse-grown cucumber in Spain since 1992. However, in the autumn of 2000 Cucumber vein yellowing virus (CVYV) was introduced, leading to mixed infections of both Bemisia tabaci-transmitted viruses. The temporal and spatial spread of disease symptoms were monitored in experimental plastic-covered greenhouses during six consecutive cucumber plantings from 2000 to 2002. Using linear regression analysis of 46 disease-progress curves, the Gompertz model best described the CYSDV epidemics in 2000, whereas the logistic model best described the development of CYSDV and CVYV epidemics in 2001 and 2002. The fitted models were used to calculate the amount of degree Celsius-days at half-maximum infection in the greenhouses (degrees D-0.5). After multiple regression analysis, 56% of the variation in degrees D-0.5 of CYSDV was related to the numbers of whiteflies infesting the cucumber crops, and was independent of the mean temperatures in the greenhouses. In contrast, 76% of the variation in degrees D-0.5 of CVYV was related to both the numbers of vectors present and maximum temperature. Symptom expression in cucumbers mechanically inoculated with CVYV was most prevalent when plants were grown at regimes of at least 28 degrees C day temperature. According to analysis of spread using Taylor's power law, beta-binomial distribution fitting, and the ordinary runs test, the prevalence of CVYV showed significant overdispersion, whereas that of CYSDV did not. The chi(2) test of independence and Spearman's rank correlation coefficient were used to measure co-occurrence and covariation, respectively, during the first half of the cultivation period. These results showed that the two diseases were not associated.
Abstract: The complete nucleotide sequence of isolates of Cucumber vein yellowing virus (CVYV) has been determined. The viral genome comprises 9734 nucleotides, excluding a 3'-terminal poly(A) sequence. The genome of CVYV has a 5'-non coding and a 3' non coding region of respectively 67 and 240 nucleotides. The RNA of CVYV encodes a single polyprotein of 3148 amino acid residues and has a deduced genome organization and motifs typical for a member of the family Potyviridae. However, CVYV is atypical because it lacks a coding sequence region for the putative helper-component as well as conserved helper-component-proteinase motifs which may account for its vector relations. All the present coding regions were compared to those from several members of the Potyviridae family. CVYV is most closely related to Sweetpotato mild mottle virus confirming its assignation to the genus Ipomovirus, despite similarities with tritimoviruses.
Abstract: Common bean (Phaseolus vulgaris L.) is grown on approximately 1,500 ha in commercial greenhouses and is of major economic importance in the Souss-Massa Region, Agadir, Morocco. Since October 2003, symptoms resembling a viral disease, consisting of pod mosaic and distortion and mild to severe mosaic in leaves, have been observed on bean plants in several greenhouses. Mechanical inoculation with symptomatic leaf extracts produced necrotic local lesions on P. vulgaris ‘Pinto’ and systemic symptoms similar to those observed in the naturally infected bean plants P. vulgaris ‘Donna’ (five plants per cultivar). Inoculated and naturally infected samples reacted positively using a double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) to Southern bean mosaic virus (SBMV) (DSMZ, Braunschweig, Germany), a member of the Sobemovirus genus that is transmitted by contact, soil, beetles, and seeds (1). Virions purified from a naturally infected ‘Donna’ plant contained a 30-kDa polypeptide that reacted positively using sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blot analysis with SBMV antiserum (DSMZ). Reverse transcription-polymerase chain reaction amplification with SMBV primers as described by Verhoeven et al. (2) produced an expected 870-bp band. The amplicon was cloned, sequenced (GenBank Accession No. AJ748276), and compared to those isolates available in GenBank and had a nucleotide sequence identity of 87% and a derived amino acid sequence identity of 95% with an SBMV isolate from Spain (2). During a survey in different areas of the Souss-Massa Region, 20 symptomatic leaf and pod samples were randomly collected from 12 greenhouses (50 ha) where significant commercial losses were suffered because of this virus disease, and all samples were positive using DAS-ELISA for SBMV. To our knowledge, this is the first report of SBMV in Morocco.
References: (1) J. H. Tremaine and R. I. Hamilton. Southern bean mosaic virus. No. 274 in: Descriptions of Plant Viruses. CMI/AAB, Kew, Surrey, England, 1983. (2) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 109:935, 2003.
Abstract: During 2001 and 2002, Pisum sativum var. vulgare plants grown as commercial crops in Almeria (southeast Spain) showed vein clearing and chlorotic mottle of leaves, leaf deformation, flower abortion, necrotic mottle and deformation of pods, and stunted plant growth. Crude sap of collected plants was mechanically inoculated on healthy pea plants which reproduced symptoms observed in the field; local necrotic lesions were produced on mechanically infected Chenopodium quinoa, C. amaranticolor, and Gomphrena globosa, systemic mosaic symptoms on Brassica napus and Nicotiana benthamiana, and local lesions plus systemic mosaic symptoms on N. clevelandii, which are all characteristic of Turnip mosaic virus (TuMV) (1). A reverse transcription-polymerase chain reaction assay using general primers for the extreme 3' end of the potyvirus genome amplified products of 750 and 1,700 bp in nucleic acid extracts from naturally infected pea plants as well as from the mechanically infected test plants. The overlapping nucleotide sequences of the products (GenBank Accession No. AJ489259) had a nucleotide sequence identity of 86.5% and a derived amino acid identity of 95.0% with several published sequences of TuMV (1). This report cites the first partial nucleotide sequence of TuMV infecting pea crops, and although natural infections of this virus in pea have been reported in Morocco (1976) and in the United States (2), to our knowledge, this is the first report of TuMV in Spain.
Notes: References: (1) P. Lehmann et al. Physiol. Mol. Plant Pathol. 51:195, 1997. (2) R. Provvidenti. Plant Dis. Rep. 62:482, 1978.
Abstract: Hybridisation of tissue prints with nonradioactive cDNA probes was developed to detect cucumber vein yellowing virus (CVYV) in cucurbit plants. Results showed irregular distribution of the virus within cucumber, zucchini or melon plants without defined tropism for a specific tissue. Therefore, reliable diagnosis of CVYV requires analysis of tissue prints from at least five different plant sites. This detection procedure allows rapid analysis of large numbers of plants and it can be useful for epidemiological studies of CVYV and to control virus spread via eradication of early foci.
Abstract: Southern bean mosaic virus (SBMV) has been identified as the cause of a new disease in greenhouse-cultivated common bean (Phaseolus vulgaris), in the south-east of Spain. The identification was based on host range comparisons, morphological and serological characteristics of the virus, the size of its dsRNA species and the nucleotide sequence of an 810-bp fragment from ORF2. The virus could be clearly discriminated from the related sobemovirus Southern cowpea mosaic virus. This is the first report of SBMV in Spain.
Abstract: A cost-efficient hybridisation assay was developed to estimate the amount of cucurbit yellow stunting disorder virus (CYSDV) in Bemisia tabaci (Gennadius) whiteflies infesting protected cucumber crops. cDNA from the coat protein (cp) gene and the hsp70 homologue protein gene from CYSDV were obtained by reverse transcriptase-PCR from viruliferous whiteflies and cloned into plasmids. Digoxigenin (DIG)-labelled cDNA probes reacted with extracts from these whiteflies applied on nylon membranes. Precision and linear ranges were established in a hybridisation analysis using known concentrations of unlabelled homologue cDNA. Extracts from non-viruliferous B. tabaci showed a concentration-dependent effect on the assay with cp-specific probes but not with hsp70-specific probes. The hsp70 probe was used to evaluate natural B. tabaci populations in commercial cucumber crops, and the estimated amounts of CYSDV per whitefly were found ranging from 5.6 fg to approximately 2.5 pg of corresponding hsp70-cDNA.
Abstract: The complete nucleotide sequence of the RNA genome of a Pepper Mild Mottle Virus (PMMoV) isolate that overcomes L3 resistance in pepper (Capsicum sp.) was determined and compared with the sequence of other Tobamoviruses. The RNA genome consists of 6357 nucleotides and contains four open reading frames. The 5' proximal ORF encodes a 128 kDa product that terminates in an amber codon which may be readthrough to produce a 180 kDa replication-associated protein (ORF 2). ORF 3 codes for the 28 kDa protein assumed to be involved in cell to cell spread of the virus. The last ORF encodes the coat protein (CP). Amino acid sequence comparison of the CP of this and other PMMoV isolates showed the same substitution (Met to Asn) as found in the Italian isolate of PMMoV and which is assumed to be responsible for L3-resistance breaking. RT-PCR using a common primer pair for PMMoV followed by restriction enzyme analysis with EcoRI allowed the discrimination of resistance breaking from non-L3 resistance breaking virus isolates.
Abstract: Since the experimental infection by hydatid cysts (Echinococcus granulosus) in mice causes immunomodulation of the host, the effects of hydatid fluid (HF) and fractions of HF were compared in vitro and in vivo. Fractions of HF were obtained using ammonium sulphate precipitation, chloroform/methanol extraction and thin-layer chromatography (TLC). HF proved to be toxic to murine peritoneal macrophages in vitro, and when macrophages were incubated with the different fractions of HF, most toxicity was found in a single TLC-purified fraction with an adjuvant-like effect on the production of specific antibodies against bovine albumin and human red blood cells in mice. Treatment of mice with the toxin caused a drop in the percentage of peripheral blood lymphocytes. Flow-cytometric analysis showed that T-cells from toxin-treated mice had lower membrane-CD3, CD4 and CD8 density, and higher percentages of CD8+ splenocytes and CD4+ thymocytes expressing CD25. The toxin caused a down-regulation of CD4 and CD8 expression on thymocytes in vitro, that was dependent on the presence of macrophages. The results may attribute to these toxins a role in the host-parasite relationship of hydatidosis.
Abstract: Diagnosis of hydatid disease in humans relies on the detection of specific antibodies against antigens of the metacestode from Echinococcus granulosus. The specificity and sensitivity of current immunological techniques based on specific serum IgG rely on the way antigens are purified. We used Western immunoblotting to detect specific IgG, IgE, and IgA antibodies in serum from patients with hydatid disease using either crude antigen preparations (total hydatid fluid), purified fractions enriched in Antigens 5 and B, and glycoproteins from hydatid fluid. Depending on whether crude HF or purified antigen fractions were used, IgG and IgE recognized specifically low-to-medium MW bands between 12 and 42 kDa. IgA recognized specifically 110 kDa band in crude hydatid fluid and in the glycoprotein fraction of hydatid fluid, and a 42 kDa band in all antigen samples used. Besides the advantage of detecting specific IgA in crude hydatid fluid, these results offer the possibility of simplifying future immunological tests if specific secretory IgA can be similarly detected.
Abstract: The specificity and sensitivity of enzyme-linked immunosorbent assays (ELISA) and Western immunoblot assays in detecting antibodies in serum from patients suffering cystic hydatid disease (Echinococcus granulosus) are compared using either crude antigen preparations (total sheep hydatid fluid and homogenates of protoscoleces), purified fractions enriched in Antigens 5 and B, and glycoproteins from hydatid fluid. Polyprotein bands of 12-14, 20, and 34 kDa, when purified from hydatid fluid by applying changes in the ionic strength, yielded a sensitive (95%) immunodiagnostic test that was also extremely specific (100%) when assayed with sera from noninfected humans and from patients suffering from other parasitic diseases. However, subjecting hydatid fluid to chromatography through a concanavilin A column rendered a 42 kDa band that was sensitive (95%) as well as highly specific (100%) for hydatidosis. Therefore purification procedures can strongly affect the diagnostic value of antigens with identical electrophoretic behavior in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Abstract: Copper-zinc superoxide dismutase was purified from Ascaris suum (Nematoda). Four benzimidazole derivatives, six recently synthesized pyrimidine derivatives and eleven recently synthesized glycine derivatives were shown to inhibit: (1) purified extracts of A. suum superoxide dismutase; (2) superoxide dismutase from host liver, and (3) purified extracts of superoxide dismutase from living A. suum incubated in the presence of these drugs. Thiabendazole compounds, with a documented effect against helminth parasites, were found to affect the superoxide dismutase. The inhibitory effects of some pyrimidine and glycine derivatives were higher than those of benzimidazoles, and the pyrimidine compounds failed to inhibit the host's enzyme. These derivatives are candidate anthelmintics, acting as inhibitors of certain metalloenzymes in parasites.
Abstract: Balb/c mice were infected intraperitoneally with protoscoleces of Echinococcus granulosus. After 15 months of infection, and by means of flow cytometry, the expression of T-cell markers CD3, CD4, and CD8 on T cells from peripheral blood, spleen, and thymus was analyzed and compared with that of age-matched controls. Infected mice had higher percentages of CD3+, and CD4+ cells in peripheral blood, and higher percentages of CD8+ cells in the spleen, when compared with control mice. CD4+ and CD8+ cells in peripheral blood and CD8+ cells in thymus also showed higher percentages of expression of interleukin-2 receptor. The results infer a role for interleukin-2 in experimental secondary echinococcosis.
Abstract: A lipid-binding protein (LBP) has been purified from the cytosol of the cestode Moniezia expansa. The native LBP was found to be an oligomer of approx. 250 kDa, consisting of 11 kDa monomers. The LBP bound saturated and unsaturated fatty acids, but not their CoA derivatives, with KD values in the range 0.68-7.8 microM. Cholesterol, dihydroergosterol, bilirubin and retinoids were also bound, but alpha-tocopherol, bile acids, alk-2-enals and alka-2,4-dienals were not. Evidence suggests that there are two binding sites per subunit, each with different specificities. The fluorescent fatty acid 11-[(5-dimethylaminonaphthalene-1-sulphonyl)amino]undecanoic acid (DAUDA) and retinol both showed an additional high-affinity binding site with a density of approximately 0.1 per subunit, suggesting specific binding to the oligomer. The amino acid composition of Moniezia LBP was distinct from that of previously characterized fatty acid-binding proteins (FABPs). The protein was not N-terminally blocked and yielded a unique amino acid sequence, unrelated to that of any known FABP; there was also evidence of microheterogeneity. Polyclonal antibodies raised to the Moniezia protein did not cross-react with mammalian, nematode or digenean FABP. The Gibbs free energy for protein folding (13.02 kJ/mol; 3.1 kcal/mol), determined by urea denaturation, was identical for both the native and ligand-bound Moniezia LBP. CD spectra showed that the Moniezia protein contained 36% alpha-helix and that the secondary structure underwent only minor changes on ligand binding. Moniezia LBP binds a range of anthelmintics, with KD values again in the range 0.66-7.3 microM. It is possible that, in helminths, binding proteins may play a role in determining the specificity and site of action of anthelmintics.
Abstract: Infections by the protozoan parasite Cryptosporidium parvum are routinely diagnosed by modified Ziehl-Neelsen (acid-fast) staining of faecal preparations despite the counterstaining and ghost-like appearance of some oocysts. Quantitative studies demonstrated that only a small percentage of oocysts excreted by naturally infected newborn calves displayed acid-fast characteristics, but that percentage increased when the time between excretion and sample staining was increased. The treatment of faecal samples with hydrogen peroxide (10 min, 5 vol. final concentration) caused all oocysts to become acid-fast, with up to 40-fold increases in test sensitivity in samples treated and stained within 3 h of excretion. Flow-cytometry analysis of hydrogen peroxide-treated oocysts also demonstrated increased labelling of oocysts by a commercial monoclonal antibody preparation commonly used for diagnosis.
Abstract: Copper-zinc superoxide dismutase from Ascaris suum (Nematoda) was purified in a new, more efficient, and faster manner. The process included differential centrifugation, fractionation with ammonium sulfate, and sodium dodecyl sulfate-polyacrylamide electrophoresis, yielding a 340-fold purification (specific activity of 47 units/mg). Optimal storage conditions, optimal pH range, thermostability, molecular weight and ultraviolet-visible absorption spectrum of the enzyme are described, and a new enzymatic model for pharmacological screening is suggested.
Abstract: Hydatid fluid (HF) from hydatid cysts of the cestode Echinococcus granulosus inhibited the phagocytosis of bacteria and yeast cells by host macrophages in vitro. Different assays were used to study the dose-dependent effect of partially purified HF toxins on peritoneal macrophages (PM phi) of the mouse. Trypan blue exclusion, as well as measuring the specific release of lactate dehydrogenase activity and 51Cr release of 51Cr-labelled PM phi, showed that incubation with HF toxins lead to the lysis of the target cells. By measuring the effect of the toxins on the reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide by PM phi, and by experimental labelling using [3H]-uridine, a decrease in metabolic activity was seen. However, at non-lytic concentrations, the PM phi showed a peak of metabolic activity. The observations of the effects on macrophages by parasite-derived toxins may be related to a mechanism by which the parasite survives within an immunized host.
Abstract: 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.
Abstract: It is obvious that during the evolution of a host-parasite relationship, in which one animal is needing the other, while the latter is trying to expel or destroy the former, both partners tend to reach an equilibrium. In many cases they are still far off, but in hydatidosis (Echinococcus granulosus) it would appear that a lot has been achieved. As the near equilibrium is mainly related to the state of the immunological relationship, it is in fact a case of immunohomeostasis. The immunological reactions of the host involve both humoral and cellular phenomena. The nature of the humoral response varies greatly according to host species, parasite strain and several factors of the infection and analysis procedures. The cell-mediated defense mechanism involves various types of cells, but activated macrophages appear to be most important. With regards to the modes of immunoevasion, attention is paid to the barrier role of the laminated layer, the inactivation of complement and the interference with the activities of host immune cells. The role of toxic substances released by protoscoleces and cysts is especially emphasized. The toxins appear extremely vulnerable to host macrophages and play a very important role in the establishment of a hydatid cyst. However, they have only a local influence and do not jeopardize the development of concomitant immunity. Observations in experimental secondary echinococcosis led to the conclusion that the establishment of a hydatid cyst depends on the speed of the parasite's toxic effect versus the host immunological reaction. Survival of the parasite is based on the continued balancing effects of both.
Abstract: Various biochemical and physiological characteristics of fresh sheep serum were estimated and compared with those of pooled hydatid cyst fluid (HCF), isolated from Echinococcus granulosus cysts of ovine origin. Concentrations of the following compounds were determined: total protein, total albumin, total lipids, triglycerides, cholesterol, high density, low density and very low density lipoproteins, glucose, bilirubin, creatinine, uric acid, urea, calcium, inorganic phosphate, iron, sodium and potassium. Total activities of the following enzymes were measured: glutaminate-oxaloacetate transaminase, glutaminate-pyruvate transaminase, L-gamma-glutamyl transferase, alkaline phosphatase and alpha-amylase. Osmolarity and pH were also determined and total proteins were subjected to acetate cellulose electrophoresis. It was shown that compounds that are related to the carbohydrate and lipid metabolism, as well as nitrogenous catabolism, were predominantly present in HCF when compared with sheep serum. In contrast, the HCF contained few proteins and showed a low activity of enzymes related to protein metabolism. HCF, although osmotically comparable to serum of a putative host, is characterized by high levels of sodium and potassium. Microscopically, ammonium urate, amorphous phosphate and calcium carbonate crystals are found. The results are discussed in view of cestode physiology and metabolical (in-)dependence of hydatid cysts.
Abstract: Vesiculated protoscoleces (VP) were produced by culturing freshly collected protoscoleces from Echinococcus granulosus horse liver hydatids in RPMI 1640 monophasic medium at 37 degrees C for 18 days. Half of the VP were used as such, the other half used after killing them by freeze-thawing. Nine-day-old chicken heart fragments (CHF) were cultured in MEM at 37 degrees C for 72 h. Subsequently, CHF were put together with live and dead VP, respectively, for up to 53 days, on a semisolid medium consisting of agar, Ringer's and MEM. Time-dependent histological observations revealed that dead VP were surrounded by CHF cells. Dead VP tissue was eventually internalized and disintegrated in about 1 week. Live VP penetrated into the CHF tissue and further developed into small hydatid cysts, located within the boundaries of the experimental 'host' tissue. The amorphous-looking contact region PAP-stained positively only with anti-E. granulosus serum and not with anti-CHF serum; it was considered identical to the normal laminated layer. The invasion of VP in CHF tissue proved to be different from a tumour or a bacterial invasion: it was concluded that the confrontation of VP and CHF had resulted in an 'in vitro cohabitation' rather than in an 'in vitro infection'.
Abstract: Echinococcosis/hydatidosis is characterized by the existence of various strains of the parasite Echinococcus granulosus. In order to study the degree of variability in Belgium, protoscoleces (PS) were isolated from infected intermediate hosts (horse, cattle, pig, goat and man). Homogenate supernatants of these PS were compared by SDS-polyacrylamide gel electrophoresis and western blotting: 15 to 26 predominantly silver staining bands (depending on the source of the parasites), were selected to study existing homologies among these possible strains. Antisera raised against PS homogenates of horse, cattle, pig and goat origin, were used to compare antigenicity by western blotting. The results indicate a closer similarity between E. granulosus of horse, goat and pig origin whereas E. granulosus derived from cattle showed less homology with any of the former. Results are discussed against the background of the occurrence of E. granulosus in Belgium.
