Na(+)-ATPase and protein kinase C are targets to 1-O-hexadecylphosphocoline (miltefosine) in Trypanosoma cruzi

Miltefosine has been shown to be a very active compound against Trypanosoma cruzi. Here, we evaluated the effects of miltefosine on the activity of the Na⁺-ATPase and protein kinase C (PKC) present in the plasma membrane of T. cruzi. Furosemide (2 mM), a specific inhibitor of Na⁺-ATPase, abolished the growth of T. cruzi showing a crucial role of this enzyme to parasite growth. Miltefosine inhibited the Na⁺-ATPase activity with IC₅₀ = 18 ± 5 μg mL⁻¹. This effect was shown to be reversible, dependent on the pH and Ca²⁺. The inhibition was not observed when the membranes were solubilized with 0.1% deoxycholate, suggesting that the interaction between the enzyme and membrane phospholipids might be important for the drug effect. Miltefosine also inhibited the parasite PKC activity, but through a Na⁺-ATPase-independent way. Altogether the results indicate that miltefosine inhibits T. cruzi growth through, at least in part, the inhibition of both Na⁺-ATPase and PKC activities.     

Trypanosoma cruzi nucleoside diphosphate kinase 1 ( TcNDPK1) has a broad nuclease activity

Here, we present the characterization of a trypanosomatid nucleoside diphosphate kinase (TcNDPK1) exhibiting nuclease activity. This is the first identification of a NDPK with this property in trypanosomatid organisms. The recombinant TcNDPK1 protein cleaves not only linear DNA, but also supercoiled plasmid DNA. Additionally, TcNDPK1 is capable of degrading Trypanosoma cruzi genomic DNA. ATP or ADP did not affect the nuclease activity, while the absence of Mg2+ completely inhibits this activity. NDPK and nuclease activities were inhibited at the same temperature, suggesting the presence of related catalytic sites. Furthermore, phenogram analysis showed that TcNDPK1 is close to Drosophila melanogaster and human NDPKs. The unspecific nuclease activity could suggest a participation in cellular processes such as programmed cell death.     

Antimicrobial peptides isolated from Phyllomedusa nordestina (Amphibia) alter the permeability of plasma membrane of Leishmania and Trypanosoma cruzi

Nature has provided inspiration for Drug Discovery studies and amphibian secretions have been used as a promising source of effective peptides which could be explored as novel drug prototypes for neglected parasitic diseases as Leishmaniasis and Chagas disease. In this study, we isolated four antimicrobial peptides (AMPs) from Phyllomedusa nordestina secretion, and studied their effectiveness against Leishmania (L.) infantum and Trypanosoma cruzi. The antiparasitic fractions were characterized by mass spectrometry and Edman degradation, leading to the identification of dermaseptins 1 and 4 and phylloseptins 7 and 8. T. cruzi trypomastigotes were susceptible to peptides, showing IC₅₀ values in the range concentration of 0.25–0.68 μM. Leishmania (L.) infantum showed susceptibility to phylloseptin 7, presenting an IC₅₀ value of 10 μM. Except for phylloseptin 7 which moderate showed cytotoxicity (IC₅₀ = 34 μM), the peptides induced no cellular damage to mammalian cells. The lack of mitochondrial oxidative activity of parasites detected by the MTT assay, suggested that peptides were leishmanicidal and trypanocidal. By using the fluorescent probe SYTOX® Green, dermaseptins 1 and 4 and phylloseptins 7 and 8 showed time-dependent plasma membrane permeabilization of T. cruzi; phylloseptin 7 also showed a similar effect in Leishmania parasites. The present study demonstrates for the first time that AMPs target the plasma membrane of Leishmania and T. cruzi, leading to cellular death. Considering the potential of amphibian peptides against protozoan parasites and the reduced mammalian toxicity, they may contribute as scaffolds for drug design studies.     

Detection of matrix metallopeptidase-9-like proteins in Trypanosoma cruzi

In this study, the cell-associated and extracellular peptidases of Trypanosoma cruzi grown in modified Roitman’s complex (MRC) medium were analyzed by measuring peptidase activity in gelatin-containing zymograms. Our results showed that the cell-associated peptidases as well as peptidases extracellularly released by T. cruzi displayed two distinct proteolytic classes: cysteine and metallopeptidase activities. The major cysteine peptidase, cruzipain, synthesized by T. cruzi cells was detected in cellular parasite content, as a 50 kDa reactive polypeptide, after probing with anti-cruzipain antibody. In addition, metallo-type peptidases belonging to the matrix metallopeptidase-9 (MMP-9) family were revealed, after Western blotting, as a 97 kDa protein band in cellular extract and an 85 kDa polypeptide in both cellular and secreted parasite extracts. The MMP-9-like activity present in cells and spent culture medium was immunoprecipitated by an anti-MMP-9 polyclonal antibody. The surface location of MMP-9-like proteins in T. cruzi was also evidenced by means of flow cytometry analysis. Furthermore, doxycycline that has direct MMP-9 inhibiting properties in vitro, inhibited MMP-9-like activities in gel zymography, immunoprecipitation and flow cytometry analyses. This is the first report of the presence of MMP-9-like molecules in T. cruzi. The presence of a matrix extracellular-degrading enzyme may play a role in the T. cruzi-host cell interaction, making this enzyme a potential target for future drug development against this pathogenic trypanosomatid.     

Experimental Chagas’ disease in orchiectomized Calomys callosus infected with the CM strain of Trypanosoma cruzi

The incidence and progression of disorders associated with an unbalanced immune response has among many factors the gender as a contributory factor. The aims of this work were to evaluate the effects of orchiectomy and the immune response during the experimental Trypanosoma cruzi infection. Young adult, male Calomys callous were i.p. inoculated with 1 × 10⁵ blood trypomastigotes of the CM strain of T. cruzi and divided in groups: Control, Sham and Castrated. Castrated group displayed significantly lower values for prostate and seminal vesicle weights indicating a drastic drop of testosterone plasmatic levels. Orchiectomized animals also displayed lesser number of blood parasites, enhanced lytic antibody percentage, splenocyte proliferation and NO concentration when compared to its sham and control counterparts, indicating that steroid gonadal ablation actually influences immune response triggering a more efficient cellular and humoral response which led animals to become more resistant against T. cruzi infection.     

Synthesis and characterization of a pyridine-2-thiol N-oxide gold(I) complex with potent antiproliferative effect against Trypanosoma cruzi and Leishmania sp. insight into its mechanism of action

In the search for new therapeutic tools against parasitic diseases caused by the Kinetoplastids Leishmania spp. and Trypanosoma cruzi, a novel gold(I) triphenylphosphine complex with the bioactive coligand pyridine-2-thiol N-oxide (mpo) was synthesized and characterized by using analytical and conductometric measurements, electrospray ionization-mass spectrometry (ESI) and electronic, FTIR and ¹H and ³¹P NMR spectroscopies. A dinuclear structure is suggested for the complex. At a 1 microM concentration the complex induced in vitro after 30 min a potent leishmanicidal effect (LD₅₀) against promastigotes of Leishmania (L.) mexicana while on Leishmania (V.) braziliensis with the same concentration only a leishmanistatic effect (IC₇₅) was observed 48 h after treatment. Similar differential susceptibilities were also found when testing the ligand mpo, but at a higher dose (5 μM). In addition, the compound showed growth inhibitory effect on Dm28c T. cruzi epimastigotes in culture (IC₅₀ 0.09 μM), being even more active than the anti-trypanosomal reference drug Nifurtimox (IC₅₀ 6 μM). DNA interaction studies showed that this biomolecule does not constitute a main target for the mpo complex currently tested. Instead, the significant potentiation of the antiproliferative effect against both Leishmania species and T. cruzi could be associated to the inhibition of NADH fumarate reductase, a kinetoplastid parasite-specific enzyme absent in the host. Furthermore, due to its low unspecific cytotoxicity on mammalian cells (J774 macrophages), the new gold complex showed a selective anti-parasite activity. It constitutes a promising new potent chemotherapeutic alternative to be evaluated in vivo in experimental models of leishmaniasis and Chagas´ disease.     

Trypanosoma cruzi: Isolation and characterization of aspartyl proteases

Two aspartyl proteases activities were identified and isolated from Trypanosoma cruzi epimastigotes: cruzipsin-I (CZP-I) and cruzipsin-II (CZP-II). One was isolated from a soluble fraction (CZP-II) and the other was solubilized with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CZP-I). The molecular mass of both proteases was estimated to be 120 kDa by HPLC gel filtration and the activity of the enzymes was detected in a doublet of bands (56 and 48 kDa) by substrate-sodium dodecyl sulphate-polyacrylamide-gelatin gel electrophoresis. Substrate specificity studies indicated that the enzymes consistently hydrolyze the cathepsin D substrate Phe-Ala-Ala-Phe (4-NO₂)-Phe-Val-Leu-O₄MP but failed to hydrolyze serine and other protease substrates. Both proteases activities were strongly inhibited by the classic inhibitor pepstatin-A (?68%) and the aspartic active site labeling agent, 1,2-epoxy-3-(phenyl-nitrophenoxy) propane (?80%). These findings show that both proteases are novel T. cruzi acidic proteases. The physiological function of these enzymes in T. cruzi has under investigation.     

Trypanosoma cruzi: Multiplex PCR to detect and classify strains according to groups I and II

A multiplex PCR was developed for simultaneous detection of Trypanosoma cruzi DNA and classification of the parasite strain into groups I and II. As little as 10 fg of T. cruzi DNA could be detected by multiplex PCR. The technique was shown to be specific for T. cruzi DNA, since no PCR amplification products were obtained with DNA from other tripanosomatid species. Multiplex PCR was validated by assaying genomic DNA from 34 strains of T. cruzi that had been previously characterized; 24 blood samples from experimentally-infected mice and non-infected controls; 20 buffy coat samples from patients in the acute phase of Chagas disease and non-infected individuals, and 15 samples of feces from naturally-infected Triatoma infestans. T. cruzi samples from patients and from Y strain-infected mice were classified by multiplex PCR as T. cruzi II and samples from T. infestans and Colombiana strain-infected mice as T. cruzi I.     

Prevalence and distribution of parasites and pathogens of Triatominae from Argentina, with emphasis on Triatoma infestans and Triatoma virus TrV

Chagas’ disease is the most important endemic arthropod-zoonosis in Argentina with an estimated 1.6 million people infected with the causative agent Trypanosoma cruzi. Triatoma infestans is the main vector of Chagas’ disease in Argentina. A survey for parasites and pathogens of Triatominae was conducted from August 2002 to February 2005. Collections of insects were made in domiciles, peridomiciles, and in the natural habitats of the Triatominae. Insects from these collections were dissected and their organs and tissues examined for flagellates. Frass from these insects was collected and examined for detection of the entomopathogenic virus Triatoma virus (TrV) using AC-ELISA and PCR. Triatominae belonging to four species, T. infestans (n = 1646), Triatoma guasayana (n = 4), Triatoma platensis (n = 1) and Triatoma sordida (n = 5) were collected from 62 sites located in 13 provinces of Argentina. Triatoma virus and two protozoan species, Blastocrithidia triatomae and T. cruzi, the etiological agent of Chagas disease, were found infecting Triatominae. The total prevalence of TrV in 1646 T. infestans analyzed by ELISA was 9.66% (159/1646) from 7 to 13 provinces where collections were made. Triatoma virus positive triatomines were found in 17 of 62 populations when examined by AC-ELISA but in 38 of 62 populations when PCR was used for detection. The prevalence of B. triatomae in T. infestans was 0.43% (7/1646), while the prevalence of T. cruzi was 1.3% (21/1646). This is the first study on the diversity, distribution and prevalence of flagellated protozoa and TrV of Triatominae in endemic Chagas’ disease regions of Argentina.     

Molecular cloning and analysis of a cDNA coding for nucleoside diphosphate kinase from ryegrass

A full-length cDNA, LpNDPK, encoding ryegrass nucleoside diphosphate kinase (EC 2.7.4.6) has been cloned and sequenced. The nucleotide sequence of the clone contains an open reading frame of 450 nucleotides encoding a protein of 150 amino acid residues with a calculated molecular mass of 16.5 kDa and a P_i of 6.62. The LpNDPK encoded protein possesses substantial homology with nucleoside diphosphate kinases (NDPKs) isolated and cloned form other sources; the highest identity (86 percnt;) was observed with NDPK from sugarcane (Saccharum officinarum). Amino acid comparisons with other NDPKs show that the presented ryegrass NDPK sequence also contains several motifs and specific residues crucial for catalytic activity which are highly conserved among other NDPKs. RT-PCR expression analysis using primers covering the coding region of LpNDPK revealed that the ryegrass NDPK gene is equally expressed in stem, leaf, and flower tissue.     

B-type natriuretic peptide and anthropometric measures in a Brazilian elderly population with a high prevalence of Trypanosoma cruzi infection

B-type natriuretic peptide (BNP) is a diagnostic and prognostic tool in heart failure and also in Chagas disease, which is caused by the protozoan Trypanosoma cruzi and has cardiomyopathy as a main feature. BNP lipolytic actions and T. cruzi infection in the adipose tissue have been recently described. We aim to investigate the relationship between BNP and anthropometric measures and whether it is influenced by T. cruzi infection. We measured BNP, body mass index (BMI), waist circumference (WC), triceps skin-fold thickness (TSF) and performed serological, biochemical and electrocardiographic exams in 1398 subjects (37.5% infected with T. cruzi) in a community-dwelling elderly population in Bambui city, Brazil. Linear multivariate regression analysis was performed to investigate determinants of BNP levels. BNP levels were significantly (p < 0.05) higher in T. cruzi-infected subjects than in the non-infected group (median = 121 and 64 pg/mL, respectively). BMI, WC and TSF in infected subjects were significantly lower than those in non-infected subjects (24.3 vs. 25.5 kg/m2; 89.2 vs. 92.4 cm; and 14.5 vs. 16.0 mm, respectively). There was an inverse relationship between BNP levels and BMI (b = −0.018), WC (b = −0.005) and TSF (b = −0.193) levels. Infected and non-infected groups showed similar inverse relationships between BNP and BMI (b = −0.021 and b = −0.015, respectively). In conclusion, there was an inverse relationship between BNP levels and the anthropometric measures. Despite the actions in the adipose tissue, T. cruzi infection did not modify the associations between BNP and BMI, suggesting that body mass does not modify the accuracy of BNP in Chagas disease.     

Cell disruption using a different methodology for proteomics analysis of Trypanosoma cruzi strains

We have developed a cell disruption method to produce a protein extract using Trypanosoma cruzi cells based on a straightforward hypoosmotic lysis protocol. The procedure consists of three steps: incubation of the cells in a hypoosmotic lysis buffer, sonication in a water bath, and centrifugation. The final protein extract was designated TcS12. The stages of cell disruption at different incubation times were monitored by differential interference contrast microscopy. After 30 min of incubation in lysis buffer at 4 °C, the T. cruzi epimastigote forms changed from slender to round-shaped parasites. Nevertheless, cell disruption took place following sonication of the sample for 30 min. The efficiency of the methodology was also validated by flow cytometry, which resulted in 72% of propidium iodide (PI)-labeled cells. To estimate the protein extraction yield and the differential protein expression, the proteomics profile of four T. cruzi strains (CL-Brener, Dm28c, Y, and 4167) were analyzed by liquid chromatography tandem mass spectrometry (LCMS/MS) on a SYNAPT HDMS system using the label-free MS^E approach. ProteinLynx Global Server (version 2.5) with Expression^E analysis identified a total of 1153 proteins and revealed 428 differentially expressed proteins among the strains. Gene ontology analysis showed that not only cytosolic proteins but also nuclear and organellar ones were present in the extract.     

An unusual thermostable aspartic protease from the latex of Ficus racemosa (L.)

The most extensively studied ficins have been isolated from the latex of Ficus glabrata and Ficus carica. However the proteases (ficins) from other species are less known. The purification and characterization of a protease from the latex of Ficus racemosa is reported. The enzyme purified to homogeneity is a single polypeptide chain of molecular weight of 44,500 ± 500 Da as determined by MALDI-TOF. The enzyme exhibited a broad spectrum of pH optima between pH 4.5-6.5 and showed maximum activity at 60 ± 0.5 °C. The enzyme activity was completely inhibited by pepstatin-A indicating that the purified enzyme is an aspartic protease. Far-UV circular dichroic spectra revealed that the purified enzyme contains predominantly β-structures. The purified protease is thermostable. The apparent T_m, (mid point of thermal inactivation) was found to be 70 ± 0.5 °C. Thermal inactivation was found to follow first order kinetics at pH 5.5. Activation energy (E_a) was found to be 44.0 ± 0.3 kcal mol⁻¹. The activation enthalpy (ΔH^∗), free energy change (ΔG^∗) and entropy (ΔS^∗) were estimated to be 43 ± 4 kcal mol⁻¹, −26 ± 3 kcal mol⁻¹ and 204 ± 10 cal mol⁻¹ K⁻¹, respectively. Its enzymatic specificity studied using oxidized B chain of insulin indicates that the protease preferably hydrolyzed peptide bonds C-terminal to glutamate, leucine and phenylalanine (at P₁ position). The broad specificity, pH optima and elevated thermal stability indicate the protease is distinct from other known ficins and would find applications in many sectors for its unique properties.     

Structural analysis of Arabidopsis thaliana nucleoside diphosphate kinase-2 for phytochrome-mediated light signaling

In plants, nucleoside diphosphate kinases (NDPKs) play a key role in the signaling of both stress and light. However, little is known about the structural elements involved in their function. Of the three NDPKs (NDPK1-NDPK3) expressed in Arabidopsis thaliana, NDPK2 is involved in phytochrome-mediated signal transduction. In this study, we found that the binding of dNDP or NTP to NDPK2 strengthens the interaction significantly between activated phytochrome and NDPK2. To better understand the structural basis of the phytochrome-NDPK2 interaction, we determined the X-ray structures of NDPK1, NDPK2, and dGTP-bound NDPK2 from A.thaliana at 1.8A, 2.6A, and 2.4A, respectively. The structures showed that nucleotide binding caused a slight conformational change in NDPK2 that was confined to helices alphaA and alpha2. This suggests that the presence of nucleotide in the active site and/or the evoked conformational change contributes to the recognition of NDPK2 by activated phytochrome. In vitro binding assays showed that only NDPK2 interacted specifically with the phytochrome and the C-terminal regulatory domain of phytochrome is involved in the interaction. A domain swap experiment between NDPK1 and NDPK2 showed that the variable C-terminal region of NDPK2 is important for the activation by phytochrome. The structure of Arabidopsis NDPK1 and NDPK2 showed that the isoforms share common electrostatic surfaces at the nucleotide-binding site, but the variable C-terminal regions have distinct electrostatic charge distributions. These findings suggest that the binding of nucleotide to NDPK2 plays a regulatory role in phytochrome signaling and that the C-terminal extension of NDPK2 provides a potential binding surface for the specific interaction with phytochromes.     

Trypanosoma cruzi: Biological characterization of a isolate from an endemic area and its susceptibility to conventional drugs

We describe some biological and molecular characteristics of a Trypanosoma cruzi isolate derived from a Triatomine captured in Nicaragua. PCR based typification showed that this isolate, named Nicaragua, belonged to the lineage Tc I. Nicaragua infected culture cells were treated with allopurinol, showing different behavior according to the cellular compartment, being cardiomyocyte primary cultures more resistant to this drug. The course of the infection in a mice experimental model and its susceptibility to benznidazole and allopurinol was analyzed. In benznidazole treatment, mice reverted the high lethal effect of parasites during the acute infection, however, a few parasites were detected in the heart of 88% of mice 1 year post-infection. Since T. cruzi is a heterogeneous species population it is important to study and characterize different parasites actually circulating in humans in endemic areas. In this work we show that T. cruzi Nicaragua isolate, is sensitive to early benznidazole treatment.     

Kinetic mechanism and catalysis of Trypanosoma cruzi dihydroorotate dehydrogenase enzyme evaluated by isothermal titration calorimetry

Trypanosoma cruzi dihydroorotate dehydrogenase (TcDHODH) catalyzes the oxidation of l-dihydroorotate to orotate with concomitant reduction of fumarate to succinate in the de novo pyrimidine biosynthetic pathway. Based on the important need to characterize catalytic mechanism of TcDHODH, we have tailored a protocol to measure TcDHODH kinetic parameters based on isothermal titration calorimetry. Enzymatic assays lead to Michaelis-Menten curves that enable the Michaelis constant (K_M) and maximum velocity (V_max) for both of the TcDHODH substrates: dihydroorotate (K_M = 8.6 ± 2.6 μM and V_max = 4.1 ± 0.7 μM s^-1) and fumarate (K_M = 120 ± 9 μM and V_max = 6.71 ± 0.15 μM s^-1). TcDHODH activity was investigated using dimethyl sulfoxide (10%, v/v) and Triton X-100 (0.5%, v/v), which seem to facilitate the substrate binding process with a small decrease in K_M. Arrhenius plot analysis allowed the determination of thermodynamic parameters of activation for substrates and gave some insights into the enzyme mechanism. Activation entropy was the main contributor to the Gibbs free energy in the formation of the transition state. A factor that might contribute to the unfavorable entropy is the hindered access of substrates to the TcDHODH active site where a loop at its entrance regulates the open-close channel for substrate access.     

Treatment of industrial wastewater with two-stage constructed wetlands planted with Typha latifolia and Phragmites australis

Industrial wastewater treatment comprises several processes to fulfill the discharge permits or to enable the reuse of wastewater. For tannery wastewater, constructed wetlands (CWs) may be an interesting treatment option. Two-stage series of horizontal subsurface flow CWs with Phragmites australis (UP series) and Typha latifolia (UT series) provided high removal of organics from tannery wastewater, up to 88% of biochemical oxygen demand (BOD₅) (from an inlet of 420 to 1000 mg L⁻¹) and 92% of chemical oxygen demand (COD) (from an inlet of 808 to 2449 mg L⁻¹), and of other contaminants, such as nitrogen, operating at hydraulic retention times of 2, 5 and 7 days. No significant (P < 0.05) differences in performance were found between both the series. Overall mass removals of up to 1294 kg COD ha⁻¹ d⁻¹ and 529 kg BOD₅ ha⁻¹ d⁻¹ were achieved for a loading ranging from 242 to 1925 kg COD ha⁻¹ d⁻¹ and from 126 to 900 kg BOD₅ ha⁻¹ d⁻¹. Plants were resilient to the conditions imposed, however P. australis exceeded T. latifolia in terms of propagation.     

Chemical evaluation of fatty acid desaturases as drug targets in Trypanosoma cruzi

Four positional isomers of Thiastearate (TS) and Isoxyl (Thiocarlide) were assayed as fatty acid desaturase inhibitors in Trypanosoma cruzi epimastigotes. 9-TS did not exert a significant effect on growth of T. cruzi, nor on the fatty acid profile of the parasite cells. One hundred micromolars of 10-TS totally inhibited growth, with an effective concentration for 50% growth inhibition (EC₅₀) of 3.0 ± 0.2 μM. Growth inhibition was reverted by supplementing the culture media with oleate. The fatty acid profile of treated cells revealed that conversion of stearate to oleate and palmitate to palmitoleate were drastically reduced and, as a consequence, the total level of unsaturated fatty acids decreased from 60% to 32%. Isoxyl, a known inhibitor of stearoyl-CoA Δ9 desaturase in mycobacteria, had similar effects on T. cruzi growth (EC₅₀ 2.0 ± 0.3 μM) and fatty acid content, indicating that Δ9 desaturase was the target of both drugs. 12- and 13-TS were inhibitors of growth with EC₅₀ values of 50 ± 2 and 10 ± 3 μM, respectively, but oleate or linoleate were unable to revert the effect. Both drugs increased the percentage of oleate and palmitate in the cell membrane and drastically reduced the content of linoleate from 38% to 16% and 12%, respectively, which is in agreement with a specific inhibition of oleate Δ12 desaturase. The absence of corresponding enzyme activity in mammalian cells and the significant structural differences between trypanosome and mammalian Δ9 desaturases, together with our results, highlight these enzymes as promising targets for selective chemotherapeutic intervention.     

Interaction and ovicidal activity of nematophagous fungus Pochonia chlamydosporia on Taenia saginata eggs

The ovicidal activity of the nematophagous fungi Pochonia chlamydosporia (isolates VC1 and VC4), Duddingtonia flagrans (isolate AC001) and Monacrosporium thaumasium (isolate NF34) on Taenia saginata eggs was evaluated under laboratory conditions. T. saginata eggs were plated on 2% water-agar with fungal isolates and controls without fungus and examined after 5, 10 and 15 days. At the end of the experiment P. chlamydosporia showed ovicidal activity against T. saginata eggs (p < 0.05), mainly for internal egg colonization with results of 12.8% (VC1) and 2.2% (VC4); 18.1% (VC1) and 7.0% (VC4); 9.76% (VC1) and 8.0% (VC4) at 5, 10 and 15 days, respectively. The other fungi showed only lytic effect without morphological damage to the eggshell. Results demonstrated that P. chlamydosporia was effective in vitro against T. saginata eggs unlike the other fungi.