Deglycosylation of Excretory-Secretory Antigens of the Second-Stage Larvae of Toxocara cati Improves Its Efficacy in the Diagnosis of Human Toxocariasis.

Background: Toxocariasis is an important zoonotic infection, especially in tropical areas. One of the significant challenges in the serodiagnosis of human toxocariasis is the cross-reaction of Toxocara antigens with other parasites due to their relatively similar glycan structures. Removing the glycan structure from Toxocara excretory-secretory (TES) antigens may increase the efficacy of these antigens in the diagnosis of toxocariasis. The current study aimed to assess the efficacy of deglycosylated Toxocara cati excretory-secretory (dTES) antigens for the serodiagnosis of human toxocariasis. Methods: Toxocara ES antigens were prepared from T. cati second-stage larvae and deglycosylated using sodium hydroxide (NaOH). The TES antigens, along with the dTES antigens, were used in an ELISA as well as a western blotting system for the detection of anti-Toxocara antibodies. Sera samples collected from 30 confirmed cases of toxocariasis, 30 patients with other diseases, and 30 healthy subjects were evaluated by both systems. Results: The sensitivity of TES and dTES ELISA for the diagnosis of human toxocariasis was 96.67% (95% CI = 82.78-99.92) and 93.33% (95% CI = 77.93-99.18), respectively, while the specificity of dTES (88.33%; 95% CI = 77.43-95.18) increased significantly compared to the TES (80.00%; 95% CI = 67.67-89.22). The sensitivity of both antigens was 100% (95% CI = 88.43-100) by the western blotting system. Moreover, the specificity of TES and dTES antigens was 95% (95% CI = 86.08-98.96) and 98.33% (95% CI = 91.06-99.96), respectively, when using the western blotting system. Conclusion: Results of the current study indicate that the chemical removal of the glycan epitopes of T. cati ES antigens significantly reduces cross-reactivity rates with other parasitic infections. Considering the findings of the present study, the dTES antigens seem to be suitable antigens for the serodiagnosis of human toxocariasis.

Very low frequency waves as selective probe for Cysticercus tenuicollis, Hydatid cyst and Coenurus cerebralis bio-analysis using single cell-signal recording.

Comparative electric behavior of Cysticercus tenuicollis, Hydatid cyst and Coenurus cerebralis at the Very Low Frequency (VLF) region has been studied in detail. This investigation could be significant, because of the economic and public health importance of these parasitic infections in domestic animals. In this report, a single cell signal recording technique has been adopted for comparison using a stainless steel (type: 316, diameter: ~ 300 µm, height: 2.00 cm) two identical electrode system, implanted on the surface of the tested cysts with inter electrode distance of 0.50 cm at a ~ 6.0 giga ohm (GΩ) sealed condition (based on the situation of the implanted electrode system). This process was achieved based on applying electrical interaction between the cysts and the VLF electrical signal. Relative to the measured time domain signal (Current-time diagram), the frequency domain (Current-frequency diagram) was estimated via applying a "Discrete Fast Fourier Transform" (DFFT) algorithm at a fixed time interval (5.0 min). Factors, having important influence on the sensitivity of the detection system including the type (waveform) of different alternating-current (AC) triggering stimulus signals (such as direct current, square wave, triangular, sin (t), etc.), the amplitude, as well as the frequency were optimized automatically through a written "Visual Basic 6" program by one-factor-at-a-time method. Direct applying this AC triggering VLF voltage to the cysts resulted in tracing an AC electrical current vs. time that considered as the time domain wave. However, this electrical current was decayed rapidly versus time during maximum 30.0 s time scale. Applying the DFFT algorithm to the measured time domain, resulted in accessing to the frequency domain at the selected frequency range between 2 and 5 kHz that was considered as the selected frequency for the selective differentiation of C. tenuicollis, Hydatid cyst and C. cerebralis. The related probable mechanism of this process may be attributed to the correlation between the triggering potential and the cyst's electrical surface charge (Zeta potential) as the current source under similar conditions. The results of this study may help to introduce a new detection system for in vivo recognition of the cysts in future.

In Vivo Chemogenetic Biocompatibility of Mercury as a Specific Hypercalcemia Actuator in Snail's Spinal Cord Cell Manipulation: An Extracellular Field Potential Biosensor.

The in vivo chemogenetic property of mercuric ions (Hg2+) was investigated as a specific hypercalcemia actuator in snail's spinal cord cell manipulation by extracellular field potential biosensing analysis. For this purpose, a three-microelectrode system with working, counter, and pseudo reference electrodes was blindly implanted into the snail's spinal cord to electrically stimulate (triggering) the action potential with a staircase electrical voltage at a very low frequency level, along with measurement of the electrical current, as a detection system. Under optimum conditions, using the one-factor-at-a-time method, a wide linear range between 1.0 × 10-14 and 1.0 × 10-1 mol L-1 with correlation coefficients (R2) >0.98 and a response time (t90) of maximum 10.0 s were approximated. Percentages of relative standard deviation were estimated to be 3.08 (reproducibility, n = 50) and 7.31 (repeatability, n = 15). The detection limit was estimated to be sub 2.1 × 10-16 mol L-1 based on the Xb- + 3Sb definition. The reliability of this phenomenon was evidenced by the estimation of recovery percentages (between 95 and 107%) during spiking Hg2+ standard solutions. The probable mechanism behind this process could be attributed to the following: (i) the neuronal ephaptic coupling during electrical synchronization by a specific brain-triggered wave as a neuronal motor toolkit and (ii) chemical synchronization using a Hg2+ hypercalcemia actuator (biosensor). Linear correlation has been evidenced during interactions between Hg2+ and a calcium ionic channel's protein with a gram molecular weight of 66.2 ± 0.3 KCU. This process, therefore, caused an opening of the Ca2+ channel gates and majorly released the Ca2+ (hypercalcemia) that was detected as the main source of the measured electrical current. At this condition, ultratrace levels of Hg2+ ions not only were considered as nontoxic reagents but also had chemically regulating effects as ephaptic synchronizers to the neuron cells. This report may pave the way for using mercury ions at an ultratrace level for clinical controlling purposes during neuronal spinal cord cell manipulation.

A new method for laboratory rearing of Galba truncatula, the intermediate host of Fasciola hepatica.

In this study a relatively large and open top aquarium was designed, constructed and introduced as a suitable habitat for nutrition, growth and development as well as for egg laying and breeding of Galba truncatula under laboratory conditions. The soil and water used in the aquarium were collected from the locality in which the snails were collected. The aquarium was placed in a laboratory with temperature of 18-32 ºC and relative humidity of 22-37% respectively, according to the season. The artificial light was controlled by a light timer, giving 12 h of light and 12 h of darkness. The snails were fed with dried lettuce leaves, Cyperus alternifolius (aquatic plant), Spirulina (algae), Orthotrichum rupestre (moss) and cuttlebone (a supplementary source of calcium). Approximately five weeks after the start of study, there was evidence of reproduction and success in rearing of G. truncatula by the appearance of eggs and small snails (0.1-0.5 mm) in the aquarium. In conclusion, large scale laboratory rearing of G. truncatula is a feasible task. The method may be improved by balancing the temperature and relative humidity as well as by optimizing the soil type, the water quality and the type of food.

In vitro ovicidal activity of Peganum harmala seeds extract on the eggs of Fasciola hepatica.

Peganum harmala seeds extract has been previously reported to have antimicrobial and other medicinal properties. The aim of this study was to evaluate the ovicidal activity of the methanolic extract of P. harmala seeds against the eggs of F. hepatica. The phenolic compounds of the methanolic extract of P. harmala seeds were identified by HPLC analysis. Catechin, rutin, p-Coumaric acid, chloregenic acid and hesperetin were found to be the major phenolic compounds. F. hepatica eggs were collected from the gall bladder of naturally infected sheep. The eggs were exposed to two concentrations of P. harmala seeds extract (1 and 3 mg/mL) for 24 and 48 h. To investigate the effect of the P. harmala seeds extract on the miracidial formation, the treated eggs were incubated at 28 °C for 14 days. The results indicated that F. hepatica eggs were susceptible to the methanolic extract of P. harmala seeds. Following 24 h exposure of the eggs to P. harmala seeds extract with concentrations of 1 and 3 mg/mL, the miracidial formation reduced to 5 and 2.2 % respectively (compared with 60 % for the control group). Following 48 h of exposure of the eggs to P. harmala seeds extract with 1 mg/mL concentration, the miracidial formation reduced to 0.5 %. In this exposure time, no miracidial formation was observed in the eggs exposed to P. harmala seeds extract with concentration of 3 mg/mL. Therefore, the results of this study indicated that P. harmala seeds extract has high ovicidal activity against the eggs of F. hepatica. Accordingly, this extract may have the potential flukicidal activity against the immature and mature F. hepatica.

Endohelminths of European pond turtle Emys orbicularis in Southwest Iran.

Very little is known about parasitic diseases of European pond turtles (Emys orbicularis) in Iran. The objective of this study is to examine parasitic fauna of European pond turtles collected from Fars province, southwest Iran. Carcasses of turtles (n = 52) which died during dredging procedure are collected from earthen fishery basins in Zarghan region. They have been died earlier during dredging procedure in different farms. Three species of helminths in total were found in gastrointestinal tract, including two nematodes (Serpinema microcephalus and Falcaustra araxiana) and one digenean trematod (Telorchis assula). Large intestines of all examined turtles were infected by F. araxiana (100 %, Mean intensity = 18) and this nematode were also found in gastric nodules. Nine turtles (17.3 %, 3 male, 6 female, Mean intensity = 3) were infected with Serpinema microcephalus. T. assula were found in 25 turtles (48.07 %, 5 male, 20 female, mean intensity = 5). Helminths were not found in any examined organs and no ectoparasite found eighter. F. araxiana is the most prevalent nematode in European pond turtles. Detection of Serpinema.microcephalus is in agreement with the fact which this parasite is common parasite of turtles in all over the world. T. assula might be transmitted between variety of reptiles so presence of the trematod should be considered as a risk factor for other reptiles.

In vivo study of the efficacy of the aromatic water of Zataria multiflora on hydatid cysts.

Gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) were employed to determine the chemical composition of the essential oil (EO) from aromatic water (AW) of Zataria multiflora. Thymol (66.9%), carvacrol (15.2%), and carvone (7.3%) were found to be the major EO constituents. Eighty laboratory BALB/c mice were infected intraperitoneally by injection of 1,500 viable protoscolices and were divided into prevention (40 mice) and therapeutic (40 mice) groups. To prove the preventive effect of the Z. multiflora AW on development of hydatid cysts, the 40 infected mice were allocated into three treatment groups, including the albendazole group (10 mice that received 150 mg/kg body weight/day for 10 days), the Z. multiflora AW group (15 mice that received 20 ml/liter in drinking water for 8 months), and a control group (15 mice that received no treatment). To estimate the therapeutic effect of the Z. multiflora AW on the hydatid cyst, after 8 months of infection, the 15 remaining mice were allocated into three experimental treatment groups of five animals each, including the albendazole group (300 mg/kg/day for 20 days), Z. multiflora AW group (40 ml/liter in drinking water for 30 days), and control group (no treatment). All mice were then euthanized, and the sizes and weights of the cysts as well as their ultrastructural changes were investigated. The weights and sizes of the hydatid cysts significantly decreased upon treatment with the Z. multiflora AW in both the preventive and therapeutic groups (P < 0.05). The results of scanning electron microscopy also showed considerable damage in the germinal layer of the hydatid cysts recovered from the treated animals.

Genetic characterization and phylogenetic analysis of Trypanosoma evansi in Iranian dromedary camels.

Whole blood samples were collected from 117 male clinically healthy Camelus dromedarius aged between 6 months to 18 years from several farms in Yazd Province of Iran. Trypanosoma evansi-affected camels were detected by Giemsa-stained blood smears, and the positive blood samples (4 out of 117) were submitted to PCR examination and phylogenetic analysis. Basic Local Alignment Search Tool data of the obtained complete internal transcribed spacer (ITS) sequences revealed that they corresponded to those of T. evansi, Thailand cattle isolate (AY912276) with the homology of 99 %. Both phylogenetic trees generated by ITS1 and complete ITS were unable to clearly show inter- and intraspecific genetic diversity of Trypanosoma spp. isolates. The phylogenetic tree inferred from the ITS2 nucleotide sequences (569 bp) clearly showed the genetic diversity of the parasites. Phylogenetic and molecular analyses of this region showed that two distinct genotypes of T. evansi in Iranian dromedary camels are present. In contrast to the ITS1 and ITS2 regions, multiple alignment of the nucleotide sequence of the 5.8S rRNA showed a high degree of sequence conservation during evolution in various Trypanosoma spp.