Introducing a simple and economical method to purify Giardia lamblia cysts

Direct microscopic examination of stool to diagnosis giardiasis (wet mount) has low diagnostic value, but immunologic methods (like IFA and especially ELISA) that are based on the determination of parasite antigens in fecal samples (antigen detection) have relatively high sensitivity and specialty. To prepare anti-Giardia lamblia antibodies needed to design diagnostic kits as well as parasite culture and other molecular studies, we require purification of the parasite cysts. In this study, we designed a rapid, simple and inexpensive method to purify parasite cysts from fecal samples of the patients suffering from giardiasis. Initially, fecal samples that the presence of G. lamblia in them was affirmed by direct microscopic observation of cysts were subjected to various purification methods like one- and twophase sucrose gradient isolation, percoll-sucrose gradient isolation, and a modified two-phase method run by 0.85 and 1.5 M sucrose. The first procedure contained some contents of bacteria and small particles of feces. In the second and third procedure, bacteria were almost removed and the cysts were intact but the suspension contained some extras and cellulose particles. The recovery rate for modified two-phase method was 1.5 × 104 cysts for each two grams of fecal sample. In this study, by using and comparing with the results of some other studies, we introduce and run a modified method that in fact is a mélange of them with some changes. So this method could be recommended as a fast, advantageous and simple method in purification of G. lamblia cysts.Key words: Giardia lamblia, cyst, purification

Designing of enzyme linked immunosorbent assay (ELISA) kit for diagnosis copro-antigens of Giardia lamblia

The sensitivity of microscopic examination of fecal samples to recognize Giardia parasites is low. In the methods based on antigen scanning of parasites such as enzyme linked immunosorbent assay (ELISA), copro-antigens of parasite will be traced and diagnosed even if the live parasite is absent in the fecal samples. To design this method, a pure antibody against parasite as well as an antibody conjugated to a proper enzyme is needed. In this study, an anti-Giardia IgG extracted from serum of contaminated rabbit was purified by ion-exchange chromatography and conjugated to the enzyme horse radish peroxidase (HRP). This antibody was used to design direct and indirect ELISA kits to measure conjugation titer. In both direct and indirect ELISA methods, optical densities (ODs) were 1 by using dilution of 1/4000 of conjugation. According to the results of both tests and the success in produced conjugate, it could be proceeded to prepare ELISA kits to diagnose giardiasis infections in various samples.Key words: Enzyme linked immunosorbent assay (ELISA), antibody, copro-antigen, Giardia lamblia

Simultaneous Modeling of Young's Modulus, Yield Stress, and Rupture Strain of Gelatin/Cellulose Acetate Microfibrous/Nanofibrous Scaffolds Using RSM.

Electrospinning is a promising method to fabricate bioengineered scaffolds, thanks to utilizing various types of biopolymers, flexible structures, and also the diversity of output properties. Mechanical properties are one of the major components of scaffold design to fabricate an efficacious artificial substitute for the natural extracellular matrix. Additionally, fiber orientations, as one of the scaffold structural parameters, could play a crucial role in the application of fabricated fibrous scaffolds. In this study, gelatin was used as a highly biocompatible polymer in blend with cellulose acetate (CA), a polysaccharide, to enhance the achievable range of mechanical characteristics to fabricated fibrous electrospun scaffolds. By altering input variables, such as polymers concentration, weight ratio, and mandrel rotation speed, scaffolds with various mechanical and morphological properties could be achieved. As expected, the electrospun scaffold with a higher mandrel rotation speed shows higher fiber alignment. A wide range of mechanical properties were gained through different values of polymer ratio and total concentration. A general improvement in mechanical strength was observed by increasing the concentration and CA content in the solution, but contradictory effects, such as high viscosity in more concentrated solutions, influenced the mechanical characteristics as well. A response surface method was applied on experimental results in order to describe a continuous variation of Young's modulus, yield stress, and strain at rupture. A full quadratic version of equations with the 95% confidence level was applied for the response modeling. This model would be an aid for engineers to adjust mandrel rotation speed, solution concentration, and gelatin/CA ratio to achieve desired mechanical and structural properties

Prevalence of Intestinal Parasitic Infections among Mentally Disabled Children and Adults of Urmia, Iran

Background: The prevalence of intestinal parasites infection in institutions for mental retarda­tion of Ur­mia City, West Azerbaijan Province, Iran was investigated.Methods: This descriptive - cross sectional study was carried out in of Urmia city in 2007-2008. Fecal samples of 225 less than 29 year old mentally disabled individu­als were examined using direct smear, formalin - ether concen­tration. Beside their scotch tape samples were observed for Enterobius eggs. Statisti­cal evaluation was per­formed by SPSS 10.Results: Of 225 mentally retarded persons, 118(52.4%) and 107(47.6%) were female and male. The over­all prevalence of infection was 20.4% and that of male, and female were 20.5% and 20.3%, respectively. 17.3% of examined individuals had protozoa infection and 3.1% showed Entero­bius vermicularis eggs. The infection rates of detected intestinal protozoa were Enta­moeba coli 9.7%, Giardia lamblia 6.2%, Io­doamoeba butschlii 5.7%, Blastocystis hominis 4%, and Entamoeba histolytica/dispar 0.4%. Forty per­cent of 1-5 year, 22.8% of 6-14 year, 22.2% of 15-18 year, and 16.8% of more than 18-year age groups, had positive results in their tests. Accord­ing to IQ test results, 23.8% of less than 25 score group, 19.6% of 25-50, 17.2% of 50-75, and 40% of 75-90 groups were infected.Conclusion: More efforts for increasing sanitation level and prompt diagnosis and treat­ment of infected persons in these institutions are necessary

sparse inflorescence1 encodes a monocot-specific YUCCA-like gene required for vegetative and reproductive development in maize

The plant growth hormone auxin plays a critical role in the initiation of lateral organs and meristems. Here, we identify and characterize a mutant, sparse inflorescence1 (spi1), which has defects in the initiation of axillary meristems and lateral organs during vegetative and inflorescence development in maize. Positional cloning shows that spi1 encodes a flavin monooxygenase similar to the YUCCA (YUC) genes of Arabidopsis, which are involved in local auxin biosynthesis in various plant tissues. In Arabidopsis, loss of function of single members of the YUC family has no obvious effect, but in maize the mutation of a single yuc locus causes severe developmental defects. Phylogenetic analysis of the different members of the YUC family in moss, monocot, and eudicot species shows that there have been independent expansions of the family in monocots and eudicots. spi1 belongs to a monocot-specific clade, within which the role of individual YUC genes has diversified. These observations, together with expression and functional data, suggest that spi1 has evolved a dominant role in auxin biosynthesis that is essential for normal maize inflorescence development. Analysis of the interaction between spi1 and genes regulating auxin transport indicate that auxin transport and biosynthesis function synergistically to regulate the formation of axillary meristems and lateral organs in maize

Investigating the Prevalence of Intestinal Parasites in Immunocompromised Patients in Bushehr Province, Southwest Iran: A Conventional and Molecular Study [İran’ın Güneybatısındaki Bushehr Eyaletindeki İmmün Yetmezliği Olan Hastalarda Bağırsak Parazitlerinin Prevalansının Araştırılması: Konvansiyonel ve Moleküler Bir Çalışma]

Objective: The aim of this study was to determine the status of intestinal parasitic infections in immunocompromised patients in Bushehr province, southwest Iran by conventional and molecular methods. Methods: A total of 201 stool samples were collected from kidney transplant recipients, AIDS patients and patients under chemotherapy. Samples were collected from healthy people as the control group. The specimens were tested using various conventional methods. Polymerase chain reaction (PCR) testing was performed on samples identified as positive for Coccidia by direct microscopic examination. Results: Approximately 32.45% were infected with at least one type of intestinal parasite. The highest (46.8%) and lowest rates of infection (24%) were observed in AIDS and chemotherapy patients, respectively, while the infection rate of the control group was 16%. Isospora spp. and Cryptosporidium spp. were observed in all patient groups, and Sarcocystis spp. sporocysts were detected in one of the transplant recipients. All identified coccidia were confirmed by PCR. There was a significant relationship between the rate of intestinal parasite infection and certain variables. Conclusion: Given the potential risk of certain intestinal parasites in people with immune deficiency, it is recommended that diagnosis of parasitic infections in such patients be based on specific parasitological methods. Thus, it is advisable that physicians refer them to a parasitology laboratory prior to drug administration. Amaç: Bu çalışmanın amacı, İran’ın güneybatısındaki Bushehr eyaletindeki immün yetmezlikli hastalarda bağırsak paraziter enfeksiyonlarının durumunu konvansiyonel ve moleküler yöntemlerle belirlemektir. Yöntemler: Böbrek nakli alıcılarından, AIDS hastalarından ve kemoterapi alan hastalardan olmak üzere toplam 201 dışkı örneği toplandı. Kontrol grubu numuneleri sağlıklı insanlardan toplandı. Numuneler çeşitli konvansiyonel yöntemler kullanılarak test edildi. Polimeraz zincir reaksiyon (PZR) testi, doğrudan mikroskobik incelemelerde tespit edilen pozitif Coccidia örnekleri üzerinde yapıldı. Bulgular: Hastaların yaklaşık %32,45’i en az bir tür bağırsak paraziti ile enfekte olmuştu. En yüksek (%46,8) ve en düşük enfeksiyon oranı (%24) sırasıyla AIDS ve kemoterapi hastalarında görülürken, kontrol grubunun enfeksiyon oranı %16 idi. Isospora spp. ve Cryptosporidium spp. tüm hasta gruplarında ve Sarcocystis spp. sporokistleri ise transplant alıcılarından birinde tespit edildi. Tanımlanan tüm Coccidialar, PZR ile doğrulandı. Bağırsak parazitlerinin oranı ile bazı değişkenler arasında anlamlı bir ilişki mevcuttu. Sonuç: İmmün yetmezliği olan kişilerde bazı bağırsak parazitlerinin potansiyel riski göz önüne alındığında, paraziter enfeksiyonların spesifik parazitolojik yöntemlere dayanarak teşhis edilmesi önerilir. Bu nedenle, doktorların ilaç vermeden önce bu hastaları parazitoloji laboratuvarına yönlendirmeleri tavsiye edilir

Comparison of various methods for DNA extraction from human isolated paraffin-embedded hydatid cyst samples

Successful molecular research with reliable results depends on achieving significant and uniform amounts of genomic DNA from the parasite as the first and most basic step. Therefore, selection of an appropriate method that minimizes damage to the DNA of the parasite, is very important. In this study, we are going to describe a method that can extract DNA from human isolated paraffin-embedded hydatid cysts with a high quality and quantity. Formalin fixed and Paraffin-embedded hydatid cyst samples isolated from human lung and archived in the pathology laboratory were used for this purpose. Several sections of the paraffin blocks were prepared with 5 micron thickness and DNA were extracted by three different methods including; modified boiling, commercial kit and the method described by Larissa A. Pikor et al. The obtained DNA were evaluated by Nanodrop in terms of the yield of DNA and possible contaminations. To compare the quality of DNA prepared, cox1 region was amplified using specific primers. It was found that the DNA extracted by modified boiling had the lowest rate of contamination and the best electrophoretic band on the gel, compared to other two performed methods. Considering the findings of this study, this simple and high throughput DNA extraction method with high yield and quality can be recommended for extraction of DNA from formalin fixed and paraffin-embedded hydatid cysts

Genotyping and phylogenetic analysis of hydatid cysts isolated from livestock in Bushehr province, Iran

Hydatid cyst is one of the parasitic zoonoses caused by infection with the larval stage of Echinococcus granulosus tapeworm. The spread of this parasite is global and is of great importance in terms of public health. To date, ten different species of this parasite have been identified that differ in characteristics such as life cycle, epidemiology and pathogenesis. The purpose of this study was to determine the genotype and phylogenetic relationship of hydatid cysts isolated from livestock of Bushehr province, Iran. About 62 samples of hepatic and pulmonary hydatid cysts were collected from slaughtered animals. DNA extracted by phenol–chloroform method was amplified by PCR using primers specific for the cox1 gene. The PCR products of 50 samples were sequenced and analyzed using BioEdit software and compared with sequences in the GenBank. The phylogenetic tree was drawn using Neighbor Joining tree-NJ method, and its reliability was evaluated. Sequencing results showed that out of 50 sequenced samples, 43 samples had the genotype of Echinococcus granulosus and 7 samples had the genotype of Taenia hydatigena. By drawing a phylogenetic tree, all 43 hydatid cyst samples belonged to G1 strain. The predominance of G1 strain of hydatid cyst in livestock of Bushehr province shows the main role of this genotype in establishing the life cycle of parasite in this region and if the genotype of the parasite in dogs and humans is determined, then these findings can be used to disrupt the life cycle of the parasite and reduce the human infections

In vitro study of antibacterial activity of the alga Sargassum oligocystum from the Persian Gulf

Background and Objectives: With due attention to the development of drug-resistant bacteria, discovering of new antibacterial compounds is needed. Algae produce numerous bioactive substances which may have pharmacological properties such as antibacterial activity. The objective of this investigation was to in vitro study of antibacterial activity of brown alga Sargassum oligocystum collected along the Bushehr coast of Persian Gulf (south west of Iran). Materials and Methods: Hot water extract, cold water extract, and hot glycerin extract were prepared. The effect of the extracts were investigated on Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 14990), Pseudomonas aeruginosa (ATCC 27853), and Escherichia coli (ATCC 25922). Results: Hot water extract exhibited antibacterial activity against Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa. Cold water extract and hot glycerin extract did not show antibacterial activity on any of the four test bacteria. The minimum inhibitory concentration (MIC) of hot water extract for both Staphylococcus aureus and Staphylococcus epidermidis was 3.175 mg/ml. However, the MIC of this extract for Pseudomonas aeruginosa was 9.556 mg/ml. Discussion: In this study gram-positive bacteria were more susceptible to hot water extract than gram-negative bacteria. Extract of Sargassum oligocystum could be a candidate for purification and further in vivo studies

Seroprevalence of Toxocariasis in schoolchildren in Bushehr, Iran

Background: Toxocariasis is an infection caused by larvae of a group of animal nematodes named Toxocara in human tissues. The disease often affects children under the age of 10 years. In this research, we decided to determine the prevalence of infection in schoolchildren of Bushehr city as a first study in the region. Methods: In this cross-sectional study, the samples were selected randomly among Bushehr primary school students between September 2011 and June 2012. ELISA method was used to measure anti-Toxocara canis IgG antibodies. Data were collected by questionnaire and analyzed using SPSS 18.0 software and Chi square test. Results: Of the 400 students studied, 48 (12%) were positive for anti-Toxocara IgG antibodies. Results showed that there is no a statistically significant relationship between infection and some of the analyzed variables such as sex, abdominal pain, anorexia, chronic cough, diffuse pain, itching and skin sensitivity. However, with the habit of playing with soil, this relationship was significant. Conclusion: Considering the importance of the disease in children and influence of harmful effects on their health, it is suggested that the methods for diagnosis be trained for clinicians in the area and periodically tests as well as teaching the prevention and control strategies should be considered to the educational staff and parents