Iranian Journal of Parasitology
Volume:6 Issue: 4, Oct-Dec 2011

The main goal of the present study was to develop a new sensitive and specific PCR based method for Identification of Cryptosporidium sp. using novel primers from 18S ribosomal RNA. Cryptosporidi­osis in high-risk host groups particularly in neonates and immuno-compromised individuals may result in death. To the best of our knowledge this is the first study regarding develop a new PCR based method to diagnose the cryptosporidiosis in Iran. A total of 850 human fecal samples from patients clinically suspected to cryptosporidiosis and 100 healthy and diarrheic cattle stool specimens were collected. The simplified formol-ether concentration method was carried out for all samples. They were then examined microscopically by modified Ziehl-Neel­sen staining method. Total DNA was extracted by QIA amp DNA stool mini kit was carried out by using designed prim­ers. Twenty nine cases of cryptosporidiosis infection in human and 30 samples from cattle microscopi­cally were posi­tive. The described primary and nested PCR method could detect all Cryptospori­dium positive samples from human and cattle. Regards to suspected negative samples in pri­mary PCR examination, the Nested PCR could ap­prove two more positive results. Furthermore, Nested PCR analysis was able to detect one more case which was nega­tive in both microscopically examination and primary PCR. Specificity of the test was 100%. Sensitivity of Nested PCR in comparison to our gold standard; microscopy after Ridley concentration modified ziehl-Neelsen, was 100 %. Our developed PCR based method by using new primers devised from 18S ribosomal RNA revealed the ability for identification of the Cryptosporidium species such as C. parvum and C. huminis with high specificity and sensitivity.

Cytoadherence of Plasmodium falciparum- infected red blood cells to endothelial cells is an important mechanism for parasite survival and a major trigger for diseases pathology. Here, we de­scribe a new adhesion assay in which different cell types (CHO, CHO/CD36 and CHO/ICAM-1) are at­tached to Cytodex beads in a mini-column format to measure the relative sizes of various binding subpopula­tions as a percentage of the total population. Relative size of CD36 and ICAM-1-binding subpopulations of erythrocytes infected with P. falcipa­rum were measured by amount of parasitemia before and after passing the infected erythrocytes through a particular column. The mini-column adhesion assay was a suitable method as parasitemia always reduced after pass­ing through a particular column in independent experiments. For example, in a typical experiment us­ing P. falciparum ITG line, 75% of the parasites are retained on a CHO/ICAM-1 while 0% of clone 3D7 is retained. This work introduced and validated a method for measuring the relative size of parasite bind­ing subpopulations and the selection of them. Also, the mini-column method is of value for assessments of cytoadherence and can be used as tool for different applications.

The purpose of this comparative study was to detect superoxide dismutase (SOD) activities in Fasciola hepatica, F. gigantica parasites, infected and healthy liver tissues in order to determine of species effects and liver infection on SODs activity level. Fasciola spp. parasites and sheep liver tissues (healthy and infected liver tissues), 10 samples for each, were collected, homogenized and investigated for protein measurement, protein detection and SOD enzyme activity assay. Protein concentration was measured by Bradford method and SODs band protein was detected on SDS-PAGE. SODs activity was determined by iodonitrotetrazolium chloride, INT, and xanthine substrates. Independent samples t-test was conducted for analysis of SODs activities difference. Protein concentration means were detected for F. hepatica 1.3 mg/ ml, F. gigantica 2.9 mg/ml, healthy liver tissue 5.5 mg/ml and infected liver tissue 1.6 mg/ml (with similar weight sample mass). Specific enzyme activities in the samples were obtained 0.58, 0.57, 0.51, 1.43 U/mg for F. hepatica, F. gigantica, healthy liver and infected liver respectively. Gel electrophoresis of Fasciola spp. and sheep liver tissue extracts revealed a band protein with MW of 60 kDa. The statistical analysis revealed significant difference between SOD activities of Fasciola species and also between SOD activity of liver tissues (P<.05). Fasciola species and liver infection are effective causes on SOD enzyme activity level.

Coccidiosis of domestic fowl, caused by species of the Genus Eimeria, is responsi­ble for important economic losses in poultry production. Because different species and/or strains can vary in pathogenicity and other biological parameters, their precise characteriza­tion is important for epizootiological studies. Fifty samples from litter, whole intestinal tract and feces were collected from poul­try houses located in different provinces of Iran. One hundred twenty male day-old broi­ler chicks were challenged with three selected isolates. Data on weight gain, Food Conversion Ratio (FCR), food intake, lesion scoring and shedding of oocysts per gram of feces were rec­orded and analyzed by the Duncan's test. In all treatments, the challenged groups had statistically significant lower weight gain than that of unchallenged control group. Isolate three caused the lowest weight gain and food intake and the worst lesion score as well as FCR. Despite originating from close geographi­cal regions for isolates 1 and 2, the difference in biopathologic factors may be either due to different proportion of identified species or the different pathogenicity of the species present in the isolates. The results highlight the importance of considering various species of Eimeria in designing the preventive, control and treatment strategies to prevent coccidiosis in different regions of Iran. Further characterization of each isolate would be the next step to provide a basis for coccidiosis research with well-characterized local isolates.

Hydatidosis is the larval stage of the Echinococcus granulosus. This disease is en­demic in Iran. There are many studies about hydatidosis in different regions of the country, but there is not any information about the disease in Kermanshah Province. This article will review all available data about hydatidosis in this province. Using web based search engines and a survey on medical student's theses, all the informa­tion about hydatid cysts in the province from 1986 -2008 was collected. During these twenty years, at least 482 proven cases of hydatid cyst have been identified in the province. Accordingly, the trend of hydatid cyst operation in the province has been grow­ing and the average annual number of cases has reached 1.41/100,000. Frequency of disease in urban areas was slightly higher than rural areas and the rate of infection in housewives was more than others. Because of the growing trend of hydatid cyst operation in Kermanshah Province, which may be due to many different reasons, this province should be considered as one of the impor­tant endemic regions of hydatidosis in Iran.

Cryptosporidium is a worldwide protozoan parasite and one of the most common causes of infection and diarrhea in humans and cattle. The aim of the present study was determina­tion of subtypes of Cryptosporidium among children with diarrhea in Tehran by se­quence analysis of the highly polymorphic 60-kDa glycoprotein (GP60) gene. Fecal samples were collected from 794 diarrheic children. Initial identification of Crypto­spo­ridium was carried out on stool samples by Ziehl-Neelsen acid-fast staining method. DNA was extracted from positive microscopically samples and Cryptosporidium genotypes and subtypes were determined, accordingly. Out of 794 collected samples, 19 (2.40 %) were positive for Cryptosporidium oocysts. Sequences analysis of GP60 gene showed that 17 (89.47 %) of the positive isolates were Crypto­spori­dium parvum and 2 (10.52 %) were C. hominis. All subtypes of C. parvum isolates belonged to allele families IIa (6/17) and IId (11/17). The most common allele in all 17 isolates belonged to IId A20G1a (41.18%). A22G1 (IF) subtype was detected in two C. hominis isolates of the chil­dren. The predominancy of C. parvum species (specially, IId A20G1a sub­type) in current study underlines the importance of zoonotic Cryptosporidium transmission in Iran.

Cystic echinococcosis (CE), caused by hydatid cysts, is a widespread and hazard­ous disease in humans and animals worldwide. The disease is very common in Turkey, causing serious economic losses and public health problem. In this study, the seroprevalence of equine CE was determined by enzyme-linked immunosorbent assay (ELISA). Partially purified cyst fluid antigen from sheep hydatid cyst fluid was used as antigen in ELISA. A total of 250 equids consisting of 206 donkeys and 44 horses from various regions of Erzurum province of Eastern Turkey. Anti- Echinococcus granulosus antibodies were detected in 78 (31.2%) of 250 equids. The prevalence rate was 20.4% for horses and 33.5% for donkeys. There was no statistically differ­ence between sex and ages groups for both horses and donkeys (P>0.05). Equine CE is quite endemic in Eastern Turkey. The high prevalence of CE suggests that equids in the transmission cycles is possible as a source of infection for definitive hosts.

New cases of visceral leishmaniasis (VL) have been reported recently in some parts of Mazandaran Province, north of Iran where the first human case of VL was reported in 1949. This study aimed to determine the present status of Leishmania infantum infection among humans and domestic dogs using serological and molecular methods in central parts of Mazandaran Province. In this cross-sectional study, blood samples were randomly collected from 402 humans and forty-nine domestic dogs throughout 2009 and 2010 in the central part of Mazandaran Province including Semeskadeh and Kiakola districts where recent cases of human visceral leishmaniasis had been reported there. All the collected samples were tested by direct agglutination test (DAT) for the detection of anti-Leishmania infantum antibodies as well as convenience PCR assay on whole blood samples for detection of leishmanial infection and identification of Leishmania species. None of 402 collected human (402) and dog (49) blood samples showed anti Leishmania infantum antibodies at titers 1:3200 and 1:320 as cut-off values of DAT, respectively but only 2 of domestic dogs (4.1 %) were found PCR-positive corresponding to L. infantum. This study confirms the circulation of L. infantum at least among domestic dogs and highlights the sporadic pattern of VL in the studied areas. Further investigations regarding to sand flies fauna and wild canines as reservoir hosts of the disease, are recommended.

Leishmaniasis is a worldwide disease prevalent in tropical and sub tropical coun­tries. Many attempts have been made and different strategies have been approached to develop a potent vaccine against Leishmania. DNA immunisation is a method, which is shown to be effec­tive in Leishmania vaccination. Leishmania Soluble Antigen (SLA) has also recently been used Leishmania vaccination. The immunity generated by SLA and L. mexicana gp63 cDNA was compared in groups of 6 mice, which were statistically analysed by student t- test with the P-value of 0.05. SLA was administered by two different methods; intramuscular injection and injection of den­dritic cells (DCs) loaded with SLA. L. mexicana gp63 cDNA was administered by the gene gun. Immunisation of BALB/c mice with L. mexicana gp63 resulted in high levels of Th1-type immune response and cytotoxic T lymphocytes (CTL) activity, which were accompanied with protection induced by the immunisation against L. mexicana infection. In contrast, administra­tion of SLA, produced a mixed Th1/Th2-type immune responses as well as a high level of CTL activity but did not protect mice from the infection. The results indicate higher protection by DNA immunisation using L. mexicana gp63 cDNA compared to SLA, which is accompanied by a high level of Th1 immune response. However, the CTL activity does not necessarily correlate with the protection induced by the vac­cine. Also, gene gun immunisation is a potential approach in Leishmania vaccination. These find­ings would be helpful in opening new windows in Leishmania vaccine research.

Food contamination may occur through production, processing, distribution and prepara­tion. In Iran especially in Khorramabad, 33° 29' 16" North, 48° 21' 21" East, due to kind of nutrition, cul­ture and economic status of people, bread is a part of the main meal and the consumption of bread is high. In this study, the bakery workers were studied for determining of intestinal parasites prevalence. The study was carried out during September to November 2010 in Khorramabad. All the 278 baker­ies and the bakery workers including 816 people were studied in a census method and their feces were examined for the presence of parasites by direct wet-mount, Lugol's iodine solution, and formalde­hyde-ether sedimentation, trichrome staining, and single round PCR (For discrimination of Entamoeba spp). Ninety-six (11.9%) stool specimens were positive for different intestinal parasites. Intestinal para­sites included Giardia lamblia 3.7%, Entamoeba coli 5.5%, Blastocystis sp. 2.1%, Entamoeba dispar 0.4%, Hymenolepis nana 0.1%, and Blastocystis sp. 0.1%. In order to reduce the contamination in these persons, some cases such as stool exam every three months with concentration methods, supervision and application of accurate health rules by health ex­perts, training in transmission of parasites are recommended.

Members of the Vannellidae family are free-living amoebae (FLA) distributed mainly in water and soil sources. The present study reports the first isolation of this genus in the biofilm source from hospital environment in Tehran, Iran. Biofilm samples were collected from hospital environment. Cultivation was performed in non-nutrient agar covered with a heat-killed Escherichia coli. Cloning of the suspected amoe­bae was done. PCR amplification and Homology analysis using the Basic Local Alignment Search Tool (BLASTn) was performed to search for the most similar reference sequences. Microscopic examination showed numerous fan-shaped amoebae and peculiar cysts differ­ent to the usual shape of typical FLA. Sequence analysis of the PCR- product revealed that the suspected amoebae are highly homologous with Vannella spp. gene (99% identity and 100% query coverage) available in the gene bank database. Although Vannella spp. is not proved to be pathogenic itself, but they are capable of har­boring pathogenic intracellular organisms such as Microsporidian parasites. Thus, identifica­tion of such amoebae can be of clinical importance, as they could lead to transmission of other pathogens to human.

Toxoplasma gondii is a zoonotic protozoan parasite found worldwide and responsi­ble for major economic losses in most classes of livestock. This study was aimed to deter­mine the prevalence of T. gondii infection in sheep, cattle and horses in Urmia, north-west of Iran, using MAT. Blood samples of 276 livestock and 26 horses were collected from July 2009 till April 2010. The data were analyzed by the Chi-square, Fisher's Exact and Cochran's and Mantel-Haen­s­zel Tests. The level of significance was set at P < 0.05. Thirty-three (21.1%) sheep, 2 (1.6%) cattle and 3 (11.5%) horses were seropositive to T. gondii. Analysis showed that sheep were 15 times more likely to be seropositive comparing to cattle also 2 times more likely to be seropositive than horses. This study showed seroprevalence of equine T. gondii infection with a considerable rate in sheep in Urmia, northwest of Iran. More comprehensive studies on livestock toxoplasmo­sis are required for further analysis of the parasite reservoir for human infection.

The aim of this study was to determine the seroprevalence of antibody to Neospora can­inum in healthy and aborted dairy cattle in Tabriz, capital of East-Azarbaijan in northwest of Iran. In this cross-sectional study serum samples were collected from 266 healthy and ab­orted Holestein-Feriesisnc cows from September 2008 to August 2009. The sera were analyzed to de­tect of antibody against N. caninum using the commercially ELISA kit. Seroprevalence of antibody to N. caninum was 10.5% in Tabriz dairy cattle. Also the abortion rate in all cattle sampled was 33.6% but percentage of seropositive aborted cattle was 18.4%. Neosporosis could be one of the possible causes of abortion in dairy cattle in Tabriz and regarding the distribution in dogs as definitive host for the parasite, further studies in dog and cat­tle are recommended.

In the urine of a patient with chronic prostatitis, renal microlithiasis and acute cystitis we found the ciliate protozoa Colpoda spp., both in vegetative and cystic form. The entry point was most likely the urinary tract. Keeping in mind that only four more cases of Colpoda spp. existent in human urine have already been described, and that in the case of our patient the ciliate was present at repeated examinations of his urine, we presumed that it is not only a spurious infection of the urogenital tract. It still remains to be analyzed whether this ciliate belongs to a species of Colpoda adapted to parasitism in homeothermae and whether it can be pathogenic for humans.

The Asian cheetah is known as Iranian panther. A four years old female cheetah was killed in a road accident by a truck in Abbas Abad (Biarjamand) County around Shahrood City in Sem­nan Province, central part of Iran. Two days after the accident the carcass of animal was autopsied and only five cestodes were obtained from its intestine. In inspection of other or­gans no other helminth was observed. Cestod samples were fixed and stained by carmine acid. Characterization of the cestodes using morphological standard key, identified the ces­todes as Taenia acinonyxi.