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Gholamhossein Edrissian, Pharm. D.
Vol 7 No 4 (2012)
Background: The main goal of present study was to detect polymorphism in MSP-1 gene which is a major blood stage candidate for vaccine in Plasmodium vivax by Single Strand Conformational Poly- morphism-Polymerase Chain Reaction (SSCP-PCR).
Methods: During 2008 to 2010 fifty samples were collected from Iranian patients with P. vivax in Hormozgan Province, southern Iran. All of the samples were detected by microscopical examination. Amplification of MSP-1 gene was done by PCR after DNA extraction. Single strand DNAs due to using in SSCP, was electrophoresed on polyacrylamid- Bisacrylamid gel then banding patterns were revealed by silver-staining method. Sequencing as a typing method was performed for some isolates.
Results: All of the 50 isolates were positive microscopically. Totally 12 (24%) isolates showed 440 bp and 38 (76%) showed 500 bp in PCR assay. SSCP analysis revealed four banding patterns. Pattern I (10/50), Pattern II (12/50), Pattern III (27/50), and Pattern IV (1/50). The results sequencing analysis of the MSP-1 gene in 19 isolates revealed diversity in nucleotides and amino acid in Iranian P. vivax isolates.
Conclusion: Our study confirms that the SSCP-PCR is a rapid method for detecting polymorphism in MSP-1 gene in P. vivax. The presence of different haplotypes in MSP-1 gene shows that several P. vivax strains exist in malaria endemic areas of Iran.
Background: The aim of the present study was to determine the molecular characteristics of Echinococcus granulosus from paraffin-embedded tissues of hydatid cysts isolated from human and protoscoleces of hydatid cysts from sheep, cattle and camel isolates using PCR- RFLP of ITS1- rDNA analysis in Golestan Province, northern Iran.
Methods: E. granulosus isolates from human patients infected with hydatid cyst and protoscoleces from hydatid cysts of sheep, cattle and camel isolates were collected from different hospitals and the abattoir throughout the Golestan Province. In all, 60 E. granulosus genomic DNA were extracted and examined by PCR - ITS1 of rDNA and amplified using BD1 / 4S and EGF1 / EGR2 primers, fol- lowed by RFLP using Alu1, Msp1 and TaqI restriction enzymes.
Results: The PCR-ITS1 products obtained from sheep, cattle and human isolates were similar to sheep strain (1000 bp and 391 bp). Majority of the camel samples yielded 295 bp DNA bands. RFLP-ITS1 of E. granulosus with Taq1 in human, sheep and cattle isolates showed similar patterns in the number and size of DNA. RFLP methods in camel isolates showed a different genotype, using Taq1, whereas no DNA bands were observed using Alu1 in camel and human isolates. Therefore, two clearly distinguishable banding patterns of E. granulosus were obtained with the three enzymes, which separating human, sheep and cattle isolates from the camel origin.
Conclusion: The results indicate the possible of transmission of the G1 and G6 genotypes of E. granulosus between livestock animals and human in Golestan Province.
Background: Toxoplasmosis is a serious disease in immunocompromised patients and pregnant women. Differentiation of acute and chronic infection is a major challenge in serodiagnosis of the disease. Since the aim of this study was to assess the diagnostic utility of recombinant SAG1 (rec- SAG1) for the detection of Toxoplasma-specific IgM antibodies in human sera, by an enzyme-linked immunosorbent assay (ELISA).
Methods: The purified recombinant protein SAG1 was applied in house ELISA test and the ability of it in binding to specific immunoglobulin M in 30 serum samples of acute infected patients was evaluated. The results obtained by assays with the recombinant SAG1 and standard commercial as- says were compared.
Result: The sensitivity and specificity of in house ELISA compared to a standard commercial ELISA (com-ELISA) were 80% and 90%, respectively.
Conclusion: It was concluded that the rec-SAG1 could be an alternative marker for detection of anti Toxoplasma-specific IgM and diagnosis of acute infection.
Background: Acanthamoeba spp. are free-living amoebae commonly found in the environmental sources such as water, soil, and air. This ubiquitous amoeba is the causative agent of amoebic kerati- tis (AK). The objective of the present study was to investigate the presence of Acanthamoeba spp. in water and soil sources in Ahvaz City, Khuzestan Province, southern Iran.
Methods: In general, 110 samples of water and soil were taken from different localities of Ahvaz including agricultural canals, rivers, and swimming pools. Filtration and cultivation were carried out on non-nutrient agar medium (NNA). Axenic cultivation was performed for all of positive isolates. PCR analysis was conducted on positive samples. Sequencing was done for 15 PCR products. Geno - types were identified by Blast search and homology analysis.
Result: Acanthamoeba spp. was found in 43 (71.6%) of samples of water and 13 (26%) soil samples. Genotyping of 15 samples proved that Acanthamoeba belonged to T4 (86.6%), T2 (6.6%), and T5 (6.6%) genotypes.
Conclusion: TYI-S-33 medium could be better than PYG medium for Acanthamoeba axenic culture.
Background: Leishmania is an intracellular parasite infecting humans and many wild and domestic animals. Recent studies have suggested an important role for cytotoxic T cells against Leishmania. Peptide-based vaccines targeting short sequences derived from known immunogenic proteins have been shown to elicit cellular immune responses against disparate pathogens.
Methods: We predicted four HLA-A2 peptides derived from L. mexican/major gp63 and tested these in HHD II mice, as well as four peptides for mouse MHC class I from the same proteins tested in BALB/ mice.
Results: The results revealed immunogenicity for three of the four peptides predicted for HLA-A2.Immunisation with these peptides, along with IFA, induced CTL responses detected by standard 4- hour cytotoxicity assay and significantly upregulated the production of IFN-γ. When HHDII mice were injected IM with L. mexicana gp63 cDNA and splenocytes were restimulated with blasts loaded with the immunogenic peptides, two of the peptides were able to induce significant level of IFN-γ detected by ELISA. None of the peptides predicted for Balb/c mouse MHC class I elicited CTL ac- tivity or significantly upregulated the IFN-γ.
Conclusion: The results may help in developing a peptide-based vaccine, which can be applied alone or in combination with drugs against Leishmania.
Background: Due to the lack of a suitable and economic test for the analysis of the polymorphism at codon 167, we developed a new PCR-RFLP technique, based on a modified forward primer (UT- HC167 MF-primer), to identify simultaneously the SNPs at codons 167 and 200 of isotype 1 β-tubu- lin gene of Haemonchus contortus.
Methods: There already are several safe and easy methods for identification of point mutations at codons 198 and 200. Due to the lack of a reliable and easy method for the detection of the single nucleotide polymorphism (SNP) at codon 167, we developed an innovative PCR-RFLP technique based on a modified forward primer (UT-HC167 MF-primer), in which the nucleotide T at the posi- tion 443 was substituted through a nucleotide A creating a restriction site for restriction endonuc- lease SnaB I in the nucleotide sequences including codon 167. A total of 138 adult male H. contortus were collected from three different geo-climatic areas of Iran. The isolated genomic DNA of each single worm was amplified by PCR using primers flanking codon 167. The PCR product (527 bp) was then amplified by semi-nested PCR using the UT-HC167 MF-primer and the reverse primer achieving a PCR product of 451 bp in length. This PCR product was subsequently digested with the restriction endonucleases SnaB I and TaaI for analysis of the mutations at codons 167 and 200, re- spectively.
Results: All worms had two alleles encoding for phenylalanine (BZss homozygote) for both codons.
Conclusion: Using the UT-HC167 MF-primer and a suitable reverse primer designed upstream from codon 200, it is possible to amplify a PCR product which can be used for analysis of the SNPs at all three mentioned codons using RFLP.
Background: Visceral leishmaniasis (VL) or kala-azar is a parasitic disease caused by the species of Leishmania donovani complex. It is endemic in some parts of provinces of Iran. According to the reported cases of VL in Kermanshah Province in recent years, this study was conducted to determine the seroprevalence of VL in high risk villages of the province.
Methods: Totally, 1622 serum samples obtained from children under 15 years old and 178 from adults in 22 villages of studied areas. Serum samples were examined by direct agglutination test (DAT) for the detection of anti-Leishmania antibodies. Data were analyzed using SPSS software ver.11.5.
Results: Only 6 serum samples (0.33%) showed anti-Leishmania antibodies against L.infantum at titers ≥1/3200. Four of the seropositive cases had a history of kala-azar and Leishman bodies were seen in their bone marrows. The highest (0.5%) and lowest (0.29%) seroprevalence was seen in the age groups of 5-9 and 10-14 years old, respectively. None of the adults were seropositive. There were not any significant differences be-tween the rate of seropositivity in males (0.36%) and females (0.31%). 66.7% of seropositive individuals showed clinical manifestations. The most important symptoms in Kala-azar patients were fever, hepato- spleenomegally and anemia.
Conclusion: Kala-azar is occurred sporadically in Kermanshah Province. But presence of significant number of positive sera confirms the necessity for attention of people and clinicians to kala-azar.
Background: The objective of this investigation was to detect the presence of Trichinella in some carnivores of Mashhad in northeast of Iran and to identify Trichinella species circulating in this area.
Methods: The present study was carried out using muscle tissue collected from 120 stray dogs, 26 wild boars, 25 rodents, two foxes and two hyenas captured in Mashhad City, province of Khorasan Razavi, Iran.
Results: Trichinella larvae were detected in three stray dogs by artificial digestion and compression.All larvae were identified as T. britovi using multiplex PCR.
Conclusion: This is the first report of identification of T. britovi in stray dog in Iran.
Background: Animal models with various combination of host-parasite have long been employed to study malaria pathogenesis. Here, we describe the combination of Plasmodium berghei ANKA infec- tion in inbred ICR mice as a model of cerebral malaria (CM).
Methods: Infection in mice was initiated by intraperitoneal injection of 2 x 107 (0.2ml) parasitized red blood cells (PRBCs).
Results: This model can produce a severe degree of infection presented by the high degree of parasitaemia followed by death 6-7 days post infection. Severe anemia, splenomegaly, hepatomegaly and discolourations of major organs were observed. Histopathological findings revealed several i m- portant features mimicking human CM including, microvascular sequestration of PRBCs in major organs, particularly in the brain, hypertrophy and hyperplasia of the kupffer cells in the liver, pulmo- nary edema and hyaline membrane formation in the lungs and haemorrhages in the kidney’s medulla and cortex. Proinflammatory cytokines TNFα, IFNγ, IL-1, IL-6 and IL-18, and anti-inflammatory cytokine IL-10 were all found to be elevated in the plasma of infected mice.
Conclusion: This model can reproduce many of the important features of CM and therefore can be used as a tool to advance our understanding of the disease pathogenesis.
Background: Mediterranean type of visceral leishmaniasis (VL) is present in different parts of Iran. Several studies have identified dogs as the main reservoirs of the VL caused by Leishmania infantum in Iran and other Mediterranean regions. This study aimed to determine the seroprevalence of canine visceral leishmaniasis as animal reservoir host for human visceral leishmaniasis in Boyer Ahmad di s- trict in southwest of Iran.
Methods: A seroepidemiological study was carried out to determine the seroprevalence of canine visceral leishmaniasis (CVL) among ownership dogs by using direct agglutination test (DAT) in 23 of 182 villages of Boyer Ahmad district, during August 2009 to August 2010. One hundred and seventy serum samples from ownership dogs were selected by multi-stage cluster sampling in villages of Boy- er Ahmad district. All samples were tested by DAT and anti-Leishmania antibodies titers at ≥ 1:320 was considered as positive.
Results: Of the 170 serum samples, 10% were positive by DAT at titers of 1:320 and higher. No statistical significant difference was found between male (10.7%) and female (8.3%) seroprevalence. The highest seroprevalence rate (15.1%) was observed among the ownership dogs of four to seven years age. Altogether, seventeen (25.4%) of the seropositive dogs had clinical signs and symptoms.
Conclusion: It seems that Boyer Ahmad district is an endemic area for canine visceral leishmaniasis in Iran.
Background: Cercarial dermatitis is known as an endemic parasitic disease in North of Iran, a hy- persensitive skin reaction to the penetration of nonhuman schistosome larvae into human skin. In recent studies in this region, final and intermediate hosts were recognized and Trichobilharzia was identified as the main causative agent of cercarial dermatitis in this region, but to date the parasite species haven’t been identified. Therefore this study was performed to species identification of nasal Trichobilharzia in infected birds for the first time.
Methods: A total of 45 Anas clypeata birds identified as final host, were collected from Sari in North of Iran and infected nasal tissues analyzed using molecular techniques. Genomic DNA was isolated by phenol/chloroform extraction method and ITS region of rDNA were amplified with specific pri- mers its5Trem and its4Trem, then sequenced area were compared with existing records in GenBank.
Results: Twelve samples were infected with Trichobilharzia and results of PCR reaction indicated that all of them belonged to T. regenti. The sequence alignment of present work isolates and those depo- sited in GenBank showed differences in nucleotide sequences of repeat region in ITS1.
Conclusion: Trichobilharzia regenti is the most frequent parasite of Anatid birds in North of Iran. This corresponds to the distribution of this parasite along the flyway of migratory birds, which annually migrate from Siberia and northern countries of Caspian Sea to wintering areas in southern regions of it.
Background: In this study the concentration of lysozyme in blood plasma of Microtus agrestis, Clethrinomys glareolus, Apodemus sylvaticus, BK rats and outbred white mice before and after infection with culture forms of Trypanosoma microti, T, evotomys, T. grosi, T. lewisi and T. musculi respectively was measured.
Methods: Blood samples of rodents, Microtus agrestis, Clethrionomys glareolus, Apodemus sylvaticus, BK rats and outbred mice infected with T. microti, T. evotomys, T. grosi, T. lewisi and T. musculi respectively were collected in heparinized micro- tubes immediately before inoculation and 3, 6, 12, 24, 48, 96 and more than 400 days after intra- perituneal inoculation with 5×105of their homologous trypano- some parasites of which more than half were metacyclic trypomastigote in 0.2 ml of culture medium. Micro- tubes were centrifuged and plasma samples were separated and the lysozyme activity was measured by the agar method.
Results: Levels of lysozyme rose rapidly three to six days after the inoculation to ten to twenty than their pre- infection levels. They then gradually decreased, although after more than one year they were still two to ten folds higher than controls. The highest level measured occurred in rats infected with T. lewisi and the lowest in A. sylvaticus infected with T. grosi. After one year the highest concentra- tion of lysozyme was in mice infected with T. musculi and lowest in A. sylvaticus.
Conclusion: Persistent enhanced lysozyme levels may prevent re- infection with trypanosomes.
Background: Anaplasma ovis infections can cause clinical symptoms in acute phase and lead to huge economic losses in flocks. The aim of the present study was to investigate the hematological and par- asitological changes in experimental anaplasmosis in sheep with Iranian strain of A. ovis.
Method: Five male sheep without any blood parasite infection were selected. One hundred ml hepa-rinized blood was collected from splenectomised sheep that showed 6% A. ovis parasitemia. Inocu- lums of 20 ml blood were administered intravenously to each test animal. Hematological, parasito- logical and clinical changes of experimental anaplasmosis were studied in 0-38 days post infection.
Result: Parasitemia was detected 3 days post infection and reached its maximum level on the day 12 of experiment in test animals. Then the parasitemia was declined, but the organism could be found persistently until the last day of study. The red cell counts, packed cell volume and hemoglobin con- centration were decreased and mean corpuscular volume was increased significantly during the infec- tion period. Reticulocytosis and basophilic stippling were also detected. No significant changes were observed in total and differential leukocyte count and animal body temperature.
Conclusion: Experimental A. ovis infection in sheep resulted in marked normocytic normochromic anemia at the beginning of the infection which became macrocytic normochromic by the develop- ment of the disease. There were negative correlations between parasitemia and RBC, PCV and Hb values, therefore hematological assessment can be considered as a practical diagnostic tool in ovine anaplasmosis.
Background: We attempted to determine the prevalence of Hepatozoon spp. infection in Mashhad, northeast of Iran, via blood smear parasitology.
Methods: The prevalence was investigated by examination of blood smear parasitology, using blood samples collected from 254 dogs (51 strays and 203 privately owned-dogs).
Results: Two stray dogs (2/51; 3.92%) and two privately-owned dogs (2/203; 0.98%) were infected with Hepatozoon spp. Therefore, as per blood smear parasitology, the prevalence of Hepatozoon spp. infection was 1.57% (4/254). Sixteen out of 254 dogs (6.29%) were infested with ticks; all of which were Rhipicephalus sanguineus. One of the dogs infected with Hepatozoon spp. exhibited ticks at the time of examination. Concurrent infection with Ehrlichia canis and Leishmania infantum was not detected in the four Hepatozoon spp. infected dogs.
Conclusion: This is the first epidemiological study on the prevalence of Hepatozoon spp. infection in dogs in Iran.
Background: Malaria is still one of the most important infectious diseases in the world. The disease also is a public health problem in south and southeast of Iran. This study programmed to show the correlation between regular malaria microscopy training and refresher training courses and control of malaria in Iran.
Methods: Three types of training courses were conducted in this programme including; five – day, ten – day and bimonthly training courses. Each of the training courses contained theoretical and practical sections and training impact was evaluated by practical examination and multiple-choice quizzes through pre and post tests.
Results: Distribution pattern of the participants in the training and refresher training courses showed that the most participants were from Sistan & Baluchistan and Hormozgan provinces where malaria is endemic and most cases of the infection come out from these malarious areas. A total of 695 identified individuals were participated in the training courses. A significant conversely correlation was found between conducting malaria microscopy training courses and annual malaria cases in Iran.
Conclusion: Conducting a suitable programme for malaria microscopy training and refresher train- ing plays an important role in the control of malaria in endemic areas. Obviously, the decrease of ma- laria cases in Iran has been achieved due to some activities that malaria diagnosis training was one of them.
Background: We tried to investigate the hair contamination of pet dogs and farm sheepdog with Toxocara eggs in terms of the different sex and age groups in north-west of Iran (Urmia and its sub- urbs).
Methods: Hair samples were collected from a total of 138 pet and farm sheepdogs from November 2008 to June 2009 in Urmia City and the suburb (West Azerbaijan-Iran) and examined for the pres- ence of T. canis eggs.
Results: T. canis eggs found in 60 samples altogether (pet and shepherd dogs) showed a contamina- tion rate of 36.2%. The number of observed T. canis eggs in each microscope field was varied from 1 to > 400. The age of the dog was found a significant factor to influence the prevalence and intensity of contamination, with 82% of all the eggs recovered from puppies (six months and younger). Addi- tionally, the numbers of eggs in farm sheepdogs were significantly higher than pet dogs (P<0.05). Conclusions: This report shows that direct contact with T. canis infected dogs, particularly puppies from shepherd dogs, may pose a serious hazard to human. Besides, as they may harbor a considera- ble number of eggs on their hair, they can contaminate the soil and the environment.