Parasitic Infection of an Endemic Fish (Blicca bjoerkna) and an Exotic Fish (Hemiculter beucisculus) In Anzali Lagoon,Caspian Sea,Iran

Background

In Anzali Lagoon, there are some endemic and exotic fishes. The present study was conducted to compare the parasitic fauna of Blicca bjeorkna, as an endemic fish and Hemiculter leucisculus, as an introduced fish to the lagoon.

Methods

A parasitological investigation was done on 78 specimens of B. bjoerkna and 114 of H. leucisculus. The fishes were collected from August 2009 to April 2010 by the electro fishing from Anzali Lagoon.

Results

Eleven parasites species were found in 192 fish samples. The prevalence and mean intensity of parasites in each host were as follows: Parasites from B. bjorkna were Trichodina perforata (53.85%); Myxobolus musayevi (27.19%, 1±0.79); Dactylogyrus difformis (88.05%, 8±7.24) and D. sphyrna (5.18%, 0.95±0.51), Diplostomum spataceum (98.72%, 9.51±9.01), Posthodiplostomum cuticula (15.38%, 4.25±2.5), Ripidocotyle sp. (1.28%, 2±0.74); Contracaecum osculatum (17.95%, 1.64±0.79), Philometra rischta (12.8%, 1.4±0.54), and Raphidascaris acus (1.04%, 0.03±0.26). The H. leucisculus were infected with T. perforata (27.19%), D. spataceum (7.89%, 1.33±0.54), Ps. tomentosa (7.02%, 1.62±0.49) and R. acus (0.88%, 3±0.28). B. bjoerkna was presented as a new host for M. musayevi and C. osculatum, while H. leucisculus was introduced as a new host for T. perforata and Ps. tomentosa.

Conclusion

The prevalence of parasites was significantly more in native fish than that of exotic fish (P<0.05). This reduction in parasitic infection in H. leucisculus may be due to its immune system resistance, well adaptation to the new environment, host-specific limitation for endemic parasites and disability of introduced parasite to complete its life cycle in the new host as well.     

Novel in Vitro Efficiency of Chitosan Biomolecule against Trichomonas gallinae

Background

Development of new natural agents for parasitic diseases treatment has unexpectedly increased to overcome effectively against emergence and re-emergence of parasitic diseases, the appearance of drug resistant organisms and toxic side effects of current agents. The aim of the study was to evaluate antiprotozoal activities of chitosan biomolecule on trophozoites of Trichomonas gallinae.

Methods

The antitrichomonal activity of various low molecular weight chitosan concentrations including 125, 250, 500 and 1250 µg ml⁻¹ against T. gallinae trophozoites cultured in trypticase-yeast extract-maltose medium supplemented with heat-inactivated cold horse serum was evaluated in vitro. Samples containing medium without chitosan were also assayed as controls.

Results

The mortality rates at 0, 3 and 6 h post treatment with all concentrations were significantly different from control group (P<0.05). Treated trophozoites showed more susceptibility to the highest concentration reaching mortality rate of 100% at 3h post inoculation. However, at this time, results for 125, 250 and 500 µg ml⁻¹ were 93%, 95% and 96.7%, respectively.

Conclusion

The results demonstrate that the application of chitosan biomolecule is a promising option for treatment of trichomoniasis in pigeons.     

Transovarial Transmission of Babesia ovis by Rhipicephalus sanguineus and Hyalomma marginatum

Background

Rhipicephalus sanguineus and Hyalomma marginatum are the most common species in sheep herds in Northeast of Iran. There is preliminary evidence that these species may be the vectors of Babesia ovis in Iran. We carried out two experiments in Mashhad area, Khorasan Razavi Province to determine whether B. ovis could be transovarially transmitted by R. sanguineus and H. marginatum.

Methods

In experiment 1, adults of laboratory reared H. marginatum and R.sanguineus were infected with B. ovis isolated from naturally infected sheep in Mashhad area by feeding the ticks on the sheep inoculated intravenously by infected blood samples. The inoculated sheep showed clinical signs with parasitaemia while the adult ticks were engorging on them. The engorged females were collected and kept at 28°C and 85% relative humidity in incubator. Then, larval, nymphal and adult stages derived from engorged females were used to infest the clean sheep. In experiment 2, two splenectomized sheep were infested only with the same adult ticks of two species.

Results

Examination of smears and PCR of blood samples to detect of B. ovis in infested sheep in two experiments were negative.

Conclusion

It seems that R. sanguineus and H. marginatum can not transovarially transmit B. ovis in sheep.     

Potential Bio-Control Agent from Rhodomyrtus tomentosa against Listeria monocytogenes

Listeria monocytogenes is an important foodborne pathogen implicated in many outbreaks of listeriosis. This study aimed at screening for the potential use of Rhodomyrtus tomentosa ethanolic leaf extract as a bio-control agent against L. monocytogenes. Twenty-two L. monocytogenes isolates were checked with 16 commercial antibiotics and isolates displayed resistance to 10 antibiotics. All the tested isolates were sensitive to the extract with inhibition zones ranging from 14 to 16 mm. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values ranged from 16 to 32 µg/mL and 128 to 512 µg/mL, respectively. Time-kill assay showed that the extract had remarkable bactericidal effects on L. monocytogenes. The extract at a concentration of 16 µg/mL reduced tolerance to 10% NaCl in L. monocytogenes in 4 h. Stationary phase L. monocytogenes cells were rapidly inactivated by greater than 3-log units within 30 min of contact time with R. tomentosa extract at 128 µg/mL. Electron microscopy revealed fragmentary bacteria with changes in the physical and morphological properties. Our study demonstrates the potential of the extract for further development into a bio-control agent in food to prevent the incidence of L. monocytogenes contamination.     

Interleukin-10 and Transforming Growth Factor-β in Early and Late Lesions of Patients with Leishmania major induced Cutaneous Leishmaniasis

Background

Cutaneous leishmaniasis is a neglected parasitic disease, which imposes massive human distress and financial costs to the endemic countries. Better understanding of host immune response to the parasite leads to helpful strategies for disease control. Interleukin (IL)-10 and transforming growth factor (TGF)-β are important immune regulatory cytokines, which appear to develop non-healing forms of leishmaniasis. However, there is little information about the function of IL-10 and TGF-β in old world cutaneous leismaniasis. The aim of this study was to analyze the role of IL-10 and TGF-β in human cutaneous leishmaniasis due to Leishmania major infection.

Methods

Biopsies were obtained from lesions of twenty proven cases of L. major induced cutaneous leishmaniasis. IL-10 and TGF-β positive cells were detected by immunofluorescence staining of frozen sections and compared between two groups of patients with early and late lesions.

Results

The mean percentage of IL-10 positive cells were significantly (P= 0.035) higher in late lesions (0.51±0.24) than early ones (0.15±0.07). Similar results were obtained for TGF-β with mean percentages of 0.16±0.05 and 0.53±0.28 in early and late lesions respectively (P= 0.008).

Conclusion

IL-10 and TGF-β are present in lesions of L. major induced cutaneous leishmaniasis and contribute to the pathogenesis of long lasting disease forms.     

Interleukin-10 and Transforming Growth Factor-β in Early and Late Lesions of Patients with Leishmania major Induced Cutaneous Leishmaniasis

Background

Cutaneous leishmaniasis is a neglected parasitic disease, which imposes massive human distress and financial costs to the endemic countries. Better understanding of host immune response to the parasite leads to helpful strategies for disease control. Interleukin (IL)-10 and transforming growth factor (TGF)-β are important immune regulatory cytokines, which appear to develop non-healing forms of leishmaniasis. However, there is little information about the function of IL-10 and TGF-β in old world cutaneous leismaniasis. The aim of this study was to analyze the role of IL-10 and TGF-β in human cutaneous leishmaniasis due to Leishmania major infection.

Methods

Biopsies were obtained from lesions of twenty proven cases of L. major induced cutaneous leishmaniasis. IL-10 and TGF-β positive cells were detected by immunofluorescence staining of frozen sections and compared between two groups of patients with early and late lesions.

Results

The mean percentage of IL-10 positive cells were significantly (P= 0.035) higher in late lesions (0.51±0.24) than early ones (0.15±0.07). Similar results were obtained for TGF-β with mean percentages of 0.16±0.05 and 0.53±0.28 in early and late lesions respectively (P= 0.008).

Conclusion

IL-10 and TGF-β are present in lesions of L. major induced cutaneous leishmaniasis and contribute to the pathogenesis of long lasting disease forms.     

Efficacy of the Bunium persicum (Boiss) Essential Oil against Acute Toxoplasmosis in Mice Model

Background:

We evaluated the in vivo activity of Bunium persicum (Boiss) essential oil on infected mice with acute toxoplasmosis.

Methods:

To evaluate prophylactic effects, male NMRI mice received B. persicum essential oil at the concentrations of 0.05 and 0.1 mL/kg for 14 days. After 24 h mice were infected intraperitonealy with 1×10⁴ tachyzoites of T. gondii, RH strain. In order to investigate therapeutic effects, mice were infected and then received B. persicum oil at the concentrations of 0.05 and 0.1 ml/kg two times a day for 5 days. The time/mean time of death in all infected mice and the number of tachyzoites from infected mice were recorded.

Results:

The time/mean time of death of infected mice was 8 and 9 days after oral administration of B. persicum oil at the concentration of 0.05 and 0.1 mL/kg, respectively (P<0.05). In contrast, the time/mean time of death control group was 5 days. In addition, B. persicum significantly reduced the mean number of tachyzoites compared with control group. The time/mean time of death of infected mice was 6 and 7 days after oral administration of B. persicum essential oil at the concentration of 0.05 and 0.1 mL/kg, respectively. In contrast, the time/mean time of death control group was 5 days. B. persicum especially at the concentration of 0.1 ml/kg significantly reduced the mean number of tachyzoites compared with control group.

Conclusion:

The results showed the potential of B. persicum essential oil as a natural source for the production of new prophylactic agent for use in toxoplasmosis.     

Haemolymph Components of Infected & None Infected Lymnaea snails with Xiphidiocercariae

Background

In this study the haemolymph components of infected and none infected Lymnaea gedrosiana with xiphidiocercaria larvae was compared.

Methods

Five hundred Fifty Lymnaea snails were collected from Ilam and Mazandaran provinces, Iran, during 2008–2009. The snails were transported to the lab at Tehran University of Medical Sciences and their cercarial sheddings were studied. Haemolmyphs of snails were extracted and cells were counted using haemocytometer and cell-surface carbohydrate were recognized by conjugated lectin (Lentil). Haemolymph protein concentrations were measured by Bradford protein assay method and soluble protein compositions were determined on sodium dodecyl sulphate polyacrilamide gel electrophoresis (SDS-PAGE).

Results

From the 550 examined Lymnaea snails for cercariae, 27 snails were infected with xiphidiocercariae. Mean of haemolymph cells (haemocyte) number were obtained 93480±2.43 (cells/ml) for none infected snails (25 snail) and 124560±2800 (cells/ml) for infected snails (25 snail). Mannose carbohydrate was recognized on haemocyte of none infected and infected snails. Mean of protein concentration of haemolymph plasma was obtained as 1354±160 µg/ml (1.4 mg/ml) for none infected snails (25 snails) and 1802±138 µg/ml (1.8 mg/ml) for infected snail (25 snails). Comparing to none infected snails, the SDS-PAGE results of haemolymph plasma of infected snails, showed an extra protein band (70 kDa). The results showed a significant difference between the amounts and the kinds of proteins in haemolymph of infected and none infected snails.

Conclusion

This information might be useful to understand of parasite detection, adhesion, engulfment and antigen agglutination by snail.     

Downregulation of Calcineurin Gene Is Associated with Glucantime? Resiatance in Leishmania infantum

Background

Pentavalent antimonials are the first line drugs for the treatment of leishmaniasis. Unresponsiveness of Leishmania spp. to antimonial drugs is a serious problem in some endemic areas. Investigations on molecular mechanisms involved in drug resistance are essential for monitoring and managing of the disease. Cal-cineurin is an essential protein phosphatase for number of signal transduction pathways in eukaryotic cells and it has a mediated role in apoptosis. This study aimed to determine of biomarker(s) in Glucantime^® resiatance strain of L. infan-tum.

Methods

We used cDNA amplified fragment length polymorphism (cDNA-AFLP) and real time-RT PCR assays to compare gene expression profiles at the mRNA levels in resistant and susceptible L. infantum field isolates.

Results

The cDNA-AFLP results showed downlegulation of calcineurin in resis-tant isolate in comparison with susceptible one. Significant downregulation of cal-cineurin (0.42 fold) (P<0.05) was found in resistant isolate compared to susceptible one by Real time-RT PCR.

Conclusion

This is the first report of calcineurin implication in Glucantime^® drug resistance of field (natural) isolate of L. infantum. Downregulation of calcineurin could protect parasites from antimonial-induced apoptosis.     

Axenic Cultivation and Pathogenic Assays of Acanthamoeba Strains Using Physical Parameters

Background

The main goal of the present study was to set up an axenic cultivation of Acanthamoeba and assess the pathogenic ability of T4 genotypes from different clinical and environmental strains of Acanthamoeba using two physical assays.

Methods

Sixteen Acanthamoeba isolates including 10 environmental and 6 clinical strains were cultured axenically. Axenic cultivation was performed using Proteosepepton, yeast extract and glucose medium and TY-I-S33culture. Pathogenic survey was done using osmotolerance and thermotolerance assay. Briefly, differentosmolarity (0.5 M and 1 M) of non-nutrient agar plates were performed. One hundred fifty µl of axenic culture were collected and were inoculated in 1% agar medium. For thermotolerance assay 150 µl of amoebas from axenic culture were divided into fresh culture mediums. Cultures were incubated at 37°C and 42 °C. All plates were monitored for 24 h, 48 h and 72 h.

Results

Overall, 16 strains of Acanthamoeba isolates previously genotyped as T4 were cultivated axenically after several months. Thermotolerance and osmotolerance assay revealed that all of clinical strains, soil and animal feces strains were highly pathogenic isolates. Two dust and water strains did not grow at high temperature (42 °C) and osmolarity (1.5 M) and thus they were classified as weak pathogens.

Conclusion

Most of T4 genotypes are highly pathogenic organisms. This is an important finding since Acanthamoeba belonging to T4 type is the predominate genotype in environmental and clinical samples. The presence of highly pathogenic Acanthamoeba may pose a risk within susceptible people.     

Possibility of CTX-M-14 Gene Transfer from Shigella sonnei to a Commensal Escherichia coli Strain of the Gastroenteritis Microbiome

Objectives

To investigated whether the CTX-M-14 gene could be transferred from a clinical Shigella sonnei strain to commensal Escherichia coli strain in the gastroenteritis microbiome.

Methods

E. coli strains were isolated from 30 stool samples of S. sonnei infected students in a gastroenteritis outbreak in 2004 and were characterized by antibiotic resistance analysis, in vitro conjugation and in vivo transfer of CTX-M-14 gene and molecular assays.

Results

One strain of Escherichia coli that had high levels of resistance to cefotaxime was isolated from a patient infected with S. sonnei. Isoelectric focusing showed that the E. coli and S. sonnei strains produced a β-lactamase with an isoelectric point of 8.1. Moreover, polymerase chain reaction analysis indicated that both strains possessed the same DNA sequences for CTX-M-14. The results of in vitro and in vivo conjugation showed that the efficiency of CTX-M-14 transfer from S. sonnei to E. coli was similar to CTX-M-14 transfer between E. coli strains.

Conclusion

The data suggest that the acquisition of the extended-spectrum β-lactamases gene by pathogenic bacteria in the human intestinal tract to commensal microbiome bacteria can cause serious infectious diseases.     

Cadmium Alters the Biotransformation of Carcinogenic Aromatic Amines by Arylamine N-Acetyltransferase Xenobiotic-Metabolizing Enzymes: Molecular,Cellular, and in Vivo Studies

Background

Cadmium (Cd) is a carcinogenic heavy metal of environmental concern. Exposure to both Cd and carcinogenic organic compounds, such as polycyclic aromatic hydrocarbons or aromatic amines (AAs), is a common environmental problem. Human arylamine N-acetyltransferases (NATs) are xenobiotic-metabolizing enzymes that play a key role in the biotransformation of AA carcinogens. Changes in NAT activity have long been associated with variations in susceptibility to different cancers in relation with exposure to certain AAs.

Objective

We explored the possible interactions between Cd and the NAT-dependent biotransformation of carcinogenic AAs.

Methods

We exposed purified enzymes, lung epithelial cells, and mouse models to Cd and subsequently analyzed NAT-dependent metabolism of AAs.

Results

We found that Cd, at biologically relevant concentrations, impairs the NAT-dependent acetylation of carcinogenic AAs such as 2-aminofluorene (2-AF) in lung epithelial cells. NAT activity was strongly impaired in the tissues of mice exposed to Cd. Accordingly, mice exposed to Cd and 2-AF displayed altered in vivo toxicokinetics with a significant decrease (~ 50%) in acetylated 2-AF in plasma. We found that human NAT1 was rapidly and irreversibly inhibited by Cd [median inhibitory concentration (IC₅₀) ≈ 55 nM; rate inhibition constant (k_inact) = 5 × 10⁴ M⁻¹ · sec⁻¹], with results of acetyl coenzyme A (acetyl-CoA) protection assays indicating that Cd-mediated inhibition was due to the reaction of metal with the active-site cysteine residue of the enzyme. We found similar results for human NAT2, although this isoform was less sensitive to inactivation (IC₅₀ ≈ 1 μM; k_inact = 1 × 10⁴ M⁻¹ · sec⁻¹).

Conclusions

Our data suggest that Cd can alter the metabolism of carcinogenic AAs through the impairment of the NAT-dependent pathway, which may have important toxicological consequences.     

PM10 Exposure and Non-accidental Mortality in Asian Populations: A Meta-analysis of Time-series and Case-crossover Studies

Objectives

We investigated the association between particulate matter less than 10 µm in aerodynamic diameter (PM₁₀) exposure and non-accidental mortality in Asian populations by meta-analysis, using both time-series and case-crossover analysis.

Methods

Among the 819 published studies searched from PubMed and EMBASE using key words related to PM₁₀ exposure and non-accidental mortality in Asian countries, 8 time-series and 4 case-crossover studies were selected for meta-analysis after exclusion by selection criteria. We obtained the relative risk (RR) and 95% confidence intervals (CI) of non-accidental mortality per 10 µg/m³ increase of daily PM₁₀ from each study. We used Q statistics to test the heterogeneity of the results among the different studies and evaluated for publication bias using Begg funnel plot and Egger test.

Results

Testing for heterogeneity showed significance (p<0.001); thus, we applied a random-effects model. RR (95% CI) per 10 µg/m³ increase of daily PM₁₀ for both the time-series and case-crossover studies combined, time-series studies relative risk only, and case-crossover studies only, were 1.0047 (1.0033 to 1.0062), 1.0057 (1.0029 to 1.0086), and 1.0027 (1.0010 to 1.0043), respectively. The non-significant Egger test suggested that this analysis was not likely to have a publication bias.

Conclusions

We found a significant positive association between PM₁₀ exposure and non-accidental mortality among Asian populations. Continued investigations are encouraged to contribute to the health impact assessment and public health management of air pollution in Asian countries.     

Rapid Detection of Toxoplasma gondii Antigen in Experimentally Infected Mice by Dot- ELISA

Background

Toxoplasmosis is a worldwide endemic disease. In congenitally infected infants and AIDS patients, toxoplasmosis causes high rates of morbidity and mortality. In these cases antibody detection is difficult; so detection of parasite or its components could be useful tool for early detection and following treatment of the infection.

Methods

Sixty-three BALB/c mice were injected intra-peritoneal with 5×10³ tachyzoites of Toxoplasma gondii RH strain, nine mice were sacrificed daily for 7 days. Fourteen mice were injected with phosphate buffer saline as control group. Dot–ELISA was performed for detection of T.gondii antigen in mice sera and capture – ELISA was done as golden standard assay too.

Results

Toxoplasma gondii antigen was detected from day 2 in mice sera; 22% of mice sera on day 2, 33% on day 3,77% on day 4 and 100% on day 5 till their death on day 7 had shown antigenemia by dot – ELISA, no positive result was detected in control mice by dot- ELISA.

Conclusion

Dot-ELISA is a sensitive method for diagnosis of T. gondii infection in the animal model; also, this technique is more rapid and easy to perform method in comparison with capture-ELISA.     

Triclabendazole Effect on Protease Enzyme Activity in the Excretory- Secretory Products of Fasciola hepatica in Vitro

Background

Fasciola hepatica is one of the most important helminthes parasites and triclabendazole (TCBZ) is routinely used for treatment of infected people and animals. Secreted protease enzymes by the F. hepatica plays a critical role in the invasion, migration, nutrition and the survival of parasite and are key targets for novel drugs and vaccines. The aim of study was to determine the protease activity of excretory- secretory products (ESP) of F. hepatica in the presence of TCBZ anthelmintic.

Methods

F. hepatica helminthes were collected and cultured within RPMI 1640 [TCBZ treated (test) and untreated (control)] for 6 h at 37 °C. ESP of treated and control were collected, centrifuged and supernatants were stored at -20°C. Protein concentrations were measured according to Bradford method. Protease enzymes activities of ESP samples were estimated by using sigma’s non-specific protease activity assay. ESP protein bands were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

Results

Mean protein concentrations in control and treated of ESP samples were determined 196.1 ±14.52 and 376.4 ±28.20 μg/ml, respectively. Mean protease enzymes activities in control and treated were 0.37 ±0.1 and 0.089 ±0.03 U/ml, respectively. Significant difference between proteins concentrations and protease enzymes activities of two groups was observed (P<0.05). SDS-PAGE showed different patterns of protein bands between treated and control samples.

Conclusion

The TCBZ reduced secreted protease enzymes activities and possibly effects on invasion, migration, nutrition and particularly survival of the parasite in the host tissues.     

Pathogenic Assays of Acanthamoeba Belonging to the T4 Genotype

Background

Acanthamoeba genus is introduced as opportunistic and cosmopolitan parasite. Monkey and wistar rat are appropriate models for experimental study on Acanthamoeba infection. In this study Acanthamoeba spp. were isolated from hot spring (HS), windows dust (WD) and a corneal sample of keratitis patient (KP) and their pathogenicity surveyed by in vitro and in vivo tests.

Methods

Isolates of Acanthamoeba were cultivated axenically for 12 months in PYG medium. Overall, 30 wistar rats, in 6 equal groups were used for developing experimental Acanthamoeba keratitis (AK) and Granulomatous Amoebic Encephalitis (GAE). The Keratitis and Granulomatous Encephalitis experiments were performed by intrastromal and intranasal inoculation of Acanthamoeba cysts, respectively. Pathogenicity of the three isolates was also evaluated by in vitro test using osmotolerance and temperature tolerance assays. Identification of genotypes were performed by PCR technique and sequencing.

Result

None of the isolates could perform AK and GAE in wistar rats, although all isolates were described as T4 genotype. Isolates obtained from KP and WD could grow only in 30 °C, but not in 37 °C and 40 °C. On the other hand, HS isolate grew in 30 °C and 37 °C but not in 40 °C. Moreover, all of isolate grew in 0.5 M mannitol but not in 1 M and 1.5 M.

Conclusion

T4 isolates with a long-term axenic culture and different factors related to host and parasite may play role in pathogenicity of these free-living amoebae.     

P-glycoprotein A Gene Expression in Glucantime-Resistant and Sensitive Leishmania major (MRHO/IR/75/ER)

Background

Leishmaniasis is a parasitic disease caused by different species of Leishmania parasites with a wide range of clinical manifestations. Antimonial compounds such as meglumine antimoniate (glucantime) are the first line drugs for the treatment of leishmaniasis. However, according to reports of the drug resistance of parasites, the efficacy of antimonial compounds is low. The ATP-binding cassette (ABC) proteins are present in all organisms and mediate the transport of vital elements through biological membranes. One of the important mechanisms of resistance in Leishmania parasites is the overexpression of ABC efflux pumps. P-glycoprotein A (pgpA) is a related gene for ABC transporter in Leishmania species. The aim of this study was to compare the pgpA expression in laboratory-induced resistant L. major (MRHO/IR/75/ER) and sensitive parasites.

Methods

RNA extraction of promastigotes of sensitive and resistant clones was performed and total RNA was reverse transcribed. The real-time quantitative polymerase chain reaction (PCR) was used to assess RNA expression profiles and the expression levels were calculated using 2^-ΔCt method.

Results

The mean expression level of pgpA mRNA was 2.70 ± 0.51 in in sensitive Leishmania clone and 6.08 ± 1.50 in resistant Leishmania clone (P = 0.021).

Conclusion

The expression of pgpA gene in resistant strains of L. major was almost fivefold higher than those in susceptible strains. Therefore, this can be used in field isolates, i.e. overexpression of the gene can prove resistance in wild type field isolates.     

Comparison of Resistant and Susceptible Strains of Trichomons vaginalis to Metronidazole Using PCR Method

Background

Metronidazole is drug of choice recommended by WHO for treatment of trichomoniasis, however, some reports claims drug resistance in Trichomonas vaginalis isolates recently. The objective of this study was to determine the minimum lethal concentration (MLC) of metronidazole in resistant and sensitive strains, as well as genetic patterns of these stains by PCR method.

Methods

From February 2006 to March 2007, in a cross sectional study, clinical and wet mount examination of vaginal smear along with culture were performed on 683 women attending to public and private outpatient clinics in Hamadan. Trichomoniasis marked based on major clinical symptoms. Diagnosis confirmed using wet mount microscopically and culture in Diamond medium. A serial concentration of metronidazole was provided and all isolated Trichomonas strains (resistant and sensitive) tested by standard method. Finally, all sensitive and resistant strains examined by PCR technique.

Results

Only 15/683, (2.2%) of patients clinically diagnosed trichomonal vaginitis were positive for T. vaginalis by wet smear and culture. The minimum lethal concentration (MLC) for clinically sensitive isolates was 25 µg/ml; however, this concentration for resistant isolates was 200 µg/ml after 24 h and 100 µg/ml after 50 h. The results of PCR examination of DNA from sensitive and resistant isolates had same pattern. The lanes appeared by two primers were 98 bp and 261 bp for both clinically sensitive and resistant strains.

Conclusion

Resistance to metronidazole in T. vaginalis has not relation to genetic variations and might be related to some physiologic pathways of organism.     

Cloning and Expression of Recombinant Plasmid Containing P36/LACK Gene of Leishmania infantum Iranian Strain

Background:

There are several methods, such as vaccination, to control visceral leishmaniasis. Although there is no efficient vaccine, it seem DNA vaccination with stimulates both cellular and humoral immunity apparently is the best way. The aim of this study was cloning and expression of LACK gene, a 36kD protein, as a candidate protein for vaccination against Iranian L. infantum.

Methods:

Iranian strain of L. infantum [MCAN/IR/07/Moheb-gh] was used as a template for PCR to amplify LACK gene. The LACK gene was cloned in pTZ57R/T vector and after confirmation it was digested by restriction enzymes (BamH1) and cloned in pcDNA3.1 expression vector. Recombinant plasmid was extracted and analyzed by sequencing, restriction digestion analysis and PCR reaction. The pc- LACK recombinant plasmid was purified from transformed E.coli (DH5α) and its expression was analyzed by SDS-PAGE and Western blot.

Results:

The results of sequencing, restriction digestion analysis and PCR reaction revealed that LACK gene was cloned correctly in pcDNA3.1 vector and the results of SDS PAGE and Western blot emphasized that LACK protein of Iranian L. infantum is a well-expressed protein.

Conclusion:

We amplified, cloned and expressed Iranian L. infantum LACK gene successfully.     

Frequency of Toxoplasma gondii in HIV Positive Patients from West of Iran by ELISA and PCR

Background

Toxoplasma gondii, the obligate intracellular parasite is life threatening in AIDS patients. Diagnosis of toxoplasmosis is based on serological methods especially increasing of IgM and IgG titers, but finding of parasite or its components (antigenemia) may be beneficial method in order to detection of acute toxoplasmosis in immunocompromised patients.

Methods

Ninety-four serum samples from HIV positive patients were collected from Sanandaj, Kordistan west of Iran. These patients were lived in Sanandaj of whom 26 were prisoners infected with HIV virus in prison. Toxoplasma gondii antibodies were determined by IgG ELISA. T. gondii antigen was identified by capture-ELISA. PCR was performed on samples with T. gondii antigenemia. CD4+ T cells counts had been determined by flowcytometry and were obtained from records of each patient.

Results

Among the examined HIV seropositive individuals, 19.1% (18/94) and 5.3% (5/94) were positive for Toxoplasma-IgG and antigenemia, respectively. Besides, one of the samples was positively detected by PCR method. Mean age of participants was 37.9 ± 9.5 year. Prevalence of IgG antibody and antgenemia was higher in age group of 40-50 years old. The Mean of CD4+ T cells counts of participants (total of HIV+ patients, IgG positive patients and patients with antigenemia) was 699.2 ± 345.2, 655.1 ± 237.9 and 620.2 ± 215.1 respectively.

Conclusion

Capture-ELISA and PCR could confirm the T. gondii acute infection in HIV positive patients. For precise diagnosis of acute toxoplasmosis in HIV positive patient, performance of more studies based on more sensitive types of PCR is suggested.