Anti Leishmanial Effect of Plantago psyllium (Ovate) and White Vinegar on Leishmania major Lesion in BALB/c Mice.

Background: Leishmania major is the etiologic agent of zoonotic cutaneous leishmaniasis in Iran, and glucantime injection is currently used for its treatment. This study aimed to evaluate the anti-leishmanial effect of topical Plantago psyllium and white vinegar in L. major infected BALB/c mice. Methods: Thirty infected mice were divided into five groups as follows: Group 1: treated with the combination of ovata powder and white vinegar, Group 2: treated with glucantime, Group 3: treated with white vinegar, Group 4: treated with the combination of ovata powder and water, and Group 5: without any treatment. All the groups were treated for 18 days. Lesion size was measured, and final smears were prepared for microscopic examination. Results: The findings indicated that the difference in the mean areas of the ulcers in all the groups before and after treatment was not significant, except for the second (glucantime) and third (vinegar) groups. Also, the results showed that in the first, second, third, and fourth group, 6 (60%), 4 (80%), 3 (60%), and 2 (40%) mice were healed, respectively. However, ulcers remained in all the five mice of the control group. Conclusion: The combination of ovata powder and white vinegar has been traditionally used to treat leishmanial lesions in Iran. It seems the most anti-leishmanial effect is related to vinegar and supported by Plantago. The route of treatment with this combination is very simple and painless in comparison with injection. Thus, further studies on this issue could help to design more effective and easy-to-use drugs.

Neospora caninum Infection in Cattle in the Province of Kohgiluyeh and Boyer Ahmad, Southwest of Iran: Seroprevalence and Molecular Assessment.

INTRODUCTION: Among the protozoa of veterinary importance, Neospora caninum is responsible for large economic and productive losses in cattle herds. Dogs are being considered the definitive hosts of the parasite. Neospora caninum has gained considerable attention through its role in the etiology of bovine abortion. The current study aimed at assessing the status of Neospora infection in cattle in Boyer-Ahmad County in Kohgiluyeh and Boyer-Ahmad province, southwest of Iran. METHODS: In this cross-sectional study, 150 cattle blood samples were collected and samples were screened for N. caninum antibodies using a modified direct agglutination test (MAT). For the same samples, 130 buffy coats were collected and tested, by PCR, for the presence of N. caninum DNA, targeting the Nc-5 gene. RESULTS: Anti-N. caninum antibodies were detected in the sera of 49 out of 150 cattle which is corresponding to a seroprevalence rate of 32.7%. Neospora DNA was detected in buffy coats of 26 out of 127 (20.47%) cattle. Even though Neospora infection was more common in females and in five-year-old cattle by serology and molecular methods, yet there was no statistically significant difference between age, sex, and Neospora infection in both molecular and serological methods (p > 0.05). CONCLUSION: Findings of the current study indicate a high rate of N. caninum infection in cattle of Boyer-Ahmad region in the southwest of Iran. This issue should be further investigated and the prevention and control of this parasite in livestock, due to the high financial burden of this parasite in the livestock industry, should be considered.

Molecular Genotyping of Toxoplasma gondii in Sheep Aborted Fetuses Reveals Predominance of Type I Infection in Southwest of Iran.

BACKGROUND: We aimed to detect Toxoplasma gondii in ovine aborted fetuses and evaluate its genetic variations in the southwest of Iran. METHODS: This cross-sectional study was performed on 100 aborted ovine fetuses collected from the different region of Kohgiluyeh and Boyer-Ahmad Province, Iran, in lambing season during 2017 and 2018. DNA was extracted from the brain samples of all of the aborted fetuses and PCR amplified, targeting a 529 bp repetitive element gene of T. gondii. Moreover, to find out the heterogeneity of the positive samples, PCR-DNA amplification of the two main genetic markers, B1 and GRA6, of T. gondii were performed. Nucleotide sequencing and phylogenetic analysis were performed, using the BLAST program and MEGA-X software. RESULTS: The 529 bp gene of T. gondii was detected in 2 out of 100 (2%) of the ovine aborted samples. The sequences analysis of GRA6 and B1 genes revealed that both isolates from the aborted fetuses of sheep belonged to type I of T. gondii. Intra-divergence was more seen in GRA6 gene whereas less divergence was observed in B1 gene. CONCLUSION: Congenital infection with Type I of T. gondii during the neonatal period is associated with abortion in ovine. Evaluation of more aborted samples from broader geographical areas is needed to elucidate the molecular epidemiology and also the genotypes of T. gondii associated with abortion.

Clinical Features, Diagnosis and Management of Patients with Suspicion of Fascioliasis in Kohgiluyeh and Boyer-Ahmad Province, Southwestern Iran.

BACKGROUND: In the current study, we described the epidemiological features, clinical presentation, diagnosis and management of patients with suspicion of fascioliasis in Kohgiluyeh and Boyer-Ahmad Province in southwest of Iran. METHODS: Overall, 56 patients with suspicion of fascioliasis, based on their clinical signs and symptoms that referred to Clinic of Internal Medicine in Yasuj city, from 2014 to 2016 were enrolled. Demographic data, history of eating aquatic local plants, the chief complains, and laboratory findings were recorded for each patient. Stool samples were obtained from each case for detection of Fasciola eggs. Moreover, blood samples were taken from each patient and evaluated for detection of anti-Fasciola antibodies by an indirect ELISA. Patients who defined as having fascioliasis were treated with triclabendazole and were followed for at least three months for clinical improvement. RESULTS: Serological test was positive in 5 patients. Of these 5 cases, three cases had a history of ingesting raw aquatic vegetables. The main clinical signs and symptoms in positive cases were; abdominal pain (60%), epigastric pain (40%), anemia (60%), and dermal pruritus (20%). Hypereosinophilia was seen in all of 5 positive cases. No Fasciola egg was found in stool specimens of any of the patients. The fascioliasis cases were treated by triclabendazole and clinical symptoms disappeared in all of 5 cases. CONCLUSION: Our observation further confirmed Yasuj district as a human endemic area for fascioliasis in Iran. The study also highlighted the importance of clinical features together with eosinophilia, as key parameters, in the diagnosis of human fascioliasis. Clinicians need to be aware of this disease and should keep in mind fascioliasis when hypereosinophilia present in patients in such endemic areas.

Prevalence of bovine fascioliasis in a new-emerging focus of human fascioliasis in BoyerAhmad district, southwest of Iran.

BACKGROUND: The prevalence of fascioliasis in a population of cattle in a new-emerging focus of human fascioliasis in Boyer-Ahmad District, in the southwest of Iran, was investigated, using an ELISA system and coprological methods. METHODS: Blood, as well as stool samples, were collected from 150 dairy cattle consisted of 82 (54.7%) males and 68 (45.3%) females, aged from 1 to 8 years, selected from different pastures. The stool samples were evaluated by direct wet mount microscopic examination as well as sedimentation methods. Moreover, modified Telemann was used for detecting of Fasciola eggs. Anti-Fasciola antibodies in the cattle sera samples were determined, using a Fasciola excretory-secretory antigen-specific ELISA. RESULTS: Anti-Fasciola antibodies were detected in 64 (42.4%) of the subjects by ELISA. Out of 64 seropositive cases, 29 (45.3%) were female and 35 (54.6%) were male. No significant association was found between sex and seropositivity to fascioliasis. Considering the age of the cattle, the highest prevalence of fascioliasis (23%) was found in the 5-year old age group. However, the difference between the age of the cattle and seropositivity to fascioliasis was not statistically significant (P > 0.05). Fasciola eggs were not detected in fecal samples of any of the studied cattle, while Paramphistomide, as well as Dicrocoelium dendriticum eggs, were detected in 6 (4%) and 17 (11.3%) of the subjects, respectively. CONCLUSION: Findings of the current study provide basic information about bovine fascioliasis in a new focus of human fascioliasis in Iran, which is needed for effective control of this parasitic infection in such areas.

Molecular genotyping and serological evaluation of Toxoplasma gondii in mothers and their spontaneous aborted fetuses in Southwest of Iran.

BACKGROUND: Given the lack of routine screening and the high prevalence of toxoplasmosis in pregnant women in Iran, the current study aimed to find out the rate and features of Toxoplasma gondii infection in the spontaneously aborted human fetuses in Kohgiluyeh and Boyer-Ahmad Province, Southwestern Iran. METHODS: This cross-sectional study was performed on 100 spontaneously aborted fetuses' tissues and their mother blood samples. The mothers' sera were evaluated for anti-Toxoplasma antibodies while their buffy coat and aborted fetuses tissues were evaluated for Toxoplasma DNA. PCR product at GRA6 locus was sequenced and phylogenetic analysis was done. Likewise, quantitative Real-Time PCR was performed to find out the parasite burdens in mothers buffy coat and fetuses tissues. RESULTS: Using serological method, anti-Toxoplasma IgG and IgM antibodies were detected in 7 (7%) and 3 (3%) out of 100 sera from women with spontaneous abortion. Real-time PCR method detected T. gondii DNA in the buffy coat of one seronegative and 2 (out of 3) IgM seropositive cases. None of the samples from aborted fetuses were infected with T. gondii. BLAST and phylogenetic analysis showed that the sequenced isolates belonged to type I of T. gondii and two identified T. gondii isolates were taxonomically grouped into one clade. CONCLUSION: Our findings revealed type I genotype of T. gondii in two mothers with spontaneous abortion, without fetus involvement. It is necessary to examine more aborted fetuses' samples from different geographical areas to determine the association between Toxoplasma genotype and abortion.

Seroepidemiological study of cystic echinococcosis in nomadic communities in the southwest of Iran: A population-based study.

The current study aimed at the seroepidemiological survey of human cystic echinococcosis (CE) in nomadic people in Boyer-Ahmad District in the southwest of Iran. One thousand and five nomads were selected by cross-sectional sample collection in nomadic tribes of Boyer-Ahmad District in the southwest of Iran. Blood samples were taken from each individual, and the sera were tested for detection of anti-hydatid cyst antibodies using antigen B-ELISA. A predesigned questionnaire which contained basic epidemiological and individual information related to hydatid cyst was filled for each subject during sample collecting. Subjects of the study were 227 males (22.6%) and 778 (77.4%) females. The mean age of the participants was 40.4 years old (±16.6). Anti-hydatid cyst antibodies were detected in 81 (8.1%) of the subjects. Seroprevalence rate for CE in females (9%) was more than males (4.8%). Multivariate logistic regression revealed significant associations between CE seropositivity and sex (odds ratio [OR] = 1.88; 95% confidence interval [CI] = 0.93-3.80) and dog ownership (OR = 8.3; 95% CI = 3.94-16.37). The rate of infection with CE in nomadic people in southwest of Iran is considerable. Treatment of dogs and increasing the level of people awareness may contribute a substantial advancement in the control of the disease in the area.

In-vitro Activity of Trigonella foenum graecum Seeds Against a Clinical Strain of Acanthamoeba Genotype T4.

Acanthamoeba keratitis (AK) is a sight-threatening infection of the cornea disease that often presents with a lengthy and not fully effective treatment. Current therapeutic options against Acanthamoeba are not very effective against the cyst. Calibrated trophozoite/cyst suspension was incubated with the same volume of serial dilutions of the Trigonella foenum graecum aqueous extract (200, 250, 350, 450, 600, and 750 mg/mL) in microcentrifuge tubes and mixed by pipetting up and down. After that, the tubes were incubated at 26 ºC for 24, 48, and 72 h. The obtained result revealed that incubation of the extract (at concentrations ranging from 200 to 750 mg/mL) with Acanthamoeba was able to decrease the number of viable trophozoites and cysts. In the presence of up to 450 mg/mL non-viable trophozoites were observed whereas cysts were only eliminated when incubated with 750 mg/mL of the extract after 24 h. Furthermore, no cytotoxicity of the extract even at the highest concentration tested in the study showed to be toxic for corneal cells. Further studies should be carried out in order to elucidate the active compounds involved in the observed anti-Acanthamoeba activities which could be used for the development of novel therapeutic approaches against Acanthamoeba infections.

Toxoplasma gondii in Blood Donors: A Study in Boyer-Ahmad County, Southwest Iran.

Toxoplasma gondii is an important foodborne protozoan that can be transmitted through infected blood containing tachyzoite form of the parasite. The current study aimed to evaluate the prevalence of T. gondii infection and related risk factors among healthy blood donors in Boyer-Ahmad County, southwest Iran. Blood samples were taken from 285 healthy blood donors who voluntarily agreed to participate in this study. Sera and buffy coat were isolated from the blood samples for serological and molecular evaluations. The sera were tested for anti-T. gondii antibodies (both IgG and IgM), using a commercial ELISA kit. The buffy coat of seropositive cases was evaluated for detection of T. gondii DNA by PCR. Moreover, a structured questionnaire, containing socioepidemiological data and possible risk factors, was filled out by each participant during sample collection. Anti-T. gondii antibodies were detected in sera of 48/285 (16.8%) participants. Only two of the subjects (0.7%) were seropositive for both IgG and IgM antibodies. T. gondii DNA was not detected in buffy coat of any of the seropositive cases. Risk factors such as contact with soil (OR, 9.7; 95% CI, 4.9-19.4) and consumption of semicooked meat (OR, 2.5; 95% CI, 1.2-5.03) were statistically associated with seropositivity to T. gondii. The seroprevalence rate of T. gondii antibodies in the blood donors of Boyer-Ahmad County was not high in comparison with other regions in Iran. In this study, consumption of undercooked meats, job, and contact with soil were independent risk factors associated with T. gondii infection, which can be considered as potential sources of T. gondii infection.

Role of environmental, climatic risk factors and livestock animals on the occurrence of cutaneous leishmaniasis in newly emerging focus in Iran.

INTRODUCTION: Occurrence of leishmaniasis is affected by various biological and environmental factors. Kohgiluye and Boyerahmad (K-B) province is an emerging focus of cutaneous leishmaniasis (CL) in Southwest Iran. SUBJECTS AND METHODS: To elucidate some angles of occurrence of CL in this province, climatic and environmental factors and close proximity to livestock were studied by univariate and two multivariate logistic regression models. The dwelling addresses of 275 CL patients were obtained from Health Centers records for a 5 years period. The effect of mean annual temperature, minimum mean annual temperature, maximum mean annual temperature, mean annual rainfall, slope, elevation, land covers and close proximity to cattle and sheep/goat sheds on the occurrence of CL were analysed using geographical information systems (GIS) approach. RESULTS: CL occurred in all counties with the most cases in Northwest, West and South semi-arid and warm regions. Land cover, slope, elevation and close proximity to cattle sheds, were the most effective factors. Urban, dry farm and thin rangeland were found as the most important land covers. Slope and elevation decreased the probability of disease. Close proximity to cattle sheds notably increased the chance of CL. The importance of other factors such as rainfall, temperature and close proximity to sheep/goat sheds only were shown when their effects were evaluated independently from other factors. So regions with urban, dry farm and thin rangeland covers with lower slope and altitude where in close proximity of cattle sheds seems to be potentially most high risk areas. CONCLUSION: Distribution of CL cases is influenced by combination of environmental, ecological factors and close proximity to livestock sheds but control programs should be focused on cities and villages in the above-mentioned most high risk regions.

Production of Monoclonal Antibody Against Excretory-Secretory Antigen of Fasciola hepatica and Evaluation of Its Efficacy in the Diagnosis of Fascioliasis.

Parasitological methods are not helpful for the diagnosis of fascioliasis in acute and invasive periods of the disease. Detection of coproantigens seems to be a suitable alternative approach in the diagnosis of fascioliasis. The present study aimed to develop a reliable antigen detection system, using monoclonal antibodies raised against excretory-secretory (ES) antigen of Fasciola hepatica, for the diagnosis of fascioliasis. Fasciola adult worms were collected from the bile ducts of infected animals. Species of the fluke was determined by polymerase chain reaction-restriction fragment length polymorphism (RFLP-PCR). ES antigen of F. hepatica was prepared. For production of monoclonal antibodies, mice were immunized with ES antigens of F. hepatica. Spleen cells from the immunized mice were fused with NS-1 myeloma cells, using polyethylene glycol. Hybridoma cells secreting specific antibody were expanded and cloned by limiting dilution. Moreover, polyclonal antibody was produced against F. hepatica ES antigen in rabbits. A capture enzyme-linked immunosorbent assay (ELISA) system, using produced monoclonal antibody, was designed and stool samples of infected animals along with control samples were tested by the system. The capture ELISA detected the coproantigen in 27 of 30 (90%) parasitologically confirmed fascioliasis cases, while 4 of 39 (10.25%) samples infected with other parasitic infections showed a positive reaction in this system. No positive reactivity was found with healthy control samples. Accordingly, sensitivity of 90% and specificity of 94.2% were obtained for the capture ELISA system. The results were compared with those obtained with commercial BIO-X ELISA, and a very good (kappa = 0.9) agreement was found between the commercial kit and the developed capture ELISA. Findings of this study showed that the produced monoclonal antibody has appropriate performance for the detection of Fasciola coproantigen in stool samples and can be appropriately used for the diagnosis of fascioliasis.

Helminth Infections of Rodents and Their Zoonotic Importance in Boyer-Ahmad District, Southwestern Iran.

BACKGROUND: Rodents are considered as reservoirs of various zoonotic diseases including helminthic infections. The current study aimed to evaluate the prevalence of helminth infections in rodents, in Boyer-Ahmad district, Southwestern Iran. METHODS: Overall, 52 rodents were captured from various areas of the district by Sherman live traps. The animals were then euthanized and dissected. During necropsy, each organ was examined macroscopically for presence of any cyst or visible parasite. The gastrointestinal tract was removed and their contents were evaluated for larva or adult worms. Trichinella larvae in the rodents' muscles were investigated by both digestion and pathological methods. RESULTS: Twenty-eight (53.8%) of the trapped rodents were male. The rodents were including 25 (48.1%) Meriones persicus, 1(1.9%) Calomyscus bailwardi, 1 (1.9%) Arvicola terresterris, 7 (13.5%) Rattus rattus, 8 (15.4%) R. norvegicus, and 10 (19.2%) Apodemus sylvaticus. Of them, 38 (73.0%) were infected with at least one helminth. Collected rodents were infected with Hymenolepis diminuta (50%), Hymenolepis nana fraterna (28.8%), Skrjabinotaenia sp. (15.4%), Anoplocephalidae sp. (15.4%), Cysticercus fasciolaris (5.8%), Trichuris muris (36.5%), Aspiculuris tetraptera (15.4%), Syphacia sp. (5.7%), Rictularia sp. (15.4%), Trichostrongylus sp. (3.8%), and Gongylonema sp. (3.8%). M. persicus was the most (84%) infected rodent, yet the differences between rodent genus and helminth infectivity were not statistically significant (P>0.05). CONCLUSION: The rodents in Boyer-Ahmad district are infected with different helminths infections that some of them are recognized as threat to human health.

Prevalence and risk factors of intestinal protozoan infections: a population-based study in rural areas of Boyer-Ahmad district, Southwestern Iran.

BACKGROUND: Parasitic infections are still a significant health problem in rural areas in developing countries including Iran. There is no recent population-based data about the prevalence of human intestinal parasites in most rural areas of Iran. The current study aimed to determine the prevalence of intestinal protozoan infection in inhabitants of rural areas of Boyer-Ahmad district, Southwestern Iran. METHODS: A total of 1025 stool samples were collected from the inhabitant of 50 randomly selected villages in Boyer-Ahmad Township. The stool samples were evaluated by parasitological methods including, direct wet-mounting, formalin ethyl acetate concentration, zinc sulfate floatation, and Trichrome permanent stain for detection of protozoan infections. Diarrheic samples were further evaluated with a modified Ziehl-Neelsen staining method for detection of coccidian parasites. RESULTS: The prevalence of both pathogenic and nonpathogenic intestinal parasites in the population was 37.5% (385 out of 1025 cases), some individual with multiple infections. Giardia lamblia was detected in 179 (17.46%), Blastocystis hominis in 182 (17.76%), Entamoeba histolytica/dispar in 9 (0.87%), Endolimax nana in 216 (21.07%), Entamoeba coli in 151 (14.73%), Ioedamoeba butschlii in 45 (4.39%), Chillomastix mesnili in 22 (2.14%), Trichomonas hominis in 2 (0.19%) and Dientamoeba fragillis in 2 (0.19%) of cases. Multivariate logistic regression revealed significant associations between protozoan infection (pathogenic protozoa) and contact with animals (OR yes/no = 2.22, p < 0.001) and educational status (OR higher/illiterate = 0.40, P = 0.01). CONCLUSION: Findings of this study demonstrated that protozoan infection rate in rural areas of southwestern Iran is still high and remained as a challenging health problem in these areas.

Molecular Evaluation of a Case of Visceral Leishmaniasis Due to Leishmania tropica in Southwestern Iran.

We describe a case of visceral leishmaniasis (VL) due to Leishmania tropica in a 50-year-old Iranian man lived in a VL-endemic area in southwest of Iran. The patient presented with a 3-month history of fever and splenomegaly. Clinical signs and serological findings were suggestive of VL. Spleen biopsy was taken from the patient and intracellular forms of Leishmania amastigotes was seen in Giemsa stained smears. The patient was treated with pentavalent antimonial compound with complete resolution of his systemic signs and symptoms. DNA was extracted from the microscopic slides of the spleen biopsy and the nagt (N-Acetylglucosamine-1-Phosphate Transferase) gene of Leishmania was PCR-amplified. Sequence analysis of the PCR product demonstrated that the case has 99% identity with those of available sequences of L. tropica. Intra-species variation within isolate was 0-0.1%; whereas, inter-species differences of the isolate with those of L. major and L. infantum was significantly higher.

Clinical and Molecular Evaluation of a Case of Giant Primary Splenic Hydatid Cyst: A Case Report.

Spleen is an unusual location for hydatid cyst. Here we report a case of primary splenic hydatid cyst in a 41-yr-old Iranian woman from Yasuj, southwest of Iran. The patient had been admitted to Shahid Beheshti Hospital because of abdominal pain. Abdominal sonography revealed a hypoechoic lesion of 150 X 130 mm in the spleen, suggestive of hydatid cyst. Splenectomy was performed for the patient and surgical interventions revealed a hydatid cyst occupying most of splenic parenchyma. She was discharged on the 5 day of her operation. Postoperative diagnosis and confirmation of hydatid cyst was done by histopathological, molecular and serological approaches. Histopathological evaluation revealed the classical laminated layer of hydatid cyst. DNA was extracted from a part of cyst and PCR amplified. Sequencing and analysis of PCR product revealed that the isolate has the most similarity with G1 strain of Echinococcus granulosus. Patient's serum was positive for IgG anti-hydatid cyst antibodies, using antigen-B ELISA.

Epidemiology of Human Fascioliasis and Intestinal Helminthes in Rural Areas of Boyer-Ahmad Township, Southwest Iran; A Population Based Study.

BACKGROUND: Fascioliasis, caused by Fasciola hepatica and F. gigantica, is one of the most important plant and water borne disease in Iran. This study aimed to determine the prevalence of fascioliasis and intestinal helminthes in inhabitants of rural areas of Boyer-Ahmad in Kohgiluyeh and Boyer-Ahmad province in Southwest of Iran. METHODS: Stool samples (1025) were collected from inhabitant of 50 randomly selected villages in Boyer-Ahmad Township. Samples were evaluated with modified Telemann and formalin-ethyl acetate methods. Blood samples of Fasciola positive cases were assessed with ELISA and Western blotting. DNA was extracted from Fasciola eggs from stool of positive individuals and evaluated by molecular (PCR) method and the PCR products were sequenced and analyzed. RESULTS: Of the 1025 participants, 473 (46.1%) were male and 552 (53.9%) were female. The mean age of the subjects was 20.25 (±15.86) years. Fasciola eggs were detected in stools of two cases (0.19%). Blood samples were obtained from the Fasciola positive cases and their infection was further confirmed by ELISA and Western blotting. Molecular analysis revealed that both cases are infected with F. hepatica. Furthermore, seven of participants (0.68%) were found to be infected with H. nana, 4 cases (0.39%) with E. vermicularis, and one case (0.09%) with Trichuris trichiura. CONCLUSION: Findings of this study showed that Boyer-Ahmad district is one of the endemic areas of human fascioliasis in Iran. The study also documented that the rate of helminthic infections in rural areas of the district has drastically declined over the past years.

Molecular and Morphological Characterization of Fasciola spp. Isolated from Different Host Species in a Newly Emerging Focus of Human Fascioliasis in Iran.

The current study aimed to find out the morphometric and genotypic divergences of the flukes isolated from different hosts in a newly emerging focus of human fascioliasis in Iran. Adult Fasciola spp. were collected from 34 cattle, 13 sheep, and 11 goats from Kohgiluyeh and Boyer-Ahmad province, southwest of Iran. Genomic DNA was extracted from the flukes and PCR-RFLP was used to characterize the isolates. The ITS1, ITS2, and mitochondrial genes (mtDNA) of NDI and COI from individual liver flukes were amplified and the amplicons were sequenced. Genetic variation within and between the species was evaluated by comparing the sequences. Moreover, morphometric characteristics of flukes were measured through a computer image analysis system. Based on RFLP profile, from the total of 58 isolates, 41 isolates (from cattle, sheep, and goat) were identified as Fasciola hepatica, while 17 isolates from cattle were identified as Fasciola gigantica. Comparison of the ITS1 and ITS2 sequences showed six and seven single-base substitutions, resulting in segregation of the specimens into two different genotypes. The sequences of COI markers showed seven DNA polymorphic sites for F. hepatica and 35 DNA polymorphic sites for F. gigantica. Morphological diversity of the two species was observed in linear, ratios, and areas measurements. The findings have implications for studying the population genetics, epidemiology, and control of the disease.

A Consistent PCR-RFLP Assay Based on ITS-2 Ribosomal DNA for Differentiation of Fasciola Species.

OBJECTIVE(S): Fascioliasis is a zoonotic parasitic disease caused by liver fluke species of Fasciola hepatica and Fasciola gigantica. Differentiation of these two species, based on their morphological characteristics, is difficult. The current study aimed to use PCR-RFLP assay to distinguish between F. hepatica and F. gigantica, based on profiles of RFLP, produced by effect of endonucleases on ITS2 of the ribosomal DNA genes from these two species. MATERIALS AND METHODS: Adult Fasciola spp. were isolated from bile duct of naturally infected animals. The species of Fasciola were confirmed by sequencing the 505 bp region of the ITS2 gene in the isolates. By running the sequences of the samples in NEBcutter, suitable restriction enzymes (MspI and KpnI) were selected. Eight F. gigantica and eighteen F. hepatica samples were evaluated. RESULTS: While RFLP pattern with MspI produced a profile by which it was difficult to differentiate these two species, KpnI along with MspI, produced a consistent pattern of a 231, 212 and 93 bp fragments in F. hepatica. This pattern was not seen in F. gigantica. CONCLUSION: Findings of this study demonstrated that RFLP with KpnI and MspI produce a suitable pattern which simply differentiates F. hepatica from F. gigantica.

An observational study on the current distribution of visceral leishmaniasis in different geographical zones of Iran and implication to health policy.

Visceral leishmaniasis is one of the most important parasitic diseases that is endemic in some parts of Iran. This study aimed to determine current distribution of visceral leishmaniasis in four distinct geographical zones of Iran. A cross-sectional study was conducted using direct agglutination test (DAT) on 9396 and 2559 serum samples collected from humans and domestic dogs, respectively during the period of 2007 through 2009. Altogether, 403 (4.3%) out of 9396 human serum samples collected from 4 distinct geographical locations showed anti-Leishmania antibodies with titers ≥ 1:3200. Physical examinations performed on 142 sero-positive cases with anti-Leishmania antibodies at titers of 1: 3200 to 1:102400 among whom fever (94.4%), paleness (67.6%) and hepato-splenomegaly (42.2%) were the predominant clinical signs and symptoms. The highest sero-prevalence rate (1.55%) was found in children ≤ 5 years old. Out of 2559 serum samples collected from domestic dogs, 212 (8.3%) were DAT positive (≥ 1:320). Leishmania infantum is the principal causative agent of the disease was isolated from both infected humans and dogs in Iran. Our findings indicate that Mediterranean visceral leishmaniasis with different distribution occurs in different geographical locations of Iran.