Iranian Journal of Parasitology
Volume:8 Issue: 3, Jul-Sep 2013

Visceral leishmaniasis (VL) is a life-threatening vector-borne parasitic dis­ease is distributed in some parts of the new world and old world. The disease is endemic in different parts of Iran. This review article has been focused on major topics of epidemio­logical aspects and clinical features of VL in Iran for the period of 2002 through 2012. For the detection of VL in humans as well as animal reservoir hosts, anti-Leishmania antibodies were detected using direct agglutination test (DAT) as a validated serological test. Parasitological examinations were performed on suspected VL patients as well as canines and rodents. Different molecular methods were used for identification of species and genotype/ or strain of Leishmania spp. isolated from infected humans, animal reser­voir hosts and vectors. Altogether, 1698 out of 36081 (4.7%) human serum samples collected from 5 distinct geographical zones showed anti-Leishmania antibodies at ti­ters≥1:3200 using DAT. The majority of VL cases in the endemic areas were found among children up to 12 years old. Almost 75% of DAT-positive cases (≥1:3200) in en­demic areas showed clinical signs and symptoms. Predominant signs and symptoms in 217 hospitalized patients with DAT positive (≥1:3200) results included paleness (99.5%), fever (96.9%), splenomegaly (91.5%), hepatomegaly (53.6%) and lymphadenopathy (21.1%). Integrated VL surveillance system in primary care using DAT, could decrease mortality and morbidity of the disease in the VL endemic areas of the northwestern Iran. Out of 7204 serum samples collected from domestic dogs in various geographical loca­tions of Iran, 879 (12.2%) were DAT sero-positive at titers≥1:320. L. infantum as the prin­cipal causative agent of the disease was isolated from infected humans, domestic and wild canines and rodents. The principal animal reservoir hosts of the infection are domestic and wild canines. Ph. kandelakii, Ph. perfiliewi transcaucasicus, Ph. tobbi in northwestern Iran; Ph. major s.l. (=Ph. neglectus), Ph. keshishiani, and Ph. alexandri in southern parts of Iran were molecularly and/or parasitologically positive for L. infantum infections. The zoonotic form of VL (ZVL) caused by L. infantum occurs sporadically in all geographical zones of Iran but in northwestern and southern parts of the country the disease is endemic. DAT as an appropriate and potential tool has been used for sero-diagnosis and sero- epidemiologi­cal of VL among humans as well as domestic and wild canines.

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

Based on recent studies, there are controversial reports on the capac­ity of tissue cyst forming of Toxoplasma gondii RH strain. In this study, the capac­ity was evaluated by in vivo and in vitro experiments. RH strain was subcutaneously inoculated to ten Wistar rats. After one month, their blood, brain, tongue and diaphragm were collected and evaluated by MAT, PCR, pathological and bioassay methods. The parasite was cultivated in the cell monolayer. To change to bradyzoite, the media pH was altered to 6.8. Biologi­cal aspect of the bradyzoites was evaluated by incubation in acidic pepsin and it''s inoculation in ten BALB/c mice. All rats showed antibodies to Toxoplasma at titers ≥1:320 but no DNA and tissue cyst were detected in the tissues. Following intraperitoneal inoculation of rats’ brain homogenate into BALB/c mice, no infection was established in none of the animals. During presence of cell culture, in acid media for a 3-5 days period, cyst-like structures were noticed when they were stained with PAS. The visible brady­zoites in the cysts that were incubated in acid pepsin medium were not able to kill any mice. This study confirmed that Iranian RH strain has lost the potential of tissue cyst forming in rats and bradyzoites cultivated in cell culture lost their resis­tance to acidic condition, so this strain can be a candidate for future vaccine re­searches.

The aim of this study was DNA extraction from protosco­lecses of Echinococcus granulosus and identification of these strains in West-Azerbai­jan Province, north western Iran. Thirty one livestock isolates from sheep and cattle were collected from abattoirs of the province. To investigate the genetic variation of the isolates, after DNA extraction by Glass beads-phenol chloroform method; PCR-RLFP analysis of rDNA-ITS1 was performed using three different restric­tion enzymes of Taq 1, Rsa 1 and Alu 1. Amplified PCR products for all isolates were 1000bp band which is expected band in sheep strains (G1-G3 complex). The results of RFLP analy­sis also were the same for all isolates. PCR-RFLP patterns restriction en­zymes were identical as follows, Rsa1 bands under UV showed two bands approximately 655bp and 345bp. Alu1 bands were as follows: two approx­imately 800bp and 200bp and Taq1 did not cut any region and bands were approximately 1000 bp in all samples. Based on PCR-RFLP patterns of ITS1 fragment produced with endonucleases enzyme digestion in animal isolates, it can be concluded that a single strain of E. granulosus (sheep strain or G1-G3 complex) is domi­nant genotype in this province.

Leishmaniasis is a group of diseases that are created by intracellular parasites of Leishmania. Cutaneous leishmaniasis is considered as one of the health problems in some provinces of Iran. In this study, a total of 178 Giemsa-stained slides from confirmed cases of cutaneous leishmaniasis were examined. The slides were prepared from the patients with cutaneous leishmaniasis that referred to health centers and infected during the epidemic of cutaneous leishmaniasis in Poldokhtar city, Lorestan Province, Iran in 2006.Genomic DNA from each slide was extracted. After DNA extraction, ITS-PCR was used. Out of 178 slides, 129 (72.47%) samples had a band in the range of 485 bp and 49 (27.53%) samples 626 bp that matched L. tropica and L. major standard samples, respectively. This study showed that Leishmania DNA could be efficiently ex­tracted and amplified even from old Giemsa-stained microscopic slides that were stored more than 6 yr. In this study was shown that both L. tropica and L. major species exist in Lorestan Province.

Giardia duodenalis is one of the most important human enteric para­sites throughout the world. Clinical symptoms of this parasite vary from asympto­matic infection to chronic diarrhea. Still it is not clear, whether different types of pathogenesis are due to different strains of organism or to variable host factors. The purpose of this study was to investigate possible correlation of clinical symp­toms with assemblages among symptomatic and asymptomatic cases collected from southwest of Iran. Fecal samples were collected from 100 symptomatic and asymptomatic cases, which were positive for G. duodenalis. The samples were subjected to semi-nested PCR and RFLP for gdh gene. Among symptomatic patients, 54% had mixed genotypes AII and BIII, 28% and 18% of samples indicated assemblages BIII and AII, respectively. In con­trast, among asymptomatic cases, 64%, 26% and 10%samples had mixed geno­types, BIII and AII assemblages, respectively. Statistical analysis using Chi-Square test showed that there was no significant correlation between assemblage and clini­cal symptoms in current study. High prevalence of mixed infection in both groups may affect this conclusion, therefore further study in more details are necessary to clarify these finding. Additionally, it is important to carry out investigations regarding human host factors as well.

Pentavalent antimonials are still the first choice treatment for leishma­niasis, but with low efficacy and resistance is emerging. In the present study, the effect of meglumine antimoniate (MA, Glucantime) combined with paromomy­cin, miltefosine or allopurinol on in vitro susceptibility of Leishmania tropica resistant isolate was evaluated. The drugs were obtained from commercial sources and diluents of each drug in medium were prepared on the day of experiment. J774 A.1 murine macrophage cell lines were attached to the cultured on slide and incubated at 37 0C with 5% CO2 for 24 h. Then the stationary phase promastigotes were added to the cells and after 4 hrs of incubation different concentrations of MA, paromomycin, miltefosine or allopurinol were added and incubated for an additional of 72 h. Then the slides were dried and fixed with methanol, stained by Giemsa and studied under a light microscope. Drug activity was evaluated by assessing the macrophage infection rate and the number of amastigotes per infected macrophage was done by examin­ing 100 macrophages. The experiment was done in triplicates. Various concentrations of MA along with paromomycin, miltefosine or allopurinol significantly inhibited (P<0.01) the proliferation of L. tropica amastigote stage in the macrophage cell line as compared with MA alone or positive control. Combination of Glucantime with paromomycin, miltefosine or allopuri­nol showed a synergistic effect on the clinical isolate of L. tropica in vitro. Use of combination therapy is a new hope and a logical basis for therapy of the pa­tients with cutaneous leishmaniasis. Further investigations are needed to evaluate the therapeutic effects of these drugs on the CL patients.

Trichomonas vaginalis causes trichomoniasis and metronidazole is its chosen drug for treatment. Ferredoxin has role in electron transport and carbohydrate metabolism and the conversion of an inactive form of metronidazole (CO) to its active form (CPR). Ferredoxin gene mutations reduce gene expression and increase its resistance to metronidazole. In this study, the frequency of ferredoxin gene mutations in clinical isolates of T.vaginalis in Tehran has been studied. Forty six clinical T. vaginalis isolates of vaginal secretions and urine sediment were collected from Tehran Province since 2011 till 2012. DNA was extracted and ferredoxin gene was amplified by PCR technique. The ferredoxin gene PCR products were sequenced to determine gene mutations. In four isolates (8.69%) point mutation at nucleotide position -239 (the translation start codon) of the ferredoxin gene were detected in which adenosine were converted to thymine. Mutation at nucleotide -239 ferredoxin gene reduces translational regulatory protein’s binding affinity which concludes reduction of ferredoxin expression. For this reduction, decrease in activity and decrease in metronidazole drug delivery into the cells occur. Mutations in these four isolates may lead to resistance of them to metronidazole.

Sparganosis is caused by the invasion of Spirometra sparganum into various tissues/organs. Subcutaneous sparganosis can be diagnosed by biopsy, while visceral/cerebral sparganosis is not easy to be diagnosed. The diagnosis de­pends largely on the detection of specific anti-sparganum antibodies. The specific­ity of the ELISA could be increased by using S. mansoni sparganum excretory–secre­tory (ES) antigens, but it also had the cross-reactions with sera of patients with cysticercosis or paragonimiasis. The aim of this study was to identify early specific diagnostic antigens in S. mansoni sparganum ES proteins. The sparganum ES proteins were analyzed by two-dimensional electrophore­sis (2-DE) and Western blot probed with early sera from infected mice at 14 days post-infection. The immunoreactive protein spots were characterized by MALDI-TOF/ TOF-MS. A total of approximately 149 proteins spots were detected with isoelectric point (pI) varying from 3 to 7.5 and molecular weight from 20 to 115 kDa and seven protein spots with molecular weight of 23-31 kDa were recognized by the infection sera. Three of seven spots were successfully identified and characterized as the same S. mansoni protein (cysteine protease), and the proteins of other 4 spots were not included in the databases. The cysteine protease from S. mansoni ES proteins recognized by early infection sera might be the early diagnostic antigens for sparganosis.

Hydatidosis is a zoonotic disease of global prevalence. It causes considerable health problems and economic losses throughout the world, including Iran. The objective of this study was to assess the current status of echinococco­sis/hydatidosis in the province of Ilam (western Iran). From April to September 2011, 65 stray dogs were collected from urban and rural areas of Ilam City. Parasites were isolated from the dogs and stained with carmine. A taxonomic study was carried out by measuring different parts of hel­minths. Meat inspection documents from slaughterhouses in Ilam were used to assess the prevalence of hydatidosis during a 3-year period in sheep, cattle, and goats. ELISA test was used to detect the presence of antibodies to hydatidosis in human sera. Clinical records from 2000 to 2010 of either treated or diagnosed pa­tients from public hospitals of this province were reviewed. The prevalence of Echinococcus granulosus infection in stray dogs was 9%. A total of 81,726 animals were assessed for hydatidosis; 2.94% (2403 cases) had liver hydatidosis and 2.34% (1918 cases) had lung hydatidosis. Within a 10-year period, 140 patients (91 females and 49 males) were treated for hydatidosis. Of 1200 hu­man sera, 2.25% (27 patients) were seropositive for hydatidosis. Hydatidosis is endemic in Ilam Province especially in rural area. The health and economic losses caused by the disease are significant; thus, our efforts need to be focused on the control of this disease.

Trematodes are a diverse group of endoparasites which require molluscan and vertebrate animals as intermediate and definitive hosts in their life cycle. The present study was carried out to determine the diversity and geographic distribution of infection with trematodes’cercariae in the snail Lymnaea gedrosiana from north-west Iran. A total number of 6759 Lymnaeidae snails were collected from 28 snail habitats; of these L. gedrosiana was the prevalent snail (74.37%) which examined for cercarial infection by shedding method. The overall infection rate was 8.03%. The most frequent trematodes cercariae in the snail were xiphidiocercariae (81.98%), furcocercariae (32.26%), echinostome cercariae (5.19%), and monostome cercariae (1.24%). The highest infection rate in L. gedrosiana (100%) was with echinostome cercariae from Golestaneh in autumn. Due to the important role of pond snails in transmission of cercariae to fish as a source of zoonotic diseases, it is essential to estimate the distribution and abundance of the snails and the rate of their infection with different trema­todes’ cercariae, and establish control programs in each region.

Cutaneous leishmaniasis (CL) is a major health problem in many parts of Iran, although diagnosis of CL especially in the endemic area is easy, but treatment and management of the disease is a global dilemma. Diagno­sis of CL in non-endemic area is not as simple as in endemic foci. In this study, the status and the proportions of CL induced by Leishmania major and L. tropica among CL suspected patients referred to the Center for Research and Training in Skin Diseases and Leprosy, (CRTSDL) during 2008 to 2011 are described. CL patients with suspected lesions were clinically examined. History of trip to zoonotic CL and/or anthroponotic CL endemic areas and the characte­ristics of their lesion(s) were recorded. Diagnosis of the lesion was done using direct smear microscopy, culture and conventional polymerase chain reaction (PCR). A total of 404 (M=256, F=148) patients with 776 lesions were re­cruited and parasitologically examined. The results showed that 255 of the pa­tients with 613 lesions; patients with lesion(s) induced by L. major=147 (M=63, 43%, F=84, 57%) and lesion(s) induced by L. tropica=108 (M=35, 32%, F=73, 68%). History of travel to endemic area was not always correlated with isolated Leishmania species. Although travel history to endemic area is an important factor to be considered for diagnosis, but parasitological confirmation is necessary initia­tion of treatment.

Infestation of the skin by the “itch mite” Sarcoptes scabiei var. hominis results in a contagious skin infection in humans called “sca­bies”. By resolving morphology issues, the present study was designed to be acquainted with itch mite by molecular markers. The mite samples were collected from scabies patients by visiting government hospitals of twin City, Pakistan. For successful molecu­lar detection approach, preparation of Sarcoptes mite DNA by commer­cial DNA extraction kit method. Furthermore, two primers i.e. Sarms 15 F/R and 16S D1/D2 were used to amplify target sequence by using PCR. The amplified products were then separated by agarose gel, electrophoresis and analyzed after staining and visualizing in UV transillu­minator. Analysis of PCR product showed one specific band of 178 bp with primer Sarms 15 F/R, while, with primer 16S D1/D2 bands of 460 bp and 600 bp were observed on 2% agarose gel. The appearance of different band of 600 bp revealed that it might be due to heteroplasmy state present in the Pakistani Sarcoptes mites population. Current study adds validity to the claim that PCR is more accurate, specific and sensitive in the detection of the ectoparasites even in smallest amount.

Serological diagnosis of Toxoplasma gondii infection using crude antigens may not be more accurate. To increase the diagnostic potency of anti­gens, isolation of their immunogenic fractions could be useful. The current re­search adopted to obtain an affinity isolated fraction from RH strain using CNBr Sepharose 4B column coupled with infected mice sera helping in detec­tion of IgM and IgG of toxoplasmosis due to RH strain and other strains. The isolated fraction was characterized by SDS-PAGE. Moreover, the diagnostic potency of the fraction was assessed by indirect ELISA in mice experimentally infected with RH strain and two other local strains; one of sheep origin and the other of human origin. The fraction was found to be consisted of a single band of 116 kDa compared with 17 bands ranged from 116 to 16 kDa associated with crude ex­tract. The fraction proved potent diagnostic potentials of acute and chronic mice toxoplasmosis. Where it was detected both IgM and IgG antibodies as early as two days and as late as 2 months post experimental infection with any of the three strains. The level of detected IgM and IgG by RH fraction was higher in mice infected with RH strain than with local strains except IgM due to sheep strain parasite. The 116 kDa fraction of T. gondii tachyzoites can be considered as a candidate in improving of serodiagnosisof Toxoplasma infections.

To control avian coccidiosis with drug-independent strategy effec­tively and safely, multivalent hyperimmune egg yolk immunoglobulin (IgY) was prepared and its ability to protect against Eimeria tenella infection was evaluated. Hens were orally immunized with live oocysts of 5 species of Eimeria for six times, antibody titers in serum and yolk were monitored by indirect enzyme-linked immunosorbent assay. The specific IgY was isolated, purified and lyophi­lized. IgY powder was orally administrated as dietary supplement in newly hatched chicks at various dosages. Birds were orally challenged with 10000 sporulated oo­cysts of E. tenella at 10 days of age, weighed and killed at 8 days post challenge, and the protective effect was assessed. The averge yeid of IgY was 9.2 mg/ml yolk, the antibody titer of IgY reached to 1:163840 per mg with the purity up to 98%. Chickens fed IgY resulted in reduced mortality, increased body weight gain (BWG), reduced oocyst shedding, reduced caecal lesion score and increased anti-coccidial index. In terms of BWG and caecal lesion, IgY significantly enhanced the resistance of bird at ≥ 0.05% of IgY in the diet when compared with the challenged control group (P<0.05). No significant difference was observed at dosage ≥ 0.5% and 1.0% when BWG and caecal lesion were compared with the sodium salinomycin control group, respec­tively (P>0.05). Supplementing newly hatched chicks with Eimeria-specific IgY represents a promising strategy to prevent avian coccidiosis.

To find out different species of helminthes and blood/tissue proto­zoan parasites of stray dogs and their potential role for transmission of zoonotic species to human in Mashhad, Khorasan Razavi Province, northeast Iran, during 2008-2009. Totally, 100 stray dogs were selected among Mashhad municipal collection from different sites of the city. Internal organs were examined for any parasites. Helminthes were identified based on morphological characteristics. Smears prepared from peripheral blood as well as liver, spleen and any skin lesion were stained by Giemsa and examined microscopically. Samples obtained from spleen were aseptically cultured in three culture media including NNN, Schneider’s Drosophila (HIMEDIA) and RPMI1640 (GIBCO) for isolation of Leishmania spp. The titer of anti-Leishmania and anti-Toxoplasma antibodies were measured by direct agglutination test (DAT) and indirect fluorescent antibody test (IFAT), respectively. 84% of dogs were infected at least with one species of intestinal helminthes. The species of parasites and rate of infection were as follows: Taenia hydatigena (61%), Dipylidium caninum (46%), Mesocestoides lineatus (19%), Echinococcus granulosus (10%), Toxascaris leonina (53%) and Toxocara canis (7%). Anti-Leishmania antibodies were detected by DAT in 8 dogs (8%) at 1:320 titers and higher. Forty seven dogs (47%) showed anti-Toxoplasma titer at 1:10 and 17 (17%) showed titer of ≥1:100. No blood parasites were found in prepared blood smears. The high rate of parasitic infection and presence of zoonotic species

In view of the massive rural-to-urban migration in Nigeria, investiga­tions on transmission of urinary schistosomiasis were carried out in ur­ban and semi-urban communities in Nike Lake area of Enugu State, Nigeria. Urine samples of school children were tested for micro-haematuria using reagent strips followed by microscopic examination for Schistosoma haemato­bium eggs. Water contact sites were also identified and sampled for snails. The overall prevalence of S. haematobium eggs in school children was 4.64%. The mean intensity of infection was 1.14 + 0.41 eggs/10ml urine. Males had insignificantly higher prevalence and intensity of S. haematobium infec­tion than females. The youngest age group (4-7 years) had no infection. The preva­lence of micro-haematuria (6.2%) was higher than that of microscopy, and this correlated positively with prevalence (r=0.65, P < 0.01) and intensity (r=0.50, P < 0.01) of the infection. Potential intermediate host of human shistosome collected were: Bulinus globosus, B. senegalensis and Biomphalaria pfeifferi. How­ever, only B. globosus shed cercariae of S. haematobium, with a snail infection rate of 0.73%. Transmission was in the dry season coinciding with the drying of wells. The results revealed that urinary schistosomiasis is prevalent, and that B. globosus and not B. truncatus as previously reported is the main interme­diate host of urinary schistosomiasis in this part of Enugu State.

The aim of the present study was the comparison of humoral and cell-mediated immunity in ‎broilers fed with different levels of zinc during a coccidiosis challenge.‎ One hundred and forty-‎four one-day-old broiler chicks were used with three ‎dietary zinc ‎(40, 120 and 200 mg/kg). At 14 d of age, all birds were inoculated orally with 5×103 sporulated oocysts of E. Tenella. ‎At ‎2, 22, 32, 42 ‎days of age, the blood serums were tested for ‎antibody titer against‎ Newcas­tle disease vaccine, using ‎the standard HI test. On day 42 the sum of nitrite ‎and nitrate based on the reduction of nitrate ‎to nitrite by cadmium ‎and white blood cell count (WBC) using a hemocytometer were measured. At 42 d, levels of ‎120 and 200 mg significantly (P< 0.05) increased the antibody titer in compare with the control. The peak response of CBH was observed at the level of 200 mg Zn/kg diet. Also both level of 120 and 200 mg Zn/kg diet increased WBC count and sum of nitrite and nitrate‎ in serum compared with the control. The levels of 120 and 200 mg Zn/kg diet could be considered as a non-pharmacologic booster of immunity in broilers chicks infected with E. Tenella.

Poor hygiene will provide good condition for corneal infections by opportunistic free-living amoebae (FLA) in soft contact lens wearers. In the present study an amoebic keratitis due to Hartmannella has been recognized in a 22-year-old girl with a history of improper soft contact lens use. She had unilateral keratitis on her left eye. Her clinical signs were eye pain, redness, blurred vision and photopho­bia. The round cysts of free-living amoebae were identified in non-nutrient agar medium by light microscopy. These cysts were suspected to be Hartmannella using morphological criteria. A PCR assay has been confirmed that the round cysts were belonged to H. vermiformis.

Rats are capable to harbor various pathogens, among which certain species of zoonotic parasites are included. A long-term detection of parasite fauna of rats has sporadically been carried out in Iran. Abundance of these vertebrate pests is of great importance as regards public health issue. The present paper is fo­cused on a digenean trematode Plagiorchis muris, obtained during a comprehensive study on rats over the decades in the country. Herein we describe this occurrence in a Rattus norvegicus in northern Tehran, with specific note on its morphological description. P. muris can infect human through consumption of infected marine food items, and has never been observed in Iran.

A known species Lutztrema (Lutziella) microacetabularae Rhode, 1966 is being described for the first time from Pakistan. This species is characterized by having body long and slender, oral sucker subterminal, acetabulum smaller than oral sucker lying in anterior third of the body, pharynx small, esophagus promi­nent which become gradually wider and bifurcates in to two rudimentary caeca. Testes symmetrical at the level of posterior margin of acetabulum separated by uterine coils, cirrus pouch median, pre-acetabular, genital opening some distance behind pharynx. Receptaculumsemin is behind ovary. Ovary submedian, post-testicu­lar, Laurer’s canal present. Vitellaria lateral from the level of testes to a short distance behind middle of the body. Uterus occupies most of the hind body, eggs small, oval, numerous. It is being reported from the rat (Rattusrattus L.) from Swat.

Genus Acanthocolpus (Trematoda: Acanthocolpiidae) is one of the most important zoonotic digenean with wide geographic distribution in the world. The purpose of the present study was to describe morphological and morphometrical characteristics of Acanthocolpus species, currently prevalent in marine fish fauna of Puri coast, Orissa, India. Gastro-intestinal organs of Leiognathus daura (Cuvier) in Puri coast, Orissa, India, were examined for infectivity with digenean trematode species. For examination and measurements of hel­minthes, acetoalum carmine staining was performed, followed by cam­era Lucida drawings of morphological characters and measure­ments of morphometrical criteria with a calibrated microscope. Us­ing valid trematode systematic keys, almost all the parasites were identi­fied at the level of species. Overall, 36 marine fishes were found infected with at least one species of Acanthocolpus. Considering morphological characteris­tics of Acanthocolpus, two species were identified as new species includ­ing Acanthocolpus durghai sp.nov. and Acanthocolpus amrawatai sp.nov. During the survey of helminth parasites, collected six different species of the genus Acanthocolpus, out of these two are new species, another is redescribed to show certain variation, the new para­site was obtain from the intestine of marine fish Leiognathus daura (Cuvier)