Molecular Characterization of Eimeria Species Naturally Infecting Egyptian Baldi Chickens

Background:

Coccidiosis is a serious protozoal disease of poultry. The identification of Eimeria species has important implications for diagnosis and control as well as for epidemiology. The molecular characterization of Eimeria species infecting Egyptian baladi chickens was investigated.

Methods:

Eimeria species oocysts were harvested from intestines of naturally infected Egyptian baldi chickens. The morphometry characterization of oocysts along with COCCIMORPH software was done. The DNA was extracted initially by freezing and thawing then the prepared samples was subjected to commercial DNA kits. The DNA products were analyzed through conventional polymerase chain reaction by using amplified region (SCAR) marker.

Results:

The PCR results confirmed the presence of 7 Eimeria species in the examined fecal samples of Egyptian baldi breed with their specific ampilicon sizes being E. acervulina (811bp), E. brunette (626bp), E. tenella (539bp), E. maxima (272bp), E. necatrix (200bp), E. mitis (327bp) and E. praecopx (354bp). A sequencing of the two most predominant species of Eimeria was done, on E. tenella and E. máxima. Analysis of the obtained sequences revealed high identities 99% between Egyptian isolates and the reference one. Similarly, E. maxima isolated from Egyptian baldi chickens showed 98% nucleotide identities with the reference strain. Only single nucleotide substitution was observed among the Egyptian E. tenella isolates (A181G) when compared to the reference one. The Egyptian isolates acquired 4 unique mutations (A68T, C164T, G190A and C227G) in compared with the reference sequence.

Conclusion:

This is the first time to identify the 7 species of Eimeria from Egyptian baladi chickens.     

Sequence Analysis of the Second Internal Transcribed Spacer (ITS2) Region of rDNA for Species Identification of Trichostrongylus Nematodes Isolated From Domestic Livestock in Iran

Background

Infectivity of herbivores with Trichostrongylus nematodes is widespread in many countries, having a major economic impact on breeding, survivability, and productivity of domestic livestock. This study was carried out on Trichostrongylus species isolated from domestic livestock in order to develop an easy-to-perform method for species identification.

Methods

Trichostrongylus isolates were collected from sheep, goat, cattle, and buffaloes in Khuzestan Province, southwest Iran. Primary species identification was carried out based on morphological characterization of male worms. PCR amplification of ITS2-rDNA region was performed on genomic DNA and the products were sequenced. Phylogenetic analysis of the nucleotide sequence data was conducted employing Bayesian Inference approach. Consequently, a restriction fragment length polymorphism (RFLP) profile was designed to differentiate Trichostrongylus species.

Results

A consensus sequence of 238 nucleotides was deposited in the GenBank for Iranian isolates of Trichostrongylus species including T. colubriformis, T. capricola, T. probolurus and T. vitrinus. The designated RFLP using restriction enzyme TasI could readily differentiate among species having different ITS2 sequence. The molecular analysis was in concordance with morphological findings.

Conclusion

Phylogenetic analysis indicated a close relationship among the sequences obtained in this study and reference sequence of relevant species. ITS2-RFLP with TasI is recommended for molecular differentiation of common Trichostrongylus species.     

Molecular Identification and Differentiation of Fasciola Isolates Using PCR- RFLP Method Based on Internal Transcribed Spacer (ITS1, 5.8S rDNA,ITS2)

Background

In this study, we used both ITS1 and ITS2 for molecular identification of Fasciola species.

Methods

The region between 18S and 28S of ribosomal DNA was used in PCR-RFLP method for molecular identification of Fasciola species. Ninety trematodes of Fasciola were collected during abattoir inspection from livers of naturally infected sheep and cattle from Khorasan, East Azerbaijan, and Fars provinces in Iran. After DNA extraction, PCR was performed to amplify region ITS1, 5.8S rDNA, ITS2. To select a suitable restriction enzyme, we sequenced and analyzed the PCR products of F. hepatica and F. gigantica samples from sheep and cattle. Tsp509I fast digest restriction enzyme was selected for RFLP method that caused the separation specifically of Fasciola species.

Results

The fragment approximately 1000bp in all of the Fasciola samples was amplified and then digested with the Tsp509I restriction endonuclease. Seventy F. hepatica and 20 F. gigantica were identified of total 90 Fasciola isolates.

Conclusion

The new PCR-RFLP assay using Tsp509I restriction enzyme provides a simple, practical, fast, low cost, and reliable method for identification and differentiation of Fasciola isolates.     

Identification of Leishmania Species Using PCR Assay on Giemsa-Stained Slides Prepared From Cutaneous Leishmaniasis Patients

Background

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.

Methods

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.

Results

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.

Conclusion

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.     

Biopathologic Characterization of Three Mixed Poultry Eimeria spp. Isolates

Background

Coccidiosis of domestic fowl, caused by species of the Genus Eimeria, is responsible for important economic losses in poultry production. Because different species and/or strains can vary in pathogenicity and other biological parameters, their precise characterization is important for epizootiological studies.

Methods

Fifty samples from litter, whole intestinal tract and feces were collected from poultry houses located in different provinces of Iran. One hundred twenty male day-old broiler chicks were challenged with three selected isolates. Data on weight gain, Food Conversion Ratio (FCR), food intake, lesion scoring and shedding of oocysts per gram of feces were recorded and analyzed by the Duncan''s test.

Results

In all treatments, the challenged groups had statistically significant lower weight gain than that of unchallenged control group. Isolate three caused the lowest weight gain and food intake and the worst lesion score as well as FCR. Despite originating from close geographical regions for isolates 1 and 2, the difference in biopathologic factors may be either due to different proportion of identified species or the different pathogenicity of the species present in the isolates.

Conclusion

The results highlight the importance of considering various species of Eimeria in designing the preventive, control and treatment strategies to prevent coccidiosis in different regions of Iran. Further characterization of each isolate would be the next step to provide a basis for coccidiosis research with well-characterized local isolates.     

Study on ITS1 Gene of Iranian Trichomonas vaginalis by Molecular Methods

Background

Trichomoniasis is a worldwide protozoan parasitic disease and metronidazole is a choice drug for its treatment. Because of disease importance in public health and its controversial ideas about the prevalence of drug resistance, this study was carried out.

Methods

Fifty-two suspected vaginal samples were collected from 2006 to 2007 in Gynecology Maryam Hospital, Tehran, Iran. All isolates were examined by microscopic, culture and PCR techniques. The PCR products were analyzed by RFLP and CSGE methods and two suspected samples were sequenced.

Results

Trichomonas vaginalis was identified from all 52 samples. Of 52 isolates, 45 samples were successfully cultured and amplified by PCR except one. Seven were positive only by PCR. Finally, ITS1 fragment was successfully amplified in 51 of 52. CSGE analysis and PCR products digestion by MspI followed by sequencing showed nucleotide mutation at position 209 (C209T) of the ITS1 fragment in two (3.9%) of them.

Conclusion

The results showed mutation in ITS1 fragment of T. vaginalis in two (3.9%) of Iranian isolates which may be related to metronidazole resistance.     

Detection of Asymptomatic Carriers of Plasmodium vivax among Treated Patients by Nested PCR Method in Minab,Rudan and Bashagard,Iran

Background

Plasmodium vivax is the most widespread species of Plasmodium in humans and causing about 80 million clinical cases annually. This study was undertaken to detect P. vivax in asymptomatic treated vivax malaria patients to trace latent/sub-patent malaria infection.

Method

The venous blood of all detected cases with P. vivax in Bashagard, Minab and Roodan Districts in Hormozgan Province from 2009 to 2010 was examined by microscopic and nested PCR methods for presence of the parasite.

Results

In microscopic examination of peripheral blood smears, all samples were negative for the presence of the parasites. But, we detected two P. vivax related bands in the electrophoresis of the nested PCR products (120 bp).

Conclusion

Following up the malaria cases after treatment by a combination of methods, or new diagnostics such as RDTs can be included in the priorities of malaria elimination program in Iran.     

Phylogenetic Study of Haemonchus Species from Iran Based On Morpho-Molecular Characterization

Background:

Haemonchosis has a negative effect on the farming industry throughout the world, especially in the tropic and sub-tropic countries. The present study was carried out to differentiate Haemonchus species from its main hosts in Iran, including sheep, goat and camel.

Methods:

The identification took place based on the morphometrics of the spicules and molecular characters. Two hundred seventy adult male nematodes were collected from the abomasums of different ruminants (90 samples from each animal) at the slaughterhouses from different localities in Iran. Samples were morphologically identified according to the spicules’ morphometric measurements. In the section on molecular study, 10 samples of each Haemonchus isolates were genetically examined. A simple PCR-restriction fragment length polymorphism (PCR-RFLP) assay of the second internal transcribed spacer of ribosomal DNA (ITS2-rDNA) were described to confirm the PCR results.

Results:

PCR-RFLP profile obtained from the restriction enzyme HPa1 in H. contortus and H. longistipes indicated 1 (278 bp) and 2 (113 and 135 bp) different fragments, respectively. The morphological parameters clearly distinguish H. contortus from H. longistipes. Moreover, regarding the ITS2-rDNA, sequences of 295 bp and 314 bp were obtained from H. contortus and H. longistipes, respectively.

Conclusion:

The genotypic results are in agreement with the phenotypic findings of both species.     

Species Identification of Birds Nasal Trichobilharzia in Sari,North of Iran

Background

Cercarial dermatitis is known as an endemic parasitic disease in North of Iran, a hypersensitive skin reaction to the penetration of nonhuman schistosome larvae into human skin. In recent studies in this region, final and intermediate hosts were recognized and Trichobilharzia was identified as the main causative agent of cercarial dermatitis in this region, but to date the parasite species haven''t been identified. Therefore this study was performed to species identification of nasal Trichobilharzia in infected birds for the first time.

Methods

A total of 45 Anas clypeata birds identified as final host, were collected from Sari in North of Iran and infected nasal tissues analyzed using molecular techniques. Genomic DNA was isolated by phenol/chloroform extraction method and ITS region of rDNA were amplified with specific primers its5Trem and its4Trem, then sequenced area were compared with existing records in GenBank.

Results

Twelve samples were infected with Trichobilharzia and results of PCR reaction indicated that all of them belonged to T. regenti. The sequence alignment of present work isolates and those deposited in GenBank showed differences in nucleotide sequences of repeat region in ITS1.

Conclusion

Trichobilharzia regenti is the most frequent parasite of Anatid birds in North of Iran. This corresponds to the distribution of this parasite along the flyway of migratory birds, which annually migrate from Siberia and northern countries of Caspian Sea to wintering areas in southern regions of it.     

The Problem of Mixing up of Leishmania Isolates in the Laboratory: Suggestion of ITS1 Gene Sequencing for Verification of Species

Background

Leishmaniasis is endemic in Iran. Different species of Leishmania (L.) parasites are causative agents of this disease. Correct identification of Leishmania species is important for clinical studies, prevention, and control of the diseases. Mix up of Leishmania isolates is possible in the laboratory, so there is need for verification of species for isolates of uncertain identity. Different methods may be used for this purpose including isoenzyme electrophoresis and molecular methods. The isoenzyme electrophoresis, due to its drawbacks, is feasible only in specialized laboratories while molecular methods may be more feasible. The aim of this research was to study the application of the internal transcribed spacer 1 (ITS1) sequencing method, in comparison to isoenzyme electrophoresis method, for verification of Leishmania species.

Methods

Six Leishmania isolates were received from different research institutions in Iran. The species of these isolates were known by donating institution according to their isoenzyme profile. The species of these isolates were re-identified in Pasteur Institute of Iran by PCR amplification of ITS1 followed by sequencing and comparison of these sequences with Leishmania sequences in GenBank. Isoenzyme electrophoresis was performed for confirmation of the results of ITS1.

Results

ITS1 sequence showed that some isolates were mixed up or contaminated with Crithidia. Isoenzyme electrophoresis confirmed the results of ITS1 sequences.

Conclusion

ITS1 sequencing is relatively more feasible than the traditional isoenzyme electrophoresis method and is suggested for verification of Leishmania species.     

Detection of Infection with Larval Stages of Ornithobilharzia turkestanicum using PCR in Field-Collected Snails of Lymnaea gedrosiana from Northwestern Iran

Background

Infection with Ornithobilharzia turkestanicum has been reported in a wide range of animals worldwide. This study was undertaken to assess the utility of polymerase chain reaction (PCR), for detecting the infection with O. turkestanicum larvae stages in Lymnaea gedrosiana.

Methods

A total of 6,759 Lymnaeidae snails were collected from six aquatic habitats in West Azarbaijan, northwest Iran. Of these, the snails of L. gedrosiana were identified. To detect infected L. gedrosiana with the larval stages of O. turkestanicum, they were subjected for cercarial shedding and molecular examinations. The genomic DNA was extracted and PCR was performed to specifically amplify a fragment of the nuclear 28SrRNA gene of O. turkestanicum.

Results

Of all collected snails, 5.4% (365/6,759) were the snails of L. gedrosiana. The cercarial shedding method revealed that 23.56% (86/365) of the snails were infected. The PCR patterns confirmed that 28.77% (105/365) snails of L. gedrosiana were infected with larval stages of O. turkestanicum. The infected snails were observed in five studied sites. The highest infection rate (66.66%, 20/30) was recorded in the snails of Ghargologh in the northern part. Only 35.24% (37/105) of the infected snails were from the plain areas, whereas the remaining existed in high altitudes.

Conclusion

It was concluded PCR method could be an efficient and fast method for uncovering the actual rate of infection with larval stages of O. turkestanicum in the snails of L. gedrosiana. This method can be also useful for the domestic animals and public health management programs in the country.     

Molecular Differentiation of Fasciola Species and Characterization of Genetic Diversity of F. gigantica Using NADH Dehydrogenase I (ND1) Gene in the Endemic Areas of Iran

Background:

Fasciola hepatica and F. gigantica are the causative agents of fasciolosis in domestic animals and humans. Based on the morphometric criteria, differential diagnosis between them is problematic. In addition, intermediate forms of Fasciola have been found in Iran, which makes the differentiation more difficult. The aim of the present study was to provide molecular evidence for the existence of F. gigantica in Iran using sequencing analysis of ND1 and PCR-RFLP analysis of ITS2 regions and to study the intraspecies variations of F. gigantica based on mitochondrial ND1 gene polymorphism.

Methods:

Forty Fasciola spp. samples collected from four distinct provinces (Fars, Khuzestan, Gilan, Khorasan Razavi) in Iran were collected for morphological and molecular characterization. In molecular method, PCR-RFLP analysis of ITS2 using pagI restriction enzyme was used as a screening approach for F. gigantica differentiation. Then mitochondrial DNA sequence variations in the ND1 gene were used for phylogenetic analysis.

Results:

Based on the morphometric criteria and RFLP analysis, 14 parasitic samples were initially identified to be F. gigantica. Phylogenetic results showed that there are at least 10 different genotypes of F. gigantica in Iran, which are different from those existing in the GenBank. Twenty-six points out of 410 base pairs of sequenced ND1 gene in 10 varieties of F. gigantica were diagnosed to be polymorphic. From 26 points of polymorphism, only eight resulted in the post-translational amino acid changes in ND1 gene product structure.

Conclusion:

Data revealed noticeable genetic diversity (up to 4.63%) between different varieties of F. gigantica in Iran.     

Detection of Acanthamoeba and Toxoplasma in River Water Samples by Molecular Methods in Iran

Background:

Free-living amoebae such as Acanthamoeba species may act as carriers of Cryptosporidium and Toxoplasma oocysts, thus, may play an important role in the water-borne transmission of these parasites. In the present study, a loop mediated isothermal amplification (LAMP) method for detection of Toxoplasma and a PCR assay were developed for investigation of Acanthamoeba in environmental water samples.

Methods:

A total of 34 samples were collected from the surface water in Guilan Province. Water samples were filtrated with membrane filters and followed by DNA extraction. PCR and LAMP methods used for detection of the protozoan parasites Acanthamoeba and Toxoplasma respectively.

Results:

Totally 30 and 2 of 34 samples were positive for Acanthamoeba and Toxoplasma oocysts respectively. Two samples were positive for both investigated parasites.

Conclusion:

The investigated water supplies, are contaminated by Toxoplasma and Acanthamoeba (oo)cystes. Acanthamoeba may play an important role in water-borne transmission of Toxoplasma in the study area. For the first time in Iran, protocol of LAMP method was used effectively for the detection of Toxoplasma in surface water samples in Iran.     

Investigation of Double-Band Electrophoretic Pattern of ITS-rDNA Region in Iranian Isolates of Leishmania tropica

Background

Leishmania tropica is a genetically divergent species. Amplification of entire internal transcribed spacer (ITS) region of L. tropica isolates obtained from Bam district, one of the well known focus of anthroponotic cutaneous leishmaniasis (ACL) in Iran, revealed a double-band pattern in agarose gel electrophoresis. This study explains how this pattern occurs.

Methods

Twenty seven L. tropica smear preparations were collected from Bam district, south east Iran, and eight L. major and one L. infantum smear preparations were gathered from Shiraz, south west Iran. Furthermore one L. major and one L. infantum cultured standard strains were tested using entire ITS-PCR to survey their electrophoretic pattern. The ITS sequences of L. tropica, L. major, and L. infantum already deposited in GenBank were analyzed. Analysis of GenBank sequences of L. tropica revealed two groups of sequences based on length size, one group having a 100 bp gap. Therefore, a new reverse primer namely LITS-MG was designed to exclude this gap in PCR products.

Results

Whole ITS fragment amplification resulted in a double-band pattern in all L. tropica cases, while a sharp single band was observed for L. infantum and L. major isolates. This result was corresponding to the result obtained from in silico analysis of GenBank sequences. Use of LITS-MG primer was expectedly resulted in a single band including ITS1, 5.8s and partial ITS2 product for L. tropica which is appropriate for following molecular studies such as sequencing or restriction analysis.

Conclusion

Sequences analysis of GenBank L. tropica sequences and following practical laboratory tests revealed at least two alleles in L. tropica which were confirmed in Bam isolates. This especial double-band pattern is because of a 100 bp fragment difference within ITS-rDNA alleles.     

Entamoeba histolytica encephalitis diagnosed by PCR of cerebrospinal fluid

Extra-intestinal amebiasis is secondary to invasive intestinal infections and usually results in an amebic liver abscess. Other organs, including lungs, brain, skin, spleen and kidneys, may be involved. Diagnosis of the cerebral infection is of the utmost importance and is most often made by detection of the organism at the time of brain biopsy or at autopsy. We report the first case of Entamoeba histolytica encephalitis diagnosed by PCR of the cerebrospinal fluid. The patient was treated successfully with metronidazole. PCR is an increasingly useful tool for the diagnosis of central nervous system infection and can provide rapid diagnosis.     

Evaluating the Resistance of Eimeria Spp. Field Isolates to Anticoccidial Drugs Using Three Different Indices

Background

In this study, the presence of resistance to diclazuril, amprolium+ethopabate and salinomycin, representing some of the commonest anticoccidials in Iran''s poultry industry, against three mixed Eimeria field isolates were investigated.

Methods

Three Eimeria field isolates, collected from typical broiler farms in Iran, were propagated once, inoculated to 480 broilers, comprising 30 chicks in each treatment. The non-medicated or medicated diets containing one of the above mentioned anticoccidials were provided ad-lib. Drug efficacy was determined using the Global index (GI), Anticoccidial Sensitivity Test (AST) and Optimum Anticoccidial Activity (OAA).

Results

None of the field isolates were fully sensitive to the selected anticoccidials. All isolates showed reduced sensitivity/partial resistance to salinomycin. Resistance to amprolium+ethopabate was evident and partial to complete resistance was recorded for diclazuril.

Conclusion

Limited efficacy of the selected anticoccidials is obvious. Considering the cost of continuous use of anticoccidials in the field, altering the prevention strategy and rotation of the anticoccidials with better efficacy, would prevent further economic losses induced by coccidiosis.     

Seroprevalence of Acanthamoeba Antibodies in Rheumatoid Arthritis Patients by IFAT,Tehran, Iran 2007

Background

This preliminary study was conducted to discriminate the prevalence of Acanthamoeba antibodies in rheumatoid arthritis (RA) patients and healthy controls to analyze the correlation between these two groups.

Methods

From October 2006 to August 2007 a total of 121 serum samples from RA patients attending the Rheumatolgy Department at Shariati Hospital in Tehran were obtained and stored at -20°C until using by indirect fluorescent-antibody test (IFAT). RA was diagnosed according to the American Collage of Rheumatology classification criteria. The organism used in this study was isolated from various water resources in Tehran, Iran cultured axenically and then went on a PCR assay based on 18S rRNA to identify the genus Acanthomoeba. Indirect immunofluorescence antibody (IFA) staining of serum samples was carried out to detect anti Acanthomoeba antibodies.

Results

In culture, out of 22 samples, 13(59%) were grown in xenic but only two in axenic medium. PCR amplified a 904bp fragment, specific for Acanthamoeba. Of examined serum samples, Acanthamoeba antibodies were present in 70 (57.8%) and 52 (41.2%), respectively. The highest titer of antibodies (1:320) was detected in one patient with RA.

Conclusion

Our study supports the hypothesis that some parasitic microorganisms can involve and contribute toward the development of rheumatoid syndromes.     

Histopathologic Effects of Dirofilaria immitis Microfilaria on Internal Organs of Dog Confirming by PCR Technique

Background

The heartworm disease is an infectious disease of dogs with Dirofilaria immitis combined with cardiovascular and circulatory abnormalities. The heartworm disease can become a serious health risk when associated with a severe infection. In this study, a male, 8 year-old dog that died suddenly was necropsied and all tissues were examined grossly.

Methods

Major organs including heart, lungs, liver, spleen, kidneys, brain, eyes, and testis were fixed in 10% neutral formalin, embedded in paraffin, sectioned at 5-µm thickness, stained with hematoxylin and eosin, and examined with a light microscope. For each examined organ, paraffin-embedded tissues were cut and placed in eppendorf tubes for genomic DNA extraction. PCR was performed using two sets of primers for amplification of a 302 bp ITS-2 gene fragment and a 203 bp cytochrome oxidase subunit 1 (CO1) gene fragment of D. immitis.

Results

During the necropsy examination, 46 adult D. immitis were found in the portal vein, right ventricle, and atrium of the heart and pulmonary trunk. Microscopically, microfilarias were found throughout the vessels of different organs including lungs, kidneys, liver, heart, brain, and spleen. All tissues examined by PCR were positive for D. immitis ITS-2 and CO1.

Conclusion

PCR technique now represents an effective method for identification of D. immitis from formalin-fixed samples.     

Genetic Diversity of Human Blastocystis Isolates in Khorramabad,Central Iran

Background

There are some genetic differences in Blastocystis that show the existence of species or genotypes. One of these genes that help in identifying Blastocystis is SSUrRNA. The aim of this study was assessment of genetic diversity of Blastocystis by PCR with seven pairs of STS primers.

Methods

This study was done on 511 stool samples collected from patients referred to the health care centers of Khorramabad, Central Iran, in 2012. Genomic DNA was extracted and in order to determine the Blastocystis subtype in contaminated samples, seven pairs of primers STS (subtype specific sequence-tagged site) were used.

Results

Out of 511 samples, 33 (6.5%) samples were infected with Blastocystis. Subtype (ST) of 30 samples was identified and three subtypes 2, 3 and 4 were determined. Mix infection was reported 10% which 3.33% of the infection was for the mixture of ST 3 and ST5 and 6.67% was for the mixture of ST 2 and ST 3.

Conclusion

The predominant subtype was ST3 that is the main human subtype. The dominance of ST2 and 5 are important in this study. This superiority has been reported in some of the studies in ST 2 which is different from the studies in other countries, because they have announced priorities of the ST1 and ST6 after ST3.