Differential detection of echinococcus Spp. copro-DNA by nested-PCR in domestic and wild definitive hosts in moghan plain, Iran

Despite Echinococcus granulosus there are merely two old reports of E. multilocularis infection among Iranian canids of Moghan Plain, the only area known endemic for the species. We detected specific DNA markers in fecal samples by PCR [Copro-PCR] for differential diagnosis of Echinococcus species in living canids. Totally 144 fecal samples from domestic dogs, red foxes and a golden jackal were examined for genus-specific Echinococcus coproantigens using ELISA. Forty two positive or ambiguous samples were further examined for Echinococcus species-specific DNA markers by two different set of nested-PCR. Twenty five out of 144 [17.4%] animals were contaminated with E. granulosus including 14 [23.7%] domestic dogs, 10 [11.9%] red foxes and one [100%] golden jackal. But none of them harboured E. multilocularis species-specific Copro-DNA. The overall prevalence of E. granulosus and E. multilocularis infections in canids of the area was estimated to be 17.4% and 0.0%, respectively. There was a significant relation between the results of Copro-PCR and CA-ELISA. The lack of E. multilocularis infection, compared to previous reports may be due to the differences in used diagnostic methods and/or recently limited territories of wild canids and altered their food resources in this particular area

Aflatoxin M1 in pasteurized milk in Babol city, Mazandaran province, Iran

Aflatoxin M1 [AFM1] is the metabolite of aflatoxin B1 [AFB1] and is found in milk when lactating animals are fed with contaminated feedstuff. The presence of AFM1 in milk, pose a major risk for humans especially kids as it can have immunosuppressive, mutagenic, teratogenic and carcinogenic effects. The present study is aimed to investigate the occurrence of AFM1 in subsidized pasteurized milk in Babol, Mazandaran Province, Iran. Some 72 pasteurized milk packages were collected from supermarkets in various districts of city during January to March 2006. Milk samples were centrifuged and amounts of 100 micro l of skimmed milk were tested for AFM1 contamination by competitive ELISA. All the samples [100%] exhibited contamination with AFM1. The contamination levels means in January, February, and March were 227.85, 229.64, and 233.1ng/l, respectively. The amount of AFM1 in all the samples were above 50ng/l, the threshold set by the European community regulations. Monitoring of AFM1 level should be part of quality control procedures in dairy factories, particularly the ones providing infant's milk. Production of safer and healthier milk and other dairy products with minimum AFM1 level can be achieved by adopting prophylactic measures including control of humidity and water content of feedstuff, which favors mould production

Comparison of PCR-based diagnosis with centrifuged-based enrichment method for detection of borrelia persica in animal blood samples

The mainstay of diagnosis of relapsing fever [RF] is demonstration of the spirochetes in Giemsa-stained thick blood smears, but during non fever periods the bacteria are very scanty and rarely detected in blood smears by microscopy. This study is aimed to evaluate the sensitivity of different methods developed for detection of low-grade spirochetemia. Animal blood samples with low degrees of spirochetemia were tested with two PCRs and a nested PCR targeting flaB, GlpQ, and rrs genes. Also, a centrifuged-based enrichment method and Giemsa staining were performed on blood samples with various degrees of spirochetemia. The flaB-PCR and nested rrs-PCR turned positive with various degrees of spirochetemia including the blood samples that turned negative with dark-field microscopy. The GlpQ-PCR was positive as far as at least one spirochete was seen in 5-10 microscopic fields. The sensitivity of GlpQ-PCR increased when DNA from Buffy Coat Layer [BCL] was used as template. The centrifuged-based enrichment method turned positive with as low concentration as 50 bacteria/ml blood, while Giemsa thick staining detected bacteria with concentrations >/- 25000 bacteria/ml. Centrifuged-based enrichment method appeared as much as 500-fold more sensitive than thick smears, which makes it even superior to some PCR assays. Due to simplicity and minimal laboratory requirements, this method can be considered a valuable tool for diagnosis of RF in rural health centers

Prevalence of zoonotic intestinal helminths of canids in Mogahan plain, Northwestern Iran

The present study was aimed to elucidate the status of intestinal helminth infections in canids of Moghan Plain, northwestern Iran. Eighty-five intestine samples from dead or shot wild canids, 59 fecal samples from sheepdogs and 5 from red foxes were collected from 2006 to 2008 and examined in Parasitology department of Pasteur Institute of Iran. Generally, adult worms, larvae, and eggs of 13 species of various parasitic helminths were recovered. Necropsy examinations showed that 96.47% animals harbored at least one helminth species. The prevalence of different species in necropsy were Mesocestoides sp. 84.7%, Rictolaria spp. 55.3%, Macranthorhynchus hirudinaceus 45.9%, Toxocara canis 43.5%, Toxascaris spp. 35.3%, Joyeuxiella sp. 34.1%; hookworms; 22.4%, Taenia spp. 11.8%, Alaria spp. 2.4% and Dipylidium caninum 1.2%. Besides, eggs belonging to 10 species of parasitic helminths were identified in 46 fecal samples and generally, 30.9% of samples harbored eggs of at least one helminth species. The high prevalence of various helminth infections among canids in Moghan plain and contamination of environment by helminths eggs may increase the risk of infection for native people

Molecular typing and phylogenetic analysis of some species belonging to phlebotomus [larroussius] and phlebotomus [adlerius] subgenera [diptera: psychodidae] from two locations in Iran

Haematophagous females of some phlebotomine sandflies are the only natural vectors of Leishmania species, the causative agents of leishmaniasis in many parts of the tropics and subtropics, including Iran. We report the presence of Phlebotomus [Larroussius] major and Phlebotomus [Adlerius] halepensis in Tonekabon [Ma-zanderan Province] and Phlebotomus [Larroussius] tobbi in Pakdasht [Tehran Province]. It is the first report of these species, known as potential vectors of zoonotic visceral leishmaniasis in Iran, are identified in these areas. In 2006-2007 individual wild-caught sandflies were characterized by both morphological features and sequence analysis of their mitochondrial genes [Cytochrome b]. The analyses were based on a fragment of 494 bp at the 3' end of the Cyt b gene [Cyt b 3' fragment] and a fragment of 382 bp CB3 at the 5' end of the Cyt b gene [Cyt b 5' fragment]. We also analysed the Cyt b Long fragment, which is located on the last 717 bp of the Cyt b gene, followed by 20 bp of intergenic spacer and the transfer RNA ser [TCN] gene. Twenty-seven P. halepensis and four P. major from Dohezar, Tonekabon, Mazanderan province and 8 P. tobbi from Packdasht, Tehran Province were identified by morphological and molecular characters. Cyt b 5 and Cyt b 3 fragment sequences were obtained from 15 and 9 flies, respectively. Cyt b long fragment sequences were obtained from 8 out of 27 P. halepensis. Parsimony analyses [using heuristic searches] of the DNA sequences of Cyt b always showed mono-phyletic clades of subgenera and each species did form a monophyletic group

Prevalence of Linguatula serrata infection in domestic bovids slaughtered in Tabriz abattoir, Iran

Linguatulosis is a rare zoonotic parasitic infection, in which human plays the role of both definitive and intermediate host and can be occasionally infected. This study determines the status of infection in livestock and its potential risk to men in the northwestern province of Azarbaijan-e-Sharghi, Iran. In a cross-sectional study from June 2007 to June 2008, 800 slaughtered animals including 400 cattle and 400 buffaloes from Tabriz abattoir in Azarbaijan-e-Sharghi Province were randomly selected and examined for L. serrata nymphs. After primary macroscopical inspection, all liver and lung samples were cut to small pieces, treated with a tissue digestion method and checked macroscopically and microscopically for free or encapsulated nymphs. Out of 800 animals, 3 [0.38%] were found to be infected with L. serrata nymphs and the prevalence of infection in cattle and buffaloes was determined to be 0.25% and 0.5%, respectively. Conclusion: Linguatula infection occurs as an endemic zoonosis in the study area and has an active transmission life cycle

Prevalence of Echinococcus spp. infection using coproantigen ELISA among canids of Moghan plain, Iran

Echinococcosis is one of the most important helminthic zoonotic diseases in Iran. Intestinal Scraping Technique [IST], the traditional method for diagnosis of the infection in definitive hosts, has many disadvantages including low susceptibility and being expensive, hazardous and laborious. Detection of coproantigens in fecal samples by enzyme-linked immunosorbent assay [CA-ELISA] is known as a useful tool for intravital mass-screening of definitive host populations. This study was performed to determine the prevalence of Echinococcus spp. infection among canids in Moghan plain, the only area in Iran known to be endemic for E. multilocularis. One hundred thirty eight fecal samples were collected from red foxes and domestic dogs in three counties of Moghan plain namely Pars Abad, Bileh Savar and Germi. The samples were examined using an ELISA, designed for the detection of Echinococcus-specific coproantigen and the formalin-ether concentration method as well. Totally, out of 138 fecal samples, 27 [21.6%] turned positive for Echinococcus. Coproantigen was detected in 16.7% and 27.1% of red foxes and domestic dogs, respectively. Formalin-ether concentration method revealed that 43 [31.2%] of samples harbored at least one parasitic helminth, but Taenia eggs were detected only in 3 fecal samples. Since coproantigen presence reflects current intestinal infection with adult worms, CA-ELISA can be regarded as one of the most useful immunological tools for diagnosis of Echinococcus infection. Besides, the high susceptibility, low cost and rapidity

study on leishmania infection rate among phlebotomus spp. collected from Abardejh district, Iran

Cutaneous leishmaniasis is a prevalent tropical parasitic disease in the Old World. The causative agents are Leishmanial parasites, which cause various forms of cutaneous leishmaniasis. The infection is commonly limited in immuno-competent individuals, but it can progress to a chronic and ulcerative disease in immunocompromised patients. The reservoirs are dogs and rodents and the vectors are different species of sandflies. In the present study, we investigated the prevalence rate of Leishmania infection among Phlebotomus mosquitoes collected from Abardejh district, Iran. Abardejh is located next to Varamin city in southeast of Tehran having a tropical ecosystem at its eastern border. Tamarisk trees and rodents have provided a suitable condition for sandfly activity. The sandflies were collected by funnel trap from rodent burrows and transferred to the Department of Parasitology, Pasteur Institute of Iran. The sampling was carried out during spring and summer [2002] with ten-day intervals. The collected sandflies were identified using discriminative morphologic features before parasitological culture on NNN medium. Analyses of the data revealed a high prevalence rate of infection among the sandflies in this region [P<0.01]. The maximum activities of Phlebotomus were in the months of June and July. Three species of sanflies were found in rodent burrow: P. papatasi, P. sergenti, and P. caucasicus. The results of blood-fed Phlebotomus culture showed that 22.07% of blood-fed females of P. papatasi and 8% of blood-fed females of P. sergenti were infected with leptomonads [P<0.05]. This could be an important issue because human and agricultural environments are located closely to this district. Therefore, use of insecticides and environmental sanitation seems to be required to prevent the transmission of infection from sandflies to human

Analysis of plasmodium vivax merozoite surface protein-1 gene sequences

The emergence of Plasmodium vivax in Dashte Moghan in northwestern Iran has become a major concern for Iranian's health officials. Knowledge of genetic make up of the P. vivax populations in this area would give us an insight into the origin of the prevalent infections and the possible routes they are introduced. This paper reports the analysis of a variable region between the two interspecies conserved blocks [ICBs] of 5 and 6 of MSP-1 gene in 18 isolates from Dashte Moghan. The results revealed that all the 18 isolates were similar to an Azari Belem-like type with 21 glutamine [Q] in the repeated residues. Our results may give a clue that the resurgent malaria in Dashteh Moghan might have primarily been introduced from Azerbaijan. However, much more molecular and epidemiological evidence are needed to confirm this hypothesis

Evaluation of a native preparation of HCV core protein [2-122] for potential applications in immunization, diagnosis and mAb production

Infection with hepatitis C virus [HCV] is a worldwide problem. Among HCV proteins, core antigen [Ag], besides its importance for diagnostic application is a prime candidate for component of a vaccine. Herein, we report results of studies on production of the hydrophilic domain of core Ag [2-122] in native conformation by an arabinose induction system in E.coli and the primary characterization of this recombinant protein for applications in diagnosis, immunization and mAb production. Recombinant core [r-Core] was able to detect anti-core antibodies in HCV positive serum samples in a dilution rate of 1/3200. It was also capable to elicit a potent anti-HCV humoral immune response in BALB/c mice. Finally, we established two stable clones of hybridoma which shown to produce specific and sensitive mAbs against the core protein. HCV core was able to elicit a broad range of antibody specificities depending on the immunogen conformation. Therefore, it may be possible to get new mAbs with higher affinities towards native conformation of core Ag

Characterization of a monoclonal antiody specific for the parasite surface antigen-2 of leishmanie major

The Leishmania major Parasite surface Antigen-2 [PSA-2] is a family of glycoinositol phospholipids anchored glycoprotoins expressed in both promastigotes and amastigotes. Promastigote PSA-2 comprises three polypeptides with approximate molecular weight of 96, 80 and 50 kDa. Amastigote express a distinct but closely PSA-2 polypeptide with molecular weight of 50 kDa. In this study fusion of SP2/0 myeloma cells with immunized mice spleenocytes infected with promastigotes of L. major intraperitoneally resulted to a clone of hybridoma producing a specific antibody that only reacts with L. major parasite surface antigen [PSA-2]. This mAb showed no crossreactivity with either other Leishmania species including L. tropica, L. donovani and L. infantum or recombinant gp63. Western blot analysis of culture supernatant revealed multiple b and s with molecular weight of 50, 58, 80 and 96 kDa only in L. major

rDNA-ITS2 based species-diagnostic polymerase chain reaction assay for identification of sibling species of Anopheles fluviatilis in Iran.

A species-specific polymerase chain reaction (PCR) assay using primers already designed, based on differences in the nucleotides of the second internal transcribed spacer (ITS2), was used to identify the species composition of the Anopheles fluviatilis complex in Iran. All the amplified DNA samples obtained from specimens collected from different areas using different collection methods yielded to a fragment of 450 bp size, a PCR product corresponding to the species denoted as Y. Some 21 ITS2 region of Iranian specimens were sequenced and compared with the already published sequence data of species Y from India. The sequence data of the Iranian specimens were 100% identical to that of the Indian specimens, and hence confirmed the PCR assay results. Species Y is presumably species T in India, which has no role in the transmission of malaria, whereas mosquitos of An. fluviatilis are known as a secondary vector in Iran. This conflict will remain to be solved by further biological and molecular studies.

Molecular characterization of Anopheles fluviatilis species complex in the Islamic Republic of Iran

A species-specific polymerase chain reaction [PCR] assay was used to identify the species composition of the Anopheles fluviatilis complex in the Islamic Republic of Iran. All the amplified DNA samples from specimens collected from different areas yielded a fragment of 450 bp size, a PCR product corresponding to that of the species denoted as Y. The sequence data from 21 ITS2 [second internal transcribed spacer] regions were compared with those publicly available in the GenBank database and confirmed that the specimens were 100% identical to species Y of India. Species Y is presumably the same as species T that has no role in transmission of malaria in India, whereas An. fluviatilis is known as a secondary vector of malaria in the Islamic Republic of Iran

Detection of toxoplasma gondii in dead fetuses by polymerase chain reaction [PCR]

Mouse inoculation and tissue culture employed in the earlier studies of toxoplasmosis, although specific and sensitive, are rather time-consuming and may require up to six weeks to reach a diagnosis. To use polymerase chain reaction [PCR] as a reliable alternative method in the detection of Toxoplasma gondii organisms in tissue samples. This method relies on the detection of B1 gene which is unique for T. gondii. After preparing the samples, DNA was separated using centrifugation, then, by application of primers and DNA polymerase, amplification was performed. PCR detected the presence of T. gondii in about 20% of formalin-fixed tissue samples from dead fetuses aborted within four months of gestation. PCR can be considered the method of choice, particularly when the parasites are already destroyed by preservatives or their viability is adversely affected by suboptimal storage and transportation conditions