First Molecular Identification of Sarcocystis ovicanis (Protozoa,Apicomplexa) in the Brain of Sheep in Iran

Background

The objective of the present study was to survey the presence of Sarcocystis in sheep’s brain in North Khorasan Province.

Methods

In general, 80 samples of sheep’s brain were collected from slaughtered sheep in slaughterhouses of North Khorasan Province. Tissue digestion method was used for observing bradyzoites in tissues. Histopathological processing tracing Sarcocystis and ensuing structural change in the brain tissue were conducted. PCR analysis was conducted on all the brain samples. Sequencing was done for one PCR product. Genotype was identified by Blast search and homology analysis.

Result

Sarcocystis spp. was found in one of the brain samples (1.25%) using tissue digestion method. The presence of bradyzoite was also confirmed in the prepared histopathological sections. PCR analysis was positive in one of samples. Genotyping of one sample proved that Sarcocystis species was Sarcocystis ovicanis and the nucleotide sequence of this parasite was deposited in the GenBank database under accession number No.{"type":"entrez-nucleotide","attrs":{"text":"KF489431","term_id":"540198437","term_text":"KF489431"}}KF489431.

Conclusion

Sarcocystis ovicanis can involve brain tissue of sheep and consequently causes clinical symptoms.     

Molecular Study of Sheep Malignant Theileriosis at Barka Region in the Sultanate of Oman

Background

We used the PCR technique based on the abovementioned primer pair and sequencing to demonstrate the Theileria infection in the sheep samples collected from Sultanate of Oman.

Methods

According to the frame work of “integrated control of ticks and tick borne diseases in globalized world managed by EU-ICTTD-3 project, the samples from blood, liver, spleen, lymph node and lung were sent to the laboratory of Iranian Research Center for Ticks and Tick-borne Diseases (IRCTTD). Samples from blood smear and impression smears from liver, spleen, lymph node, and lung were analyzed by Geimsa staining. The DNA was extracted from the abovementioned samples and analyzed by PCR technique using specific primers derived from the nucleotide sequences of 18S rRNA gene of T. lestoquardi, which can amplify the common region in other Theileria and Babesia spp. Subsequently the amplified DNA was sequenced.

Results

The analysis of blood smears of the sheep was negative for piroplasmosis performed through the Giemsa staining. The impression smears prepared from liver, spleen, lymph node, and lung showed suspicious structures mimicking Theileria schizonts in some cells. The results showed an expected PCR product of 428 bp in length, which is specific for Theileria spp. The PCR products were subsequently sequenced. The corresponding nucleotide sequence is registered under accession number {"type":"entrez-nucleotide","attrs":{"text":"JF309152","term_id":"323695911","term_text":"JF309152"}}JF309152 in GenBank. The sequence alignment in GenBank showed that the PCR products had 99% homology to the known T. lestoquardi registered under accession number {"type":"entrez-nucleotide","attrs":{"text":"AF081135","term_id":"7576455","term_text":"AF081135"}}AF081135 in the GenBank.

Conclusion

Oman sheep are highly susceptible for Theileria infection and the infected sheep mostly die before the microschizonts or erythrocytic form of Theileria appears in the nucleated or erytrocytic cells respectively.     

Isolation and Identification of Naegleria Species from Environmental Water in Changchun,Northeastern China

Background

Naegleria is a free-living amoeba, and pathogenic Naegleria may pose a health risk to people exposed to recreational water. Our objective in this study was to determine if there are pathogenic amoebae in environmental water samples from Changchun, Northeastern China.

Methods

During July to September 2012, a total of 70 water samples were collected from Changchun, Northeastern China, and Naegleria was enriched by in vitro culture and detected by PCR using Naegleria genus-specific primers. Resulting PCR products were sequenced and phylogenetically analyzed to identify Naegleria species.

Results

Naegleria was detected in 65 (92.9%) of 70 water samples. DNA sequence and phylogenetic analyses based on the internal transcribed spacer (ITS) rDNA sequences revealed four Naegleria species, including N. pagei (n = 24) and N. Australiensis (n = 18), N. clarki (n = 13) and N. gruberi (n = 10), in which N. australiensis is pathogenic to mice. But the pathogenic species N. fowleri was not detected.

Conclusion

This is the first report on Naegleria species in Northeastern China, showing that almost all environmental water samples were contaminated with Naegleria, including N. pagei, N. Australiensis, N. clarki and N. gruberi, which should be considered a potential public health threat.     

Development of an Indirect ELISA Using Different Fragments of Recombinant Ncgra7 for Detection of Neospora caninum Infection in Cattle and Water Buffalo

Background:

Dense granules are immunodominant proteins for the standardization of immunodiagnostic procedures to detect neosporosis. In the presented study different fragment of a dense-granule protein was evaluated for serodiagnosis of Neospora caninum in cattle and water buffalo.

Methods:

NcGRA7, from N. caninum tachyzoites was amplified. PCR product and pMAL-c2X plasmid were digested with EcoR1 restriction enzyme and expressed in Escherichia coli to evaluate its competence for detection of anti- N. caninum antibodies with ELISA in comparison with commercial IDEXX ELISA. Furthermore, 230 sera of presumably healthy cattle and water buffaloes (108 cattle and 122 water buffaloes) were analyzed by both tests to determine the agreement of these two procedures.

Results:

Sensitivities and specificities of NcGRA7-based ELISA were 94.64% and 90.38% respectively using sera of cattle, but were 98.57% and 86.54% in the case of buffaloes respectively. A good correlation between the results of IDEXX ELISA and ELISA based on recombinant NcGRA7 for detecting N. caninum antibodies was appeared. Analyzing by Mc Nemar′s showed that NcGRA7-based ELISA has acceptable capability to differentiate the positive results in comparison with IDEXX ELISA.

Conclusion:

NcGRA7-based ELISA considering utilized new fragment of genomic DNA is a good tool for serodiagnosis of anti- N. caninum antibodies for screening and epidemiological purposes on cattle herd and water buffaloes as well.     

Seroprevalence of Neospora caninum Infection in Dairy Cattle in Tabriz,Northwest Iran

Background

The aim of this study was to determine the seroprevalence of antibody to Neospora caninum in healthy and aborted dairy cattle in Tabriz, capital of East-Azarbaijan in northwest of Iran.

Methods

In this cross-sectional study serum samples were collected from 266 healthy and aborted Holestein-Feriesisnc cows from September 2008 to August 2009. The sera were analyzed to detect of antibody against N. caninum using the commercially ELISA kit.

Results

Seroprevalence of antibody to N. caninum was 10.5% in Tabriz dairy cattle. Also the abortion rate in all cattle sampled was 33.6% but percentage of seropositive aborted cattle was 18.4%.

Conclusion

Neosporosis could be one of the possible causes of abortion in dairy cattle in Tabriz and regarding the distribution in dogs as definitive host for the parasite, further studies in dog and cattle are recommended.     

Application of Multiplex PCR for Detection and Differentiation of Entamoeba histolytica,Entamoeba dispar and Entamoeba moshkovskii

Background

Entamoeba moshkovskii and E. dispar are impossible to differentiate microscopically from the pathogenic species E. histolytica. Multiplex polymerase chain reaction (Multiplex PCR) is a widespread molecular biology technique for amplification of multiple targets in a single PCR experiment.

Methods

For detection and differentiation of the three-microscopy indistinguishable Entamoeba species in human, multiplex PCR assay using different DNA extraction methods was studied. A conserved forward primer was derived from the middle of the small-subunit rRNA gene, and reverse primers were designed from signature sequences specific to each of these three Entamoeba species.

Results

A 166-bp PCR product with E. histolytica DNA, a 580-bp product with E. moshkovskii DNA and a 752-bp product with E. dispar DNA were generated in a single-round and multiplex PCR reaction.

Conclusion

We recommend this PCR assay as an accurate, rapid, and effective diagnostic method for the detection and discrimination of these three Entamoeba species in both routine diagnosis of amoebiasis and epidemiological surveys.     

Acanthamoeba species in Swimming Pools of Cairo,Egypt

Background

The free-living amoebae Acanthamoeba spp. have been recognized as etiologic agents of amoebic encephalitis, keratitis, otitis, lung lesions and other skin infections mainly in immuno-compromised individuals. The purpose of this study is to detect the presence of Acanthamoeba in swimming pools in Egypt using a polymerase chain reaction (PCR) method.

Methods

Water samples were collected from 10 different swimming pools in Cairo, Egypt. Samples were cultured on non-nutrient agar for the detection of Acanthamoeba isolates that were confirmed by PCR amplification using genus specific primers. The molecularly confirmed Acanthamoeba isolates were morphologically identified to the species level.

Results

Members of genus Acanthamoeba were detected in 49.2% of the examined swimming-pool water samples. Morphologically, six Acanthamoeba species were isolated from the examined swimming pool water namely A. polyphaga, A.castellanii, A. rhysodes, A. mauritaniensis, A. royreba and A. triangularis. All the identified species of Acanthamoeba were molecularly confirmed to be related to the genus Acanthamoeba.

Conclusion

The isolated species of Acanthamoeba could provoke variable degrees of infections to the swimmers. The culture method is cheaper and easier than PCR techniques that are faster for the detection of free-living amoebae     

Neospora caninum - Associated Abortions in Slovak Dairy Farm

Background:

Neospora caninum is considered one of the major causes of repeated abortions in livestock. This study aimed to determine the seropositivity to N. caninum using indirect ELISA and the influence of the infection on the occurrence of abortions in selected dairy herd in Slovakia.

Method:

Blood samples were obtained from 490 cattle over a period of two years and were tested for N. caninum antibodies using indirect ELISA.

Results:

The presence of specific antibodies in the herd was detected in 118 (24.1%) cows. According to selected groups; 117 (41.0%) cows with a history of abortion, 65 (43.3%) heifers and 223 (2.2%) cows without abortions were tested positive to Neospora. Vertical transmission of N. caninum dominated in examined herd and the relative risk (RR) of dam-daughter seropositivity in progenies of seropositive mothers was 2.1 times higher than in progenies of seronegative dams. Molecular analyses of aborted foetuses of seropositive mothers showed the presence of Neospora DNA. However, 23 (28.1%) of heifers born to seronegative cows were seropositive, indicating also the postnatal transmission of the infection from the environment.

Conclusion:

Study revealed significant correlation between the presence of specific antibodies and the occurrence of abortions, the risk of abortion in seropositive animals was 3.8 times higher than in seronegative ones. Incorrect farm management contributed to spread and circulation of neosporosis in entire dairy herd what could significantly impair the reproduction and economic parameters of breeding.     

Epidemiological and Diagnostic Features of Blastocystis Infection in Symptomatic Patients in Izmir Province,Turkey

Background

The aims of this study were to identify Blastocystis subtypes (STs) in a cohort of Turkish patients with various gastrointestinal symptoms using a novel Real Time PCR method developed recently for Blastocystis detection and assess the relationship between Blastocystis STs and patient symptoms.

Methods

Totally, 617 stool samples of patients with gastrointestinal symptoms were examined with microscopy and inoculated in Jones medium. Blastocystis-positive samples were further assessed to identify coinfections with other possible pathogens, including bacteria and viruses. Diagnostic efficacies of microscopy, culture and Real-Time PCR were compared. PCR products were sequenced to identify the subtypes of Blastocystis isolates.

Results

Totally 94 (15.24%) samples were positive for Blastocystis after all methods. Among these, 83 of 94 (88.3%) samples were identified with all methods, while 11 were positive only with Real Time PCR. Diarrhea and abdominal pain were the leading symptoms in the patients. The only pathogenic agent identified in 76 of 94 (80.9%) patients was Blastocystis. Subtype 3 was the leading Blastocystis subtype (44.6%), while subtypes 6 and 7 were firstly isolated from symptomatic patients in our region.

Conclusion

Comparison of three diagnostic methods indicated Real Time PCR as the most sensitive and specific method. Blastocystis was the only pathogenic agent among symptomatic patients, with subtype 3 being predominant. Patients with subtypes 6 and 7 need further assessments concerning the zoonotic potential of Blastocystis.     

Evaluation of Alum-Naltrexone Adjuvant Activity,on Efficacy of Anti-Leishmania Immunization with Autoclaved Leishmania major (MRHO/IR/75/ER) Antigens in BALB/C Mice

Background

Naltrexone, an opioid receptor antagonist shifts the immune response toward a Th1 profile. In the current study, we evaluated the efficacy of the mixture of NTX and alum, as a new adjuvant, to enhance immune response and induce protection against Leishmania major in a mouse model.

Methods

BALB/c mice were immunized three times either autoclaved L. major promastigotes’ antigens alone or in combination with the adjuvant alum, naltrexone or the alum–naltrexone mixture. Both humoral and cellular immune responses were assessed two weeks after the last immunization and compared with control mice.

Results

The administration of alum- NTX in combination with the parasite antigen, significantly increased production of IFN-γ IFN-γ /IL-5 ratio, lymphocyte proliferation and improved DTH response against L. major. There was no significant difference in survival following challenge among groups.

Conclusion

Immunization with the alum– naltrexone mixture as an adjuvant, in combination with the autoclaved L. major promastigotes antigens, can enhance cellular immunity and shift the immune responses to a Th1 pattern.     

Molecular and Serological Detection of Acute and Latent Toxoplasmosis Using Real-Time PCR and ELISA Techniques in Blood Donors of Rafsanjan City,Iran, 2013

Background

The differentiation between acute and latent forms of the Toxoplasma gondii (T. gondii) infection is still considered as a complicated issue. This study was aimed to elucidate the status of infection in the blood donors and the probable importance of blood transfusion in the transmission of the infection through detecting both immunological and genetic markers of acute and latent infection.

Methods

Totally 235 blood samples from blood donors were collected. The levels of anti-T. gondii IgG and IgM antibodies were examined by specific ELISA kits. cDNA were synthesized from total extracted mRNA molecules from the serum samples and SAG1 gene, specific for tachyzoite form, were amplified using Real-Time PCR technique. Demographic information of study subjects including their gender, age, job, and habitat were recorded.

Results

Out of 235 serum samples, 80 (34.04%) and 4 (1.71%) were positive regarding anti-T. gondii IgG and IgM antibodies, respectively. Real-Time PCR results showed that 14 out of 200 (6.97%) of blood donor had mRNA molecules of SAG1 gene. The positive results of Real-Time PCR of SAG1 in female gender and housekeepers were significantly higher than those of male gender and other job categories.

Conclusion

The prevalence of chronic and acute infection is high in Iranian blood donors. Additionally, evaluation of antibodies could not be reliable, because several donors negative for anti-T. gondii IgM antibodies had detectable SAG1 mRNA molecules. Hence, it seems that molecular diagnostic tests are essential to detect acute infections.     

Induction of Oxidative Stress in Skin and Lung of Infected BALB/C Mice with Iranian Strain of Leishmania major (MRHO/IR/75/ER)

Background

The aim of this study was to investigate the effects of Leishmania major infection on the induction of oxidative stress in skin and lung of female mice.

Methods

BALB/c mice were randomly divided into the control and experimental groups. The experimental groups were subcutaneously infected with inoculums promastigotes of L. major. The animals were sacrificed at 20, 40, 60, 90 and 120 days post-infection, and tissues were isolated and analyzed.

Results

Superoxide dismutase activity, percent of DNA fragmentation and superoxide anion production levels were increased in skin and lung of infected mice. Lung catalase activity and skin malondialdehyde level were also increased. The decreased glutathione level was observed in both tissues. The highest alteration in these parameters in both tissues was observed at 90 days post-infection.

Conclusion

L. major infection induces the production of free radicals and oxidative stress in a time-dependent manner in mice skin and lung by depletion of glutathione and increasing lipid peroxidation. The elevated DNA fragmentation may be related with increased oxidative stress. The skin is more sensitive to the effects of L. major infection on oxidative stress induction compared to the lung.     

Canine Babesiosis in China Caused by Babesia gibsoni: A Molecular Approach

Background

To provide a point of reference to study the epidemiology and clinical expression of canine babesiosis in China.

Methods

A total of 30 dogs infected with canine babesiosis were evaluated by mean of clinical history, physical examination, hematological, restriction fragment length polymorphism of PCR products (PCR-RFLP) and sequencing analysis.

Result

The most prevalent clinical abnormalities were lethargy (100%), anorexia (100%), pale or icteric mucous membranes (80%), fever (70%) and dark urine (70%). Hematology parameters revealed that anemia and thrombocytopenia were the major abnormalities in blood of dogs infected with canine babesia. The results of PCR-RFLP and sequencing analysis indicated that B. gibsoni was the main species responsible for canine babesiosis cases at the time of the study in Nanjing, China.

Conclusions

The results provide valuable information for better understanding of the epidemiology of canine babesiosis in China.     

Molecular Identification of Giardia duodenalis Isolates from Fars Province,Iran

Background

Giardia duodenalis is one of the most common human intestinal protozoan parasites worldwide and is endemic throughout the world with a vast range of mammalian hosts. The present study aimed to identify the prevalence of G. duodenalis isolates and determine the most common of its assemblages in the patients referring to health centers and hospitals in Fars province, Iran that will be subjected to further molecular investigation.

Methods

We collected 1000 human fecal samples from health centers and hospitals in Shiraz, Iran in a one year period from September 2009 to August 2010. Microscopic examination for the presence of G. duodenalis cysts and trophozoites was performed by direct wet mount before and after the concentration techniques. Extraction of DNA was performed by Phenol-Chloroform-Isoamylalcohol (PCI). G. duodenalis-positive specimens were analyzed by PCR. A fragment of the SSU-rDNA (292 bp) gene was amplified by PCR using the forward primer RH11 and the reverse primer RH4. Genotyping was performed using sequence analysis of G. duodenalis glutamate dehydrogenase gene using primers GDHeF, GDHiF, and GDHiR.

Results

The prevalence of Giardia infection was 10.7% (107/1000) examined based on microscopic examination. PCR identified 80% (40/50) of the samples as positive for G. duodenalis based on SSU-rDNA amplification on sucrose gradient samples. Besides, genotyping results indicated 32 isolates (80%) as assemblage AII and 8 isolates (20%) as assemblage BIII and BIV based on the DNA sequence analysis of the glutamate dehydrogenase locus of G. duodenalis.

Conclusion

The findings of this study emphasize that Iran (Fars Province) is a favorable area for giardiasis with an anthroponotic infection route.     

Cryptosporidium Infection in Patients with Gastroenteritis in Sari,Iran

Background

Cryptosporidiosis is a common coccidian parasite infection in patients with diarrhea that has worldwide distribution especially in developed countries. Therefore, the aim of this study was to determine the occurrence of Cryptosporidium infection in patients with gastroenteritis admitted to hospitals of Mazandaran University of Medical Sciences by parasitological and molecular methods in Sari, Iran.

Methods

Stool samples were collected from 348 patients with gastroenteritis admitted to the hospitals of Medical University in the Sari and Ghaemshahr cities in Mazandaran Province, Northern Iran in 2010-2011. Oocysts of Cryptosporidium identified using Formalin-Ether concentration method and stained by Aacid-fast staining (AFS) and Auramine phenol fluorescence (APF). Genomic DAN extracted from microscopically positive samples and nested PCR -RFLP by using SSU rRNA that identifies of the species of cryptosporidium.

Results

In 348 patients with gastroenteritis, the most clinical symptoms were diarrhea, nausea, vomiting, dehydration, fever and weight loss. 2.3% (8 cases) of diarrheal samples tested by both microscopy and molecular methods were positive for the presence of cryptosporidium. Nested PCR products yielded unique bands of 846 bp, correspond to cryptosporidium. Species diagnosis carried out by digesting the secondary PCR product with SspI restriction enzyme, which noted 3 clearly bands of 449, 254, and 108 bp correspond to Cryptosporidium spp.

Conclusion

The results of present study on Cryptosporidium spp. in this area can make a background data for control programs and further molecular analyses. Thus, further work needs to determine the origin of Cryptosporidium species in this area.     

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.     

PCR-RFLP Analysis of 28 SrDNA for Specification of Fasciola gigantica (Cobbold, 1855) in the Infected Lymnaea auricularia (Linnaeus, 1785) Snails from Northwestern Iran

Background

Fasciolosis in livestock is a crucial concern in the globe, mainly due to its impact on human health. The aim of this study was to determine the rate of infection with Fasciola gigantica (Cobbold, 1855) larvae in the field-collected snails of Lymnaea auricularia (Linnaeus, 1785) from northwestern Iran using a molecular approach.

Methods

A total of 6,759 pond snails were collected from 28 freshwater bodies in West Azarbaijan. PCR was performed to amplify a 618-bp fragment of the nuclear 28 SrRNA gene of Fasciola. The PCR products were digested by AvaII restriction enzyme to create restriction fragment length polymorphism (RFLP) patterns specific for the detection of F. gigantica.

Results

Of the total collected snails 496 (7.34 %) were L. auricularia, among which 4.64% (23 out of 496) were infected with a Fasciola species according to the PCR analysis. Only 2.22% (11 out of 496) of the infected snails were from the mountainous areas. The highest Fasciola infection rate recorded in the snails of a single site was 1.81% (9 out of 496 snails). Based on the RFLP pattern, F. gigantica accounted for 2.42% of the infection rates in the study sites.

Conclusion

Application of PCR-RFLP was proven to be a useful approach for valid and robust detection of the infection with F. gigantica in its intermediate host snails. These findings may therefore be applicable for establishment of the control programs against dissemination of the infection in different regions.     

Molecular Detection of Malaria in South Punjab with Higher Proportion of Mixed Infections

Background

Malaria is well known for its fatalities worldwide, Plasmodium vivax and the Plasmodium falciparum are the two important species of malaria reported from Pakistan and creating lots of morbidities across the country.

Method

Study was conducted to determine the Surveillance of malaria in South Punjab by microscopy and Polymerase chain reaction (PCR).

Result

samples out of 100 patients were found positive for malarial parasites. One patient was found with mixed infection, whereas P. falciparum and P. vivax infections were detected in 17 and 22 patients, respectively. In nested PCR, genus-specific primers for Plasmodium species. in round 1 and species-specific primers for P. falciparum and P. vivax in round 2 were used. By the application of PCR 41% were found to be infected by Plasmodium spp. Among Plasmodium positive patients: mixed, P. falciparum and P. vivax infection were detected in 10, 15 and 16 patients, respectively. Thirty nine microscopically positive patients confirmed to have Plasmodium spp. One negative by PCR, 2 microscopically negative patients had shown Plasmodium spp. infection (P. falciparum and P. vivax) by PCR. In total samples, P. falciparum, P. vivax and mixed infection accounted for 36.6%, 39.0% and 24.3%, respectively.

Conclusion

Microscopy was found deficient for interpretation of mixed infections, low parasitaemia, and species specific diagnosis. The sensitivity, specificity and efficacy of nested PCR was calculated 95%, 98% and 97%, respectively, showing PCR as a more effective and efficient diagnostic tool for malaria.     

Comparison of Three Staining Methods for the Detection of Intestinal Microspora Spp

Background

This study aimed to compare three staining methods including: Calcofluor white, Chromotrope and Quick Hot Gram chromotrope used in diagnosis of intestinal microsporidial spores.

Methods

One hundred and seventy five stool specimens were collected from patients referred to Laboratory of Intestinal Protozoology at the School of Public Health, Tehran University of Medical Sciences during 2012-2013. All of specimens were evaluated by nested PCR. The formalin–fixed stool samples were prepared from each specimen and dried at room temperature for 10 min, followed by 10 min methanol fixation. All the collected stool samples were evaluated blindly by calcofluor white, Chromotrope and Quick Hot Gram chromotrope staining methods separately.

Results

Microsporidial spores were recognized using Chromotrope, Quick Hot Gram chromotrope and Calcofluor white, in16 of 18 (88.8%), 17 of 18 (94.4%) and 18 of 18(100%) samples that were positive by nested PCR respectively. Regarding 14 stool samples that were negative by nested PCR, 14 cases were negative by chromotrope and Quick hot Gram chromotrope and 13 samples were negative by Calcofluor white. One discordant sample interpreted as false positive.

Conclusion

Calcofluor white staining had the best performance for the detection of intestinal Microsprora spores and can be used as initial screen test for the detection of intestinal Microspora spp.     

Molecular and Parasitological Study of Cryptosporidium Isolates From Cattle in Ilam,West of Iran

Background

Cryptosporidiosis is one of the most important parasitic infections in human and animals. This study was designed for survey on the prevalence of Cryptosporidium infection in farms of Ilam, west of Iran, using parasitology method and genotyping by Nested PCR-RFLP.

Methods

Fecal samples of 217 cattle were collected fresh and directly from the rectum of cattle. All of the samples were examined by microscopic observation after staining with modified Ziehl-Neelsen (MZN). Genomic DNA extracted by using EURx DNA kit. A Nested PCR-RFLP protocol amplifying 825 bp fragment of 18s rRNA gene conducted to differentiate species and genotyping of the isolates using SspI and VspI as restriction enzymes.

Results

The prevalence of Cryptosporidium infection in cattle using both methods is 3.68%. Most of the positive cattle were calves under six months. Species diagnosis carried out by digesting the secondary PCR product with SspI that C. parvum generated 3 visible bands of 448, 247 and 106 bp and digested by VspI restriction enzyme generated 2 visible bands of 628 and 104bp. In this investigation all of the positive samples were Cryptosporidium parvum.

Conclusion

C. parvum (bovine genotype) detected in all positive cattle samples in Ilam, west of Iran. The results of the present study can help for public health care systems to prevention and management of cryptosporidiosis in cattle and the assessment of cattle cryptosporidiosis as a reservoir for the human infection.