Molecular and Seroepidemiological Survey of Visceral Leishmaniasis among Humans and Domestic Dogs in Mazandaran Province,North of Iran

Background

New cases of visceral leishmaniasis (VL) have been reported recently in some parts of Mazandaran Province, north of Iran where the first human case of VL was reported in 1949. This study aimed to determine the present status of Leishmania infantum infection among humans and domestic dogs using serological and molecular methods in central parts of Mazandaran Province.

Methods

In this cross-sectional study, blood samples were randomly collected from 402 humans and forty-nine domestic dogs throughout 2009 and 2010 in the central part of Mazandaran Province including Semeskadeh and Kiakola districts where recent cases of human visceral leishmaniasis had been reported there. All the collected samples were tested by direct agglutination test (DAT) for the detection of anti-Leishmania infantum antibodies as well as convenience PCR assay on whole blood samples for detection of leishmanial infection and identification of Leishmania species.

Results

None of 402 collected human (402) and dog (49) blood samples showed anti Leishmania infantum antibodies at titers 1:3200 and 1:320 as cut-off values of DAT, respectively but only 2 of domestic dogs (4.1%) were found PCR-positive corresponding to L.infantum.

Conclusion

This study confirms the circulation of L. infantum at least among domestic dogs and highlights the sporadic pattern of VL in the studied areas. Further investigations regarding to sand flies fauna and wild canines as reservoir hosts of the disease, are recommended.     

Absence of Asymptomatic Malaria Infection in Endemic Area of Bashagard District,Hormozgan Province,Iran

Background

A successful malaria elimination program calls for enough attention to parasite carriers, especially asymptomatic malaria, as well as the diagnosis and treatment of clinical cases. Asymptomatic malaria is an infection that patients do not show any symptom; thus, these patients play critical role in the concept of an elimination program. The current investigation was conducted to evaluate the presence of these cases in Bashagard District, formerly a high malaria transmission area in Hormozgan Province, Iran.

Methods

Blood samples (n = 500) were collected from symptomless individuals residing in Bashagard to evaluate Plasmodium infection by using microscopic, serological and nested-PCR techniques.

Results

Regarding the microscopic and nested-PCR analysis, no asymptomatic infection was detected among studied individuals. Totally, 1% of the studied population (5 of 500) had anti PvMSP-1₁₉-specific IgG antibody; however, only 0.2% (1 of 500) of the individuals was seropositive to recombinant PfMSP-1₁₉, using ELISA.

Conclusion

This study showed no asymptomatic malaria infection in the studied population; hence malaria elimination is feasible and can be successfully carried out in this region.     

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.     

In vivo Susceptibility of Plasmodium vivax to Chloroquine in Southeastern Iran

Background

Plasmodium vivax is the predominant species causes of malaria with about 90% total annual reported malaria in Iran. This study conducted to determine the susceptibility of Plasmodium vivax isolates to chloroquine in Sistan and Balochistan Province, southeastern Iran.

Methods

A total 270 subjects with symptomatic malaria and confirmed P. vivax infection completed the designed 28-day in vivo study. The thick and thin film blood smears were screened for malaria parasites by microscopy. The nested PCR was applied using the Plasmodium 18 subunit ribosomal ribonucleic (Ssr RNA) genes for detecting mixed infections and diagnosis of parasites in the samples with low parasite on days 0, 5, 6, 7, and 28.

Results

P. vivax was cleared in 15%, 50%, 95%, and 100% of patients on days 1, 2, 3, 4 respectively by microscopy assessment. Six patients were exhibited specific P. vivax band in nested PCR on day 5. No recurrence was observed on days 7, 14 and 28. Mean (±standard deviation) parasite clearance time was 2.41 (±0.8) days.

Conclusion

P. vivax is still susceptible to chloroquine in Southeatern Iran. This finding is compatible with results of neighboring countries Pakistan and Afghanistan.     

Helminth Parasites of Eastern European Hedgehog (Erinaceus concolor) in Northern Iran

Background

Recently there is a high tendency among exotic pet owners for keeping hedgehogs. This mammal can transfer some significant zoonotic pathogens to human. Hence, the present study was conducted for the first time to prepare a list of helminth parasites of hedgehogs (Erinaceus concolor) in North of Iran.

Methods

Ten (four males and six females) road killed hedgehogs were collected during April to January 2011 in rural areas of Babol city, Mazandaran province, Iran. All of internal organs were scrutinized for helminth burden. The extracted specimens were fixed and preserved in 70% ethanol and then cleared in Lacto-phenol solution. Helminth identification was carried out according to available systematic keys.

Results

All the examined hedgehogs (100%) were infected with parasitic helminth as following: two hedgehogs (20%) were infected with Crenosoma striatum, four hedgehogs (40%) harbored Physaloptera clausa, one (10%) host had Hymenolepis erinacei and three (30%) of them were infected with Nephridiacanthus major.

Conclusion

This is noteworthy that the current survey is the first report of helminth parasites fauna of Eastern European Hedgehog in Iran. Since, this is the first such investigation in our country, more researches are required to perform on unexplored areas of Iran in order to increase our knowledge regarding hedgehog parasitic diseases     

Survey of Migratory Birds (Anatidae: Anas platyrhynchos) for Schistosome Parasites from Mazandaran Province,Northern Iran

Background

The aim of the present study was to survey birds’ schistosomes in migratory birds (Anatidae: Anas platyrhynchos) which are the source of the disease in Mazandaran Province, Northern Iran.

Methods

A number of mallards were bought from the markets of hunted birds. The respiratory tracts (nasal mucosa) and intestinal blood vessels were studied for adult worms. The nasal mucosa was separated and observed by a microscope. In order to separate the visceral schistosomes, after separating intestine, vessel mesenteric was studied under the lamp light and then in saline. The parasite sample was collected for subsequent observation.

Results

Fifteen (13.6%) cases out of 110 studied birds had nasal mucosa contaminated with Trichobilharzia sp. egg. Besides that, two birds had adult worms schistosome visceral i.e. Bilharziella sp.

Conclusion

The elements that cause cercarial dermatitis in aforementioned region are Trichobilharzia sp. and Bilharziella sp. parasites. Thus, it is necessary for the authorities of health, environmental and agricultural organization of the province to cooperate in order to control this disease.     

First Detection of Nosema ceranae,a Microsporidian Protozoa of European Honeybees (Apis mellifera) In Iran

Background

Nosemosis of European honey bee (Apis mellifera) is present in bee colonies worldwide. Until recently, Nosema apis had been regarded as the causative agent of the disease, that causes heavy economic losses in apicultures. Nosema ceranae is an emerging microsporidian parasite of European honeybees, A. mellifera, but its distribution is not well known. Previously, nosemosis in honeybees in Iran was attributed exclusively to N. apis.

Methods

Six Nosema positive samples (determined from light microscopy of spores) of adult worker bees from one province of Iran (Savadkouh- Mazandaran, northern Iran) were tested to determine Nosema species using previously- developed PCR primers of the 16 S rRNA gene. As it is difficult to distinguish N. ceranae and N. apis morphologically, a PCR assay based on 16 S ribosomal RNA has been used to differentiate N. apis and N. ceranae.

Results

Only N. ceranae was found in all samples, indicating that this species present in Iran apiaries.

Conclusion

This is the first report of N. ceranae in colonies of A. mellifera in Iran. It seems that intensive surveys are needed to determine the distribution and prevalence of N. ceranae in different regions of Iran.     

Incidence and Genetic Characterization of Gongylonema pulchrum in Cattle Slaughtered in Mazandaran Province,Northern Iran

Background

The gullet worm, Gongylonema pulchrum Molin, 1857, is a thread-like spirurid nematode found in a variety of mammals worldwide. Its incidences in Iranian cattle of different breed or age have not been reported. The aims of the present study are to disclose the infection status of G. pulchrum in cattle slaughtered in northern region of Iran.

Methods

Full-length esophagi of cattle of 97 native dairy breed and 41 Holstein-Friesian breed were collected at four local abattoirs in Mazandaran Province, northern Iran, from March 2006 to August 2007, and were examined parasitologically. Eight overlapping segments of the small- and large-subunits of rDNA were amplified by PCR, and the obtained nucleotide sequences were characterized.

Results

The incidences of G. pulchrum in female and male native dairy breed were 38.9% and 24.0%, respectively, whereas those in female and male Holstein-Friesian breed were 4.2% and 0%, respectively. The first internal transcribed spacer (ITS1) region of G. pulchrum rDNA showed an intra-individual variation in the sequence and length, and the variation was ascribed to some unstable repeats of "A" or "CA".

Conclusion

Distinct incidences of G. pulchrum infection in native dairy breed and Holstein-Friesian breed might be ascribed to different animal husbandry manners for each breed in Iran; the former breed grazes freely in the pasture, but the latter breed is usually held in a pen. The rDNA sequence of Iranian G. pulchrum, obtained for the first time by us, might facilitate a reliable species identification of the parasite with a wide spectrum of morphological variations.     

Molecular Diagnosis of Trichomoniasis in Negative Samples Examined by Direct Smear and Culture

Background

Trichomoniasis is an extremely common sexually transmitted infection (STI) worldwide and is associated with important public health problems, including amplification of HIV transmission. This disease is in forms of symptomatic and asymptomatic in women and may depend on host as well as parasite variables. Most of the studies reported from females are based on examination of vaginal secretions and urine samples by direct smear and culture in modified Diamond''s media. The aim of this study was checking the samples, which were negative by direct smear and culture, with PCR technique.

Methods

The urine samples and vaginal discharge of patients attending Gynecology Clinics of Mazandaran Province, Iran with different symptoms rechecked for Trichomonas vaginalis by PCR technique using primers targeting a conserved region of the beta-tubulin genes of the parasite. Data were analyzed by Epi Info software program

Results

Out of 161 negative samples by direct smear and culture, seven samples (4.3%) were positive by PCR technique.

Conclusion

Diagnosis of trichomoniasis by PCR is a sensitive and specific method that could play important role to help the physicians for properly treatment and control of infection.     

Seroprevalence of Human Hydatidosis Using ELISA Method in Qom Province,Central Iran

Background

The objective of this study was to determine the prevalence of cystic echinococcosis (CE) in Qom Province, central Iran using ELISA test.

Methods

Overall, 1564 serum samples (800 males and 764 females) were collected from selected subjects by randomized cluster sampling in 2011-2012. Sera were analyzed by ELISA test using AgB. Before sampling, a questionnaire was filled out for each case. Data were analyzed using Chi-square test and multivariate logistic regression for risk factors analysis.

Results

Seropositivity was 1.6% (25 cases). Males (2.2%) showed significantly more positivity than females (0.9%) (P= 0.03). There was no significant association between CE seropositivity and age group, occupation, and region. Age group of 30-60 years encompassed the highest rate of positivity. The seropositivity of CE was 2.1% and 1.2% for urban and rural cases respectively. Binary logistic regression showed that males were 2.5 times at higher risk for infection than females.

Conclusion

Although seroprevalence of CE is relatively low in Qom Province, yet due to the importance of the disease, all preventive measures should be taken into consideration.     

Malaria Epidemiology in Mersin Province,Turkey from 2002 to 2011

Background

Malaria is an infectious disease caused by Plasmodium spp. with high morbidity and mortality in human in tropical and subtropical regions. In recent years, number of malaria cases has been significantly reduced because of fight with the disease in Turkey. This study intended to investigate the malaria epidemiology in Mersin Province from 2002 to 2011 using data from the provincial Public Health Directorate.

Methods

Over ten years, 303573 blood samples were taken from the people by active and passive surveillance methods and blood smears were prepared. Smears were stained with Giemsa and examined under the microscope.

Results

Totally, 73 people including 44 male and 29 female were positive in terms of Plasmodium spp. It was determined that P. vivax observed in 67 cases while P. falciparum in 6 cases. Cases were mainly observed in 15 to 44 years old range, showed an increase between June-September periods and a significant decrease after 2006. Out of the 73 malaria cases, 54 cases were from Mersin Province and 13 cases were imported from another province of Turkey. Six cases were transmitted from abroad.

Conclusion

These results provide information about malaria epidemiology in an endemic area in Turkey and contribute its prevention in Mersin Province.     

In Vivo Antimalarial Effects of Iranian Flora Artemisia khorassanica against Plasmodium berghei and Pharmacochemistry of its Natural Components

Background

The aim of this study was to evaluate the antimalarial effects of Iranian flora Artemisia khorassanica against Plasmodium berghei in vivo and pharmacochemistry of its natural components.

Methods

The aerial parts of Iranian flora A. khorasanica were collected at flowering stage from Khorassan Province, northeastern Iran in 2008. They were air-dried at room temperature; powder was macerated in methanol and the extract defatted in refrigerator, filtered, diluted with water, then eluted with n-hexane and finally non-polar components were identified through Gas Chromatography and Mass Spectroscopy (GC-MS). Toxicity of herbal extracts was assessed on naïve NMRI mice, and its anti-malarial efficacy was investigated on infected Plasmodium berghei animals. This is the first application on A. khorssanica extract for treatment of murine malaria. The significance of differences was determined by Analysis of Variances (ANOVA) and Student''s t-test using Graph Pad Prism Software.

Results

The herbal extract was successfully tested in vivo for its anti-plasmodial activity through artemisin composition, which is widely used as a standard malaria treatment.

Conclusion

Although, this study confirmed less anti-malarial effects of A. khorssanica against murine malaria in vivo, however there are some evidences on reducing pathophysiology by this medication. In complementary assay, major components were detected by GC-MS analysis in herbal extract including chrysanthenone (7.8%), palmitic acid (7.4%) and cis-thujone (5.8%). The most retention indices of the component are given as n-eicosane, palmitic acid and n-octadecane.     

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 Variation of MSP-1 Gene in Plasmodium vivax Isolated from Patients in Hormozgan Province,Iran using SSCP-PCR

Background

The main goal of present study was to detect polymorphism in MSP-1 gene which is a major blood stage candidate for vaccine in Plasmodium vivax by Single Strand Conformational Polymorphism-Polymerase Chain Reaction (SSCP-PCR).

Methods

During 2008 to 2010 fifty samples were collected from Iranian patients with P. vivax in Hormozgan Province, southern Iran. All of the samples were detected by microscopical examination. Amplification of MSP-1 gene was done by PCR after DNA extraction. Single strand DNAs due to using in SSCP, was electrophoresed on polyacrylamid- Bisacrylamid gel then banding patterns were revealed by silver-staining method. Sequencing as a typing method was performed for some isolates.

Results

All of the 50 isolates were positive microscopically. Totally 12 (24%) isolates showed 440 bp and 38 (76%) showed 500 bp in PCR assay. SSCP analysis revealed four banding patterns. Pattern I (10/50), Pattern II (12/50), Pattern III (27/50), and Pattern IV (1/50). The results sequencing analysis of the MSP-1 gene in 19 isolates revealed diversity in nucleotides and amino acid in Iranian P. vivax isolates.

Conclusion

Our study confirms that the SSCP-PCR is a rapid method for detecting polymorphism in MSP-1 gene in P. vivax. The presence of different haplotypes in MSP-1 gene shows that several P. vivax strains exist in malaria endemic areas of Iran.     

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.     

High Seroprevalence of Toxoplasma gondii Antibody in HIV/AIDS Individuals from North of Iran

Background:

Toxoplasmosis in immunocompetent people is generally asymptomatic but in immunocompromised patients including HIV/AIDS, cancer patients, and organ transplant recipients, etc. it can lead to serious pathological problems. The objective of current study was to determine the seroprevalence of T. gondii IgG and IgM antibodies in HIV/AIDS patients using ELISA technique in Mazandaran Province, northern Iran.

Methods:

Overall, 82 serum samples (61 males and 21 females) were collected from HIV/AIDS patients in Mazandaran Provinces, in 2013. Sera were surveyed employing ELISA assay. Data were analyzed using Chi-Square or Fisher exact test. In addition, before sampling a questionnaire was filled out for each subject.

Results:

Overall seroprevalence of examined sera was 96.3% for IgG antibody but none of the sera shown IgM antibody against T. gondii. The seroprevalence of toxoplasmosis in males and females was 96.7% and 95.2%, respectively. An antibody titer of >1 IU/ml was considered as positive. Furthermore, none of the included variables statistically was significant.

Conclusions:

Seroprevalence of chronic (latent) toxoplasmosis in HIV/AIDS patients in Mazandaran Province is high compared to toxoplasmosis in general population. Consequently, the risk of acquiring Toxoplasma encephalitis in examined seropositive HIV/AIDS patients of Toxoplasma is high.     

Trichinella Infection in Wildlife of Northeast of Iran

Background

The objective of this investigation was to detect the presence of Trichinella in some carnivores of Mashhad in northeast of Iran and to identify Trichinella species circulating in this area.

Methods

The present study was carried out using muscle tissue collected from 120 stray dogs, 26 wild boars, 25 rodents, two foxes and two hyenas captured in Mashhad City, province of Khorasan Razavi, Iran.

Results

Trichinella larvae were detected in three stray dogs by artificial digestion and compression. All larvae were identified as T. britovi using multiplex PCR.

Conclusion

This is the first report of identification of T. britovi in stray dog in Iran.     

Seroprevalence of Neospora spp. in Horses in North East of Iran

Background

Neospora caninum, an obligate intracellular protozoan parasite, is recognized as a major cause of abortion in cattle, while limited information is presently available on the seroprevalence of Neospora antibodies in horses’ worldwide. The aim of the present study was to determine serologic prevalence of Neospora infection in horses in Iran.

Methods

Sera from 150 horses from Mashhad suburb in Razavi Khorasan Province, northeast Iran were examined for antibodies to Neospora spp. using Neospora modified direct agglutination test (N-MAT).

Results

Antibodies to this parasite were detected in 45 (30%) of the examined serum samples. Thirty four percent of the samples had titer of 1:40 while then reduced to 30% when 1:80 serum dilution was applied as significant cut off titer.

Conclusion

This study is the first investigation carried out on the Neospora in horses in Iran and indicates that horses in Iran are exposed to this parasite.     

Radular Teeth Morphology in Limax (Caspilimax) keyserlingi (Martens, 1880) and Parmacella ibera (Eichwald, 1841) from Northern Iran

Background

Slugs have been known worldwide as important pests of agricultural and horticultural production. They also play a role as intermediate or definitive hosts of helminths parasite. In this purpose, current study was carried out to examine slug radular teeth structure and slug infection with helminths larvae in north of Iran.

Methods

A total number of 114 slugs were collected from center and east parts of Mazandaran province from May 2011 to June 2012. The specimens were rinsed, measured, and identified. The radula of all collected slugs was extracted and stained by using Mallory II. For detecting the helminths parasite infection, foot- head and viscera of examined slugs were removed, minced, and digested with 4.5% acid pepsin.

Results

Two species of Limax (Caspilimax) keyserlingi (Martens 1880) (11.4%, 13/114) and Parmacella ibera (Eichwald 1841) (88.6%, 101/114) were prevalent in the region. There was significant difference between body length and shell size. P. ibera had the highest number of teeth rows (145±5). The radular teeth formula was approximately similar in both identified slugs. In P. ibera, there was no significant difference in the average length and width of radula. The radular teeth in L. keyserlingi were larger and thicker than P. ibera. In all examined slugs for helminths larvae infection, P. ibera (7.69%, 1/13) was infected with Strongyloid larvae from Fereidonkenar area.

Conclusion

Two prevalent species of slugs exist in the same region of which P. ibera has capability to play a role as intermediate host of nematode helminths. Radular morphology within the slug species may be also systemically informative.