Genotyping of Echinococcus granulosus isolated from canine in Northwest Iran.

Echinococcus granulosus is a parasitic tapeworm that causes cystic echinococcosis, a potentially life-threatening zoonotic infection affecting humans and animals across the globe. In Iran, the prevalence of this parasite remains a significant public health concern, particularly in the northwest region. This study aimed to investigate the genotypes of E. granulosus isolated from canines in the northwest of Iran. A total of 87 samples were collected from the Mughan plain area in Ardabil province, including 47 stray dogs (Canis familiaris), 25 golden jackals (Canis aureus), and 15 red foxes (Vulpes vulpes), and molecular analysis was performed for partial cytochrome c oxidase subunit 1 and nad1 genes. Phylogenetic analyses were carried out on the obtained sequence. The findings revealed that 9 out of 87 (10.3%) samples were infected with Echinococcus parasites, with a frequency of 1 (4%) and 8 (17%) among golden jackals and stray dogs, respectively. Overall, all (100%) E. granulosus adult samples were related to the G1 genotypes. This study provides comprehensive data regarding the epidemiological and molecular characteristics of echinococcosis in canines in northwest Iran.

The Association of Human Leucocyte Antigen (HLA) Class I and II Genes with Cutaneous and Visceral Leishmaniasis in Iranian Patients: A Preliminary Case-Control Study.

Background: Leishmaniasis is currently considered a re-emerging or emerging infection based on the geographic region. The outcome of leishmaniasis vastly depends on Leishmania-host interaction. This preliminary study aimed to show the association of human leukocyte antigen (HLA) class I and II genes with healed and non-healed cutaneous leishmaniasis (CL), and symptomatic and asymptomatic visceral leishmaniasis (VL) compared with control groups in Iran. Methods: Ninety-five people, including 31 patients versus 64 individuals in the control group, were enrolled. Among them, 20 patients had confirmed CL based on amastigote observation, 10 had improved CL and 10 non-healed CL. Eleven patients were suffering from confirmed VL based on direct agglutination test (Five asymptomatic and six symptomatic VL cases). Besides, they were residents in an endemic area of VL in the northwest of Iran. To select a control group, it was ensured that they had no history of leishmaniasis. Peripheral blood samples were collected from each patient. After DNA extraction, HLA typing was conducted using polymerase chain reaction - sequence-specific priming (PCR-SSP). Subsequently, data were statistically analyzed by SPSS. Results: There was a statistical relationship between the presence of HLA-A26 and CL, healed CL and the existence of the B38 allele, C1 allele and symptomatic VL, as well as B1.4 allele and asymptomatic VL (P<0.05). Conclusion: This primary finding indicates that several HLA genes have a potential role in the susceptibility of Iranian people to CL and VL.

Sergentomyia species identification and their screening for possible infection to Leishmania spp. in Kaleybar, East-Azerbaijan province, Iran.

Leishmaniasis is a protozoal and vector-borne disease. World health organization has considered the disease as a neglected tropical disease. Phlebotomus and Lutzumyia species (order: Diptera, family: Psychodidae) are human leishmaniasis vectors in new and old worlds. Sergentomyia spp. (Diptera, Psychodidae) are proven vectors of lizard leishmaniasis. Although some studies have identified human Leishmania parasites in Sergentomyia, their role in parasite circulation is unknown yet. Hence, the parasitological and molecular methods were used to study the possible Leishmania infection of Sergentomyia spp., in the human and canine visceral leishmaniasis endemic area in North West of Iran. Even though Sergentomyia specimens were caught in a dominant number compared to Phlebotomus spp., no Leishmania promastigote or DNA was detected in live-caught or sticky trap-caught specimens, respectively. Sergentomyia spp. are proven vectors of sauroleishmaniasis, and despite several global reports of Leishmania infection in Sergentomyia spp., such findings should be carefully interpreted to avoid false vector incriminations.

Assessment of Recombinant A2-Latex Agglutination Test (RA2-LAT) and RA2-ELISA for Detection of Canine Visceral Leishmaniasis: A Comparative Field Study with Direct Agglutination Test in Northwestern Iran.

BACKGROUND: This study aimed to set-up latex agglutination test (LAT) and ELISA based on recombinant A2 from Iranian strain of Leishmania (L.) infantum (rA2-Ag) and evaluated for detection of anti-Leishmania antibodies in dogs compared to standard direct agglutination test (DAT). METHODS: The rA2-Ag was synthesized under a part of the A2 gene sequences which contain immune dominant sequences and less number of repetitive sequences. Latex beads, 0.8 µm (Sigma, USA) were sensitized with rA2-Ag. The tests were carried out on sera collected from 350 ownership dogs including symptomatic (n=67), asymptomatic (n=230) canine visceral leishmaniasis (CVL), and (n=53) uninfected domestic dogs as control group. RESULTS: Anti-leishmanial antibodies were detected in 97 (27.7%), 96 (27.4%) and 29 (%9) of the serum samples by using DAT, rA2-ELISA, and rA2-latex, respectively with ≥1:320 as a cut-off titer when DAT-confirmed cases were compared with the control groups. A combined sensitivity of 52% and specificity of 82.40% for rA2-ELISA and 23.8% and specificity 95.38%, respectively were found with ≥1:320 as a cut-off titer when DAT-confirmed cases were compared with the control groups. The concordance between rA2-ELISA and rA2 latex compared with DAT as a gold standard serological test for VL were found 73.7% and 77.5%, respectively. CONCLUSION: A good degree of agreement was found between rA2-ELISA and DAT (73.7%). rA2-ELISA could detect more seropositive serum samples than rA2-LAT and it may be recommended as an alternative tool for the diagnosis of CVL.

High resolution melting analysis as an accurate method for identifying Leishmania infantum in canine serum samples.

BACKGROUND & OBJECTIVES: Leishmania (L.) infantum is the principal agent of visceral leishmaniasis (VL) in the Mediterranean and American regions. So far different molecular methods including high resolution melting (HRM) analysis have been developed for detecting and identifying L. infantum infection. HRM assay is an automted molecular method which detects and identifies different genus and species of infectious agents. This study aimed to diagnose and identify Leishmania infection caused by L. infantum species using real-time PCR coupled with HRM assay in the serum samples in comparison with anti-L. infantum antibodies obtained using direct agglutination test (DAT), in domestic and wild canines of northeastern Iran. METHODS: Serum samples of 15 foxes, 14 jackals, seven domestic dogs and three wolves were collected in some villages around Shirvan and Bojnourd districts from the northeast regions of Iran during 2014-15. Initially, all the collected serum samples were tested by DAT for the detection of anti-L. infantum antibodies. Afterwards, genomic DNA was extracted from the samples and tested by real-time PCR-HRM analysis targeting hsp70, ITS1 and gp63 genes. The level of agreement between DAT and HRM assay were analysed statistically. RESULTS: Out of the 39 serum samples, eight showed anti-L. infantum antibodies at titre 1: 80 while only one of them showed anti-L. infantum antibodies at titre 1 : 160. All the nine seropositive samples showed positive results with HRM analysis. Additionally, three DAT negative serum samples were also found positive in the HRM technique. Altogether, 12 out of the 39 DNA samples showed positive results in HRM analysis. Among the three gene sequences used, gp63 was best for separation and identification of species. INTERPRETATION & CONCLUSION: HRM analysis targeting hsp70, ITS1 and gp63 genes can be used as a highly sensitive technique for the screening and early detection of L. infantum infection in the wild and domestic canines. It has higher accuracy than DAT and allows detection and discrimination of different Leishmania species responsible for the Leishmaniases.

Cytokine Profile of Leishmania Infantum Fucose-Mannose Ligand in Vaccinated Dogs in the Northwest of Iran.

BACKGROUND: Canine visceral leishmaniasis (CVL) caused by Leishmania infantum is endemic in the northwest and south of Iran. An appropriate vaccine can help to prevent and control visceral leishmaniasis in both humans and animals. Few studies have confirmed that the fucose-mannose ligand (FML) antigen of Leishmania donovani produced protective immunity in dogs against CVL. OBJECTIVE: To evaluate the immune responses of vaccinated dogs against FML antigen of L. infantum. METHODS: We isolated the FML antigen from native L. infantum and vaccinated the dogs with FML-saponin in an endemic area of VL in Iran to evaluate the immune responses of vaccinated dogs against this antigen. RESULTS: Our results indicated a significant increase in the expression of IFN-γ, IL-10 and IL-13, but not IL-12A, gene transcripts in PBMCs of FML-saponin vaccinated dogs in comparison with controls. Our findings showed a significant difference in the ratio of IFN-γ/IL-10 mRNA expression in FML-saponin vaccinated dogs in comparison with two control groups. Moreover, a significant level of anti-FML antibodies was detected in serum of vaccinated dogs. CONCLUSION: These findings showed that FML-saponin stimulates both Th1 and Th2 immune responses with predominant Th1 and strong humoral immune responses to produce protective immunity against CVL.

Modification on Direct Agglutination Antigen Preparation for Simplified Sero-Diagnosis of Human and Canine Visceral Leishmaniasis.

BACKGROUND: Visceral leishmaniasis is systematic serous parasitic disease with public health importance. Zoonotic form of visceral leishmaniasis is wide spread in Mediterranean basin and South America regions. Direct agglutination test (DAT) is an accurate, reliable and non-expensive serological test for the diagnosis of visceral leishmaniasis in human and canines but the antigen preparation involves some limitations. This study aimed to compare the conventional production of DAT antigen with our modified DAT antigen and then assessed on human and dog pooled sera. METHODS: Conventional DAT antigen has been prepared at the School of Public Health, Tehran University of Medical Sciences and some modifications were carried out on it, which named as modified DAT antigen. Three positive and one negative human and dog pooled serum were separately used for the comparison of modified DAT with conventional DAT antigen batches with one-month interval for a period of 9 months. RESULTS: A good concordance was observed between modified DAT compared to conventional DAT antigens for the detection of visceral leishmaniasis on human (100%) and dog (94.4%) pooled sera, respectively. CONCLUSION: Since the modified DAT antigen could be reduced the preparation time from 3 days to several hours and a good degree of agreement was found between modified DAT and convention DAT antigen batches, it can be used as a simple and easy tool for screening and serodiagnosis of human and canine L. infantum infection.

Molecular Detection and Identification of Zoonotic Microsporidia Spore in Fecal Samples of Some Animals with Close-Contact to Human.

BACKGROUND: Microsporidia species are obligatory intracellular agents that can infect all major animal groups including mammals, birds, fishes and insects. Whereas worldwide human infection reports are increasing, the cognition of sources of infection particularly zoonotic transmission could be helpful. We aimed to detect zoonotic microsporidia spore in fecal samples from some animals with close - contact to human. METHODS: Overall, 142 fecal samples were collected from animals with closed-contact to human, during 2012-2013. Trichrome - blue staining were performed and DNA was then extracted from samples, identified positive, microscopically. Nested PCR was also carried out with primers targeting SSU rRNA gene and PCR products were sequenced. RESULTS: From 142 stool samples, microsporidia spores have been observed microscopically in 15 (10.56%) samples. En. cuniculi was found in the faces of 3 (15%) small white mice and 1 (10%) laboratory rabbits(totally 2.81%). Moreover, E. bieneusi was detected in 3 (10%) samples of sheep, 2 (5.12%) cattle, 1 (10%) rabbit, 3 (11.53%) cats and 2 (11.76%) ownership dogs (totally 7.74%). Phylogenetic analysis showed interesting data. This is the first study in Iran, which identified E. bieneusi and En. Cuniculi in fecal samples of laboratory animals with close - contact to human as well as domesticated animal and analyzed them in phylogenetic tree. CONCLUSION: E. bieneusi is the most prevalent microsporidia species in animals. Our results can also alert us about potentially zoonotic transmission of microsporidiosis.

Natural infection and phylogenetic classification of Leishmania spp. infecting Rhombomys opimus, a primary reservoir host of zoonotic cutaneous leishmaniasis in Northeast Iran.

BACKGROUND: In the northeast and central parts of Iran, Rhombomys opimus (great gerbil) is the primary reservoir host of zoonotic cutaneous leishmaniasis (ZCL). This study used both parasitological and molecular methods to identify Leishmania spp. and their different haplotypes that were circulating in the great gerbil populations in ZCL foci from northeastern Iran. METHODS: A cross-sectional study using microscopy, culturing and molecular methods was conducted to detect Leishmania parasites in 194 live R. opimus in ZCL foci from northeastern Iran during 2010-2011. RESULTS: Leishmania spp. were found in 38.1% (74/194) of the samples by microscopy and in 41.2% (80/194) by culturing. Small papules and skin thickening on the upper edge of the ears were observed in 25 (12. 9%) of the R. opimus. PCR-RFLP and PCR direct sequencing of internal transcribed spacer 1 (ITS1) rRNA showed similar infection rates for L. major and L. turanica in 60 eligible R. opimus, only one mixed infection containing both L. major and L. turanica was found. Phylogenetic analysis was conducted using the ITS1 sequences of 32 isolates that were successfully aligned by comparison of their base sequences with the ITS1 DNA sequence database using ClustalW and MEGA5. The samples were classified into monophyletic clusters (>97% bootstrap). Six haplotypes were observed for L. major and seven for L. turanica. CONCLUSION: In northeast Iran, L. major, and L. turanica naturally circulate in R. opimus, and L. major/L. turanica co-infections also exist. Phylogenetic analysis suggested that Leishmania spp. isolated from R. opimus are not a monophyletic group.

An observational study on the current distribution of visceral leishmaniasis in different geographical zones of Iran and implication to health policy.

Visceral leishmaniasis is one of the most important parasitic diseases that is endemic in some parts of Iran. This study aimed to determine current distribution of visceral leishmaniasis in four distinct geographical zones of Iran. A cross-sectional study was conducted using direct agglutination test (DAT) on 9396 and 2559 serum samples collected from humans and domestic dogs, respectively during the period of 2007 through 2009. Altogether, 403 (4.3%) out of 9396 human serum samples collected from 4 distinct geographical locations showed anti-Leishmania antibodies with titers ≥ 1:3200. Physical examinations performed on 142 sero-positive cases with anti-Leishmania antibodies at titers of 1: 3200 to 1:102400 among whom fever (94.4%), paleness (67.6%) and hepato-splenomegaly (42.2%) were the predominant clinical signs and symptoms. The highest sero-prevalence rate (1.55%) was found in children ≤ 5 years old. Out of 2559 serum samples collected from domestic dogs, 212 (8.3%) were DAT positive (≥ 1:320). Leishmania infantum is the principal causative agent of the disease was isolated from both infected humans and dogs in Iran. Our findings indicate that Mediterranean visceral leishmaniasis with different distribution occurs in different geographical locations of Iran.

Canine visceral leishmaniasis: asymptomatic infected dogs as a source of L. infantum infection.

Clinically infected dogs have been identified as the main reservoir hosts of visceral leishmaniasis (VL) caused by Leishmania infantum in the Mediterranean region. The objective of this study was to determine the potential of asymptomatic infected dogs compared with symptomatic ones as a source of L. infantum infection to golden hamster. For this purpose, anti-Leishmania antibodies were detected with direct agglutination test (DAT) in 13 symptomatic (7 seropositive =>or=1:320) and 53 asymptomatic (9 seropositive =>or=1:320 and 44 seronegative =<1:320) ownership dogs. DNA of Leishmania sp. was extracted from skin and peripheral blood tissues of each dog and tested by PCR. Sixty-six Syrian golden hamsters (Mesocricetus auratus) were used for the determination of infectivity and pathogenicity of L. infantum, isolated from the dogs. We used the internal transcribed spacer 2 (ITS 2) rDNA sequence analysis. The results showed that 22 and 11 out of 66 inoculated golden hamsters were positive by PCR and parasitological examinations, respectively. From 22 PCR positive hamsters, 17 were related to asymptomatic dogs and 5 were from symptomatic ones. There was no significant difference between symptomatic and asymptomatic dogs in producing Leishmania infection in the susceptible animal model (P=0.66). Smears and cultures of 5 dogs from 13 symptomatic dogs (38.5%) and 6 dogs from 53 asymptomatic ones (11.3%) were found to be positive at parasitological examination. All the L. infantum isolates from symptomatic and asymptomatic dogs were similar in sequencing. In conclusion, asymptomatic infected dogs as well as symptomatic ones can harbor L. infantum in their blood and skins which are virulent and infectious for inoculated golden hamster.

Epidemiological aspects of canine visceral leishmaniosis in the Islamic Republic of Iran.

An epidemiological study to examine the sero-prevalence of zoonotic visceral leishmaniosis (ZVL) among domestic and wild canines in endemic foci of Iran was carried out during 1999-2003 to assess the distribution of the disease and the possible association between infection in dogs, wild canines and people. Anti-leishmanial antibodies were detected by the direct agglutination test (DAT). Parasitological study was performed for all captured wild canines and were detected in some of the seropositive dogs with specific clinical signs (n=107). Serum samples (n=1568) were collected from domestic dogs in villages that are known endemic foci of human visceral leishmaniosis (HVL). Wild canine sera were collected from jackals (Canis aureus, n=10), foxes (Vulpes vulpes, n=10) and wolves (Canis lupus, n=10). Of the 1568 serum sampled collected from domestic dogs, 222 (14.2%) were positive by DAT (1:320 and above). No statistically significant difference was found between male (15.2%) and female (11.8%) sero-prevalence (P=0.083). Dogs of 8 years and above showed the highest sero-prevalence (40.6%). Only 23.9% of the seropositive domestic dogs had clinical signs. Parasitology and serology tests that were performed in 30 wild canines showed 10% these animals were infected by Leishmania infantum. Ten out of 11 Leishmania spp. isolated from the dogs and wild canines were identified as L. infantum and one other as L. tropica by molecular and biochemical techniques. For the first time in Iran, L. infantum and L. tropica were isolated from viscera of both a wolf and a domestic dog.

Double-blind randomized efficacy field trial of alum precipitated autoclaved Leishmania major vaccine mixed with BCG against canine visceral leishmaniasis in Meshkin-Shahr district, I.R. Iran.

This study was designed to evaluate the efficacy of a single dose of aluminum hydroxide (alum) precipitated Leishmania major (Alum-ALM) vaccine plus BCG against canine visceral leishmaniasis. Three hundred and forty-seven healthy dogs with no anti-Leishmania antibodies were double-blind randomly injected intradermally with either 0.1ml of Alum-ALM (200 microg protein) mixed with BCG (182 dogs) or injected with 0.1ml of normal saline (165 dogs). The results of 16 months follow-up showed that the vaccine was safe and well-tolerated. Strong seroconversion using DAT and ELISA techniques at 16 months post-vaccination was considered as an indication of Leishmania infection. The incidence rate was 3.7% (6/162) in vaccinated group and 12.0% (17/141) in control group using DAT technique. The efficacy of the vaccine was calculated to be 69.3%.