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Leishmaniasis refers to a disease with a wide range of manifestations; and there are three main forms of disease, cutaneous, mucocutaneous, and visceral. Leishmaniasis is one of the diseases with a protozoan agent which is vector-borne. Visceral leishmaniasis (VL) is the most severe form that can be fiercely life-threatening if left untreated. VL can be caused by members of Leishmania donovani complex, in Iran, Leishmania infantum is considered the primary causative agent of VL, resulting in a zoonotic form of VL. The two main goals of our work, which followed our prior sero-epidemiological and entomological survey, were to characterize and conduct a phylogenetic analysis of the Leishmania species that infect people, dogs, and sandflies. The samples were collected throughout 2017, from January to December, so blood samples were collected from humans and dogs, while sandfly samples were collected with sticky traps. DNA extracted from all seropositive samples of humans and dogs, 10% of sero-negative human samples, and all collected sandflies were subjected to kDNA-nested-PCR for tracing parasites. A total of 30 samples, including 20 human samples, 8 dog samples, and 2 sandfly samples, were found positive for the kDNA gene of L. infantum. Sequences were evaluated to study the genetic diversity among the six discovered L. infantum. Based on kDNA, the phylogenetic study of L. infantum demonstrated a high level of genetic variety and a relationship between the host, the parasite's geographic origin, and its genetic diversity., Competing Interests: The authors declare that they have no conflict of interest., (Copyright: © 2024 Behniafar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Purpose: Visceral leishmaniasis (VL) is a systemic and parasitic disease that is usually fatal if left untreated. VL is endemic in different parts of Iran and is caused mainly by Leishmania infantum. This study aimed to recognition immunoreactive proteins in amastigote-like and promastigote stages of L. infantum (Iranian strain) by antibodies present in the sera of VL patients., Methods: Total protein extract from amastigote-like and promastigote cells was separated by two-dimensional electrophoresis (2DE). To detect the immunoreactive proteins, 2DE immunoblotting method was performed using different pools of VL patients' sera., Results: Approximately 390 and 430 protein spots could be separated in 2DE profiles of L. infantum amastigote-like and promastigote stages, respectively. In immunoblotting method, approximately 295 and 135 immunoreactive proteins of amastigotes-like reacted with high antibody titer serum pool and low antibody titer serum pool, respectively. Approximately 120 and 85 immunoreactive proteins of promastigote extract were recognized using the high antibody titer sera pool and low antibody titer sera, respectively., Conclusion: The present study has recognized a number of antigenic diversity proteins based on the molecular weight and pH in amastigote-like and promastigote stages of L. infantum. These results provide us a new concept for further analysis development in the field of diagnosis biomarkers and vaccine targets., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

This study was designed to evaluate the safety, immunogenicity, and efficacy of a single dose of L. infantum (LiCen -/- ) live attenuated candidate vaccine against canine leishmaniasis (CanL). Eighteen healthy domestic dogs with no anti-Leishmania antibodies and negative leishmanin skin test (LST) were randomly inoculated intravenously with either L. infantum (LiCen -/- ) vaccine candidate in 10 dogs or phosphate-buffered saline (PBS) in 8 dogs. The safety, immunogenicity, and efficacy rate of L. infantum (LiCen -/- ) vaccine candidate against CanL were evaluated by different criteria, including clinical manifestations, injection-site lesion, hematology and biochemistry values, anti-Leishmania antibodies using direct agglutination test (DAT), delayed-type hypersensitivity (DTH) using LST, and CD4 + and CD8 + T-cells subsets, as well as by measuring interferon (IFN-γ), interleukin (IL-23), IL-17, and IL-10 cytokines. Spleen aspiration and detection of Leishmania parasite using parasitological examinations (microscopy and culture) were performed in both vaccinated and control groups. Two months after intervention, each dog was challenged intraperitoneally (IP) with wide type (WT) L. infantum. Two-month follow-up post vaccination showed no clinical signs and serious side effects associated with the vaccination. A significant increase was found in the expression of IL-17, CD4 + , and CD8 + gene transcripts in PBMCs, as well as increased levels of Th1 cytokines, and reduction of Th2 cytokine. The efficacy of the vaccine candidate was calculated to be 42.85%. While the time window for assessing the vaccine's effectiveness was too limited to draw any real conclusions but the preliminary results showed a moderate efficacy rate due to inoculation a single dose of L. infantum (LiCen -/- ) vaccine candidate. Further investigations with more sample sizes and multiple doses of the vaccine candidate using natural challenges in the endemic areas of CanL are recommended., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or persona,relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: In recent years, cases of human visceral leishmaniasis (HVL) have been reported in some districts of Golestan Province, northeastern Iran, particularly in rural areas. Recent epidemiological evidence in Leishmania infantum endemic regions of in Iran indicates approximately 50%-80% of seropositive dogs are asymptomatic for Leishmania infection., Objectives: The goal in this study was to determine Leishmania species infecting domestic dogs in Golestan Province, Iran., Methods: Between 2015 and 2016, blood samples were obtained from 100 domestic dogs in rural regions of Golestan Province, northeastern Iran. All samples were tested for anti-Leishmania antibodies using a direct agglutination test (DAT), and for Leishmania spp. kinetoplast DNA (kDNA) using PCR., Results: Seven (7%) dogs were antibody positive and 25 dogs (25%) were Leishmania spp. DNA positives by PCR positive for leishmaniasis. Four of the seven (71%) antibody-positive dogs and 19 of the 25 (76%) PCR-positive dogs were asymptomatic. The rate of infection detected by PCR was significantly higher in male dogs (21/75, 28%) than that in female dogs (4/25, 16%). The ITS1 PCR-RFLP assay identified the presence of L. infantum, L. tropica or Crithidia spp. in the 25 PCR-positive samples., Conclusions: The high proportion of asymptomatic dogs in the study areas represent they act as potential reservoirs in the transmission cycle of Leishmania spp. and also Crithidia fasciculata as an emerging agent for the first time. Moreover, our data showed that PCR is a more reliable assay than DAT for detecting Leishmania spp. infection among asymptomatic dogs., (© 2022 The Authors. Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

Mediterranean type of visceral leishmaniasis (VL) is a zoonotic parasitic infection. Some provinces of Iran are endemic for VL while other parts are considered as sporadic areas. This study aimed to assess a combination of recombinant K26 and rK39 antigens as well as crude antigen (CA), derived from an Iranian strain of L. infantum, compared to direct agglutination test (DAT) for the detection of VL in humans and domestic dogs as animal reservoir hosts of the disease. A combination of rK26 and rK39 antigens and also CA was evaluated using indirect ELISA on serum samples of 171 VL confirmed humans (n = 84) and domestic dogs (n = 87) as well as 176 healthy humans (n = 86) and domestic dogs (n = 90). Moreover, 36 serum samples of humans (n = 20) and canines (n = 16) with other potentially infectious diseases were collected and tested for finding cross- reactivity. The results of ELISA were compared to DAT, currently considered as gold standard for the serodiagnosis of VL. The sensitivity and specificity, positive predictive and negative predictive values were calculated compared to DAT. The positive sera had previously shown a positive DAT titer ≥ 1:800 for humans and ≥ 1:80 for dogs. Analysis was done by MedCalc and SPSS softwares. Using the combination of rK26 and rK39 in ELISA, a sensitivity of 95.2% and a specificity of 93.0% % were found in human sera at a 1:800 (cut-off) titer when DAT-confirmed cases were compared with healthy controls; a sensitivity of 98.9% and specificity of 96.7%% were found at a 1:80 (cut-off) titer compared with DAT. A good degree of agreement was found between the combined rK39 and rK26-ELISA with DAT in human (0.882) and dog serum samples (0.955) by kappa analysis (p < 0.05). The ELISA using the CA test showed 75% sensitivity in human and 93.1% in dog serum samples as well as 53.5% specificity in human and 83.3% in dog,s sera, respectively. The combination of rK26 and rK39 recombinant antigen prepared from Iranian strain of Leishmania infantum showed high accuracy for the serodiagnosis of VL in human and domestic dogs. Further extended field trial with a larger sample size is recommended., (© 2022. The Author(s).)

Background: Recent global evidences showed that asymptomatic blood donor carriers of Leishmania infection will appear as a threat for blood transfusions recipients in endemic areas. As yet, there is no appropriate diagnostic procedure for detecting infection of blood donors in blood banks., Subjects and Methods: The present study was aimed to apply various current diagnostic tests among blood donors in an endemic area of visceral leishmaniasis (VL), Ardabil Province, northwestern Iran. Blood samples were gathered from 860 blood donors in endemic areas of the province between 2017 and 2018, at eight blood donation centers. These samples was assessed using microculture, serological (DAT and rK39-ICT) and molecular based (conventional kDNA-PCR and HRM-PCR) tests., Results: Of 860 eligible donors, 24 (2.8%) were seropositive for VL by DAT, and 388 (45%) were positive by kDNA-PCR. Moreover, 19 (19/860) were positive for both of them. Out of 19 subjects, 5.3% (1/19) was positive by rK39-ICT, 10.5% (2/19), and 79% (15/19) were detected positive in microculture and HRM-PCR methods, respectively. Nineteen donors were followed up for 2 years, of which 16 (84.2%) had a serological conversion, and 4 (21%) were positive by kDNA-PCR. The sensitivity of kDNA-PCR, and HRM-PCR procedures in detecting Leishmania parasite was found to be 98.7%, and 79%, respectively., Conclusions: Our findings justify the use of kDNA-PCR as a convenient and sensitive tool for screening subjects with leishmanial latent infection in blood banks at least in endemic regions. In these areas, however, a PCR-based test should be used to validate Leishmania infection among seropositive donors., (© 2022. The Author(s) under exclusive licence to Witold Stefański Institute of Parasitology, Polish Academy of Sciences.)

Canine leishmaniasis (CanL) is an important parasitic e disease caused by Leishmania infantum and is transmitted by female phlebotomine sand flies primarily between canines and secondarily to humans. Recently, we showed that immunization with Leishmania major p27 gene knockout (Lmp27 -/- ) as a live attenuated vaccine was safe, induced immunogenicity, and protected against the development cutaneous and visceral leishmaniasis in mice. The p27 protein is a component of the COX protein complex which is responsible for ATP production. In this study, we analyzed the Lmp27 -/- candidate vaccine potential with this regard to the safety and induction of immunogenicity and protection against CanL. Variables such a clinical manifestation, anti-Leishmania antibodies using direct agglutination test (DAT), lymphocyte proliferation, delayed-type hypersensitivity (DTH), bone marrow aspiration (BMA) and parasite burden using parasitological and molecular examinations were measured. The results demonstrated that the Lmp27 -/- vaccinated group showed no clinical signs after inoculation with Lmp27 -/- mutant during a 12-month follow-up, and had significantly higher T-cell responses (Lymphocyte proliferation and DTH), lower seroconversion and parasite burdens following a challenge inoculation with L. infantum after 6-mounth. In conclusion, vaccination with Lmp27 -/- parasites would be safe and provide significant immunoprotectivity and efficacy against infection with wild type (WT) L. infantum., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Objective: Canine visceral leishmaniasis (CVL) is the main source of human visceral leishmaniosis (HVL) in Mediterranean region, including Iran and is spread from domestic dogs to Phlebotomine sand flies vectors to humans. To control the transmission of HVL, early and accurate detection of infected dogs is paramount importance despite it remains a confronting challenge. Herein, we evaluated the performance of direct agglutination test (DAT) against gold standard nested polymerase chain reaction (nested-PCR) for CVL diagnosis in symptomatic and asymptomatic domestic dogs from endemic areas of Iran., Results: Venous blood samples were collected from dogs without clinical signs (n  =  30) and with clinical signs (n  =  35) suggestive of Leishmania infantum infection. Among 65 samples examined, Leishmania DNA was detected by nested-PCR in 89.23% (58/65). Furthermore, 86.15% (56/65) nested-PCR positive samples were also DAT positive. The results of the DAT sensitivity test were 96.43% and 96.67% in symptomatic and asymptomatic dogs, respectively, while the specificity was 100.00% and 60.00% in symptomatic and asymptomatic dogs, respectively. The results of this study also pointed out substantial concordance between DAT test and nested-PCR method in both symptomatic dogs (Κ  =  0.783; P  <  0.001) and asymptomatic dogs (Κ  =  0.618; P  <  0.001). Thus, DAT represents as a simple and economic tool for initial diagnosis of CVL particularly in endemic areas of the disease., (© 2021. The Author(s).)

Background & Objectives: Visceral leishmaniasis (VL),a protozoan disease caused by Leishmania infantum is a major public health problem and cause of death among infants aged under 1 year and the elderly in endemic foci of Iran. The aim of this study is to determine the status of L.infantum infection in stray dogs from Meshkin-Shahr, a typical endemic area of VL in Iran., Methods: Sixty-eight randomly trapped stray dogs in Meshkin-Shahr area were tested for L. infantum infection using the direct agglutination test (DAT) from June to October 2016. The confirmation of seropositive samples was performed by Microscopic slides of spleen, culture and then PCR. The molecular methods performed by ITS1-PCR, RFLP-PCR and kDNA-PCR. The allof kDNA -PCR products were sequenced., Results: Out of 68 examined stray dogs, 17 (25.0%) were positive for L. infantum by DAT (1:320 titers or higher). Parasite test showed that all of seropositive samples have amastigote forms in their spleens but only 3 out of them could be cultured. The kDNA-PCR confirmed all of seropositive samples but ITS1-PCR and RFLP-PCR only confirmed 3 out of 17 (17.6%) seropositive samples. The sequenced products showed 94% homology with L. infantum., Interpretation & Conclusion: The results showed a high prevalence of L. infantum infection in dogs in an endemic area of CVL and it provided key information for designing control programs against canine and human leishmaniasis., Competing Interests: None

Background: Leishmania parasites express various essential proteins in different growth phases (logarithmic/stationary) and forms (promastigote/amastigote). Targeting the genes encoding such proteins paves the way for controlling these parasites. Centrin is an essential gene, which its protein product seems to be vital for the proliferation of Leishmania parasites. Herein, this study was contrived to analyze the expression level of the centrin gene in different growth phases and forms of Leishmania infantum (L. infantum) parasites isolated from various endemic areas of canine leishmaniasis (CanL) in Iran., Results: All three collected isolates were identified as L. infantum using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Real-time reverse transcription (RT)-PCR revealed a statistically significant up-regulation (3.13-fold) in the logarithmic phase promastigotes compared to stationary ones (p < 0.01), whereas centrin was expressed equally in intracellular amastigotes at different time points during cell culture. Also, our finding revealed a slight up-regulation of the centrin gene (1.22-fold) in the intracellular amastigotes compared to logarithmic phase promastigotes, which was found statistically non-significant (p > 0.05)., Conclusions: Centrin gene in Iranian isolates of L. infantum is more expressed in exponential than stationary phases and seems to be considered as a promising target in the development of a genetically modified live attenuated vaccine for CanL control.

Visceral leishmaniasis (VL) is a protozoan disease caused by Leishmania infantum in the Mediterranean region including Iran. In 95% of cases, the disease can be fatal if not rapidly diagnosed and left untreated. We aimed to identify immunoreactive proteins of L. infantum (Iranian strain), and to design and evaluate a recombinant multi-epitope antigen for serodiagnosis of human VL. To detect the immunoreactive proteins of L. infantum promastigotes, 2DE immunoblotting technique was performed using different pooled sera of VL patients. The candidate immunoreactive proteins were identified using MALDI-TOF/TOF mass spectrophotometry. Among 125 immunoreactive spots detected in 2-DE gels, glucose-regulated protein 78 (GRP78), ubiquitin-conjugating enzyme E2, calreticulin, mitochondrial heat shock 70-related protein 1 (mtHSP70), heat shock protein 70-related protein, i/6 autoantigen-like protein, ATPase beta subunit, and proteasome alpha subunit 5 were identified. The potent epitopes from candidate immunodominant proteins including GRP78, mtHSP70 and ubiquitin-conjugating enzyme E2 were then selected to design a recombinant antigenic protein (GRP-UBI-HSP). The recombinant antigen was evaluated by ELISA and compared to direct agglutination test for detection of anti L. infantum human antibodies. We screened 34 sera of VL patients from endemic areas and 107 sera of individuals without L. infantum infection from non-endemic area of VL. The recombinant protein-based ELISA provided a sensitivity of 70.6% and a specificity of 84.1%. These results showed that GRP78, ubiquitin-conjugating enzyme E2, and mtHSP70 proteins are potential immunodominant targets of the host immune system in response to the parasite and they can be considered as potential candidate markers for diagnosis purposes., (Copyright © 2021. Published by Elsevier Inc.)

Visceral leishmaniasis is a neglected disease caused by Leishmania infantum and transmitted via female sand flies. Canine visceral leishmaniasis diagnosis should be performed as soon as possible, even on the basis of only a few or even a single clinical sign, to enhance the prediction of disease and to avoid both dog and human transmission and unnecessary euthanasia of apparently positive dogs. In the present work, we examined whether PQ10 recombinant protein could be suitable for immunological detection of Leishmania infantum infection. The coding sequence of PQ10 recombinant protein was sub-cloned in pET28 expression vector and was commercially synthesized by GENERAY Biotechnology, China. In the following process, sequencing with proper primers was done and the expression, optimization of expression and protein purification were performed. The efficacy of PQ10 for serodiagnosis was evaluated with 100 serum samples collected from dogs living in the visceral leishmaniasis endemic areas of Iran. Samples (n=20) of the dogs with other infectious disease were also be collected. The synthesized colones verified by the sequencing with proper primers. In the following process, expression, optimization of expression and protein purification performed and the purified recombinant protein confirmed by western blot. The ELISA was performed with PQ10 recombinant protein. The sensitivity of ELISA that was evaluated with sera from naturally infected dogs was 94%. The specificity value of the ELISA determined with sera from healthy dogs and from dogs with other infectious diseases was 86%. The positive predictive value (PPV) and negative predictive value (NPV) determined 87.03% and 93.47% respectively. Our findings indicated to the potential use of this recombinant protein in the diagnosis of canine visceral leishmaniasis., (Copyright © 2020, Archives of Razi Institute. Published by Kowsar.)

Canines are proven reservoir hosts of Leishmania infantum, the causative agent of human zoonotic visceral leishmaniasis, and therefore domestic dogs play a central role in transmitting the disease to humans. Studies on the effect of insecticide-impregnated dog collars for controlling canine visceral leishmaniasis (CanL) have been increasing; however, meta-analysis has not been conducted. This study assessed the effectiveness of insecticide-impregnated dog collars for preventing CanL. We searched (PubMed, Web of Science, Scopus, Embase, Ovid Medline(R), and Cochrane library), from inception until 2 May 2020. Two authors independently performed articles screening and data extraction. We applied the RoB 2.0 tool to evaluate the risk of bias in randomized trials, while the ROBINS-I tool was used for non-randomized trials. I-squared statistics(I2) and funnel plot and Egger's test, respectively, were used to assesses heterogeneity between studies and publication bias. Relative Risk (RR) and 95% Confidence Interval (CI) were calculated using the random-effects model in Stata 14 software. Out of 242 citations identified, 14 studies comprising 3786 collared dogs and 3428 uncollared dogs were eligible for meta-analysis. The use of deltamethrin-impregnated dog collars(DMC) showed an overall effectiveness of 54% (95%CI: 35-65%, I2 = 63.2%, P = 0.002) in decreasing incidence of CanL, while 10% imidacloprid and 4.5% flumethrin collars provided an overall effectiveness of 90% (95%CI: 80-96%, I2 = 0.0%, P = 0.376). DMC efficacy stratified by follow-up duration was estimated to be 58% (RR = 0.42, 95%CI: 0.20-0.87), 54% (RR = 0.46, 95%CI: 0.31-0.68), 53% (RR = 0.47, 95%CI: 0.29-0.82) for follow-up periods of 5 to 6 months, 1 year and 2 years, respectively. The current evidence indicates that using insecticide-impregnated dog collars can reduce the risk of CanL caused by L. infantum. Therefore, insecticide-impregnated dog collars could be a viable alternative for inclusion as a public health measure for controlling CanL., Competing Interests: The authors have declared that no competing interests exist.

Visceral leishmaniasis (VL), the most severe form of leishmaniasis, is caused by Leishmania donovani and Leishmania infantum . The infected dogs with canine visceral leishmaniasis (CVL) are important reservoirs for VL in humans, so the diagnosis, treatment and vaccination of the infected dogs will ultimately decrease the rate of human VL. Proteomics and immunoproteomics techniques have facilitated the introduction of novel drug, vaccine and diagnostic targets. Our immunoproteomic study was conducted to identify new immunoreactive proteins in amastigote form of L. infantum . The strain of L. infantum (MCAN/IR/07/Moheb-gh) was obtained from CVL-infected dogs. J774 macrophage cells were infected with the L. infantum promastigotes. The infected macrophages were ruptured, and pure amastigotes were extracted from the macrophages. After protein extraction, two-dimensional gel electrophoresis was employed for protein separation followed by Western blotting. Western blotting was performed, using symptomatic and asymptomatic sera of the infected dogs with CVL. Thirteen repeatable immunoreactive spots were identified by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Some, including prohibitin, ornithine aminotransferase, annexin A4, and apolipoprotein A-I, have been critically involved in metabolic pathways, survival, and pathogenicity of Leishmania parasites. Further investigations are required to confirm our identified immunoreactive proteins as a biomarker for CVL.

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., Competing Interests: None

Visceral leishmaniasis (VL) is an important parasitic disease which is endemic in different parts of Iran; and domestic and wild canines are principal reservoir hosts of the disease. The objective of this study was to review the spatial distribution of canine VL (CVL) caused by Leishmania infantum in domestic and wild canines in different geographical areas of Iran. An extensive literature search was conducted in different international and national databases, including Cochrane, MEDLINE/PubMed, Scopus, Web of Science and Iran Medex to find articles with the words "visceral leishmaniasis in Iran" in their titles and "canine visceral leishmaniasis in Iran" or "feline visceral leishmaniasis in Iran" or "accidental reservoir hosts of visceral leishmaniasis in Iran" in their subtitles, irrespective of the type and duration of study. Screening of the irrelevant articles from total 36,342, yielded 61 eligible articles. More than 93% of the studies were carried out on domestic dogs (Canis familiaris, n = 57) and the remaining were on other carnivores such as wild canines including foxes (Vulpes vulpes, n = 4), jackals (C. aureus, n = 6) and wolves (C. lupus, n = 6); while studies on domestic cats (Felis catus, n = 3) as well as desert rodents (n = 2) were rare. The average rate of L. infantum infections reported among domestic dogs using direct agglutination test (DAT) in Iran was 12.5%. The highest prevalence rate (14%) was reported from the northwest regions of the country where VL is endemic. The review indicates that CVL is endemic in various parts of Iran and domestic dogs are the main and potential reservoir hosts of the disease. Other carnivores, such as domestic cats and some species of desert rodents (Cricetulus migratorius, Mesocricetus auratus and Meriones persicus) seem to be playing a role in the maintenance of transmission cycle of L. infantum in the endemic areas of the disease., Competing Interests: None

Visceral leishmaniasis, a neglected tropical disease, is the third most common opportunistic disease in immunosuppressed patients, such as those affected by the human immunodeficiency virus (HIV)/acquired immune deficiency syndrome. Although the reports have been characterized as Leishmania /HIV coinfections, the occurrence of a mixed infection by two Leishmania species in HIV-positive patients is rare. Here, we present an atypical case of disseminated cutaneous leishmaniasis (DCL) in a 26-year-old HIV-positive man. The diagnosis of DCL was established using skin biopsy and histopathology examinations and confirmed by molecular techniques. This is the first case of a Leishmania /HIV coinfection due to a mixed infection of Leishmania infantum / Leishmania major in 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.

Background: Little is known regarding transfusion-transmitted leishmaniasis (TTL) and the real global incidence of visceral leishmaniasis (VL) among blood donors as carriers of Leishmania spp. in endemic VL foci including Iran. Recent epidemiological evidences indicate that blood donor may be harbor of Leishmania infantum (L. infantum) infection in Iran., Methods: The aim of the present study was to determine the prevalence of Leishmania infection among blood donors in a main endemic focus of VL in Iran using DAT and polymerase chain reaction (PCR) based methods Between July-September 2016, blood samples were collected from 600 apparently healthy blood donors from six blood donation centers and blood donation mobile unit in Ardabil Province, where VL cases had been recorded. Each of these samples was tested for anti-Leishmania antibodies, in direct agglutination test (DAT), and for L. infantum kDNA, the PCR-based assay., Results: Of 600 blood donors, which were examined, 23 (3.8%) blood donors were seropositive by DAT and of 23 seropositive subjects, 82.6% (19/23) were positive by PCR. All the seropositive cases were males except one of them was female., Conclusions: Our findings showed that many asymptomatic human carriers of L. infantum live in the endemic regions of northwestern Iran and potentially act as reservoirs of infection; those must be considered carefully by arrangement VL control strategies in the country., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Leishmania RNA virus (LRV) was first detected in members of the subgenus Leishmania (Viannia), and later, the virulence and metastasis of the New World species were attributed to this virus. The data on the presence of LRV in Old World species are confined to Leishmania major and a few Leishmania aethiopica isolates. The aim of this study was to survey the presence of LRV in various Iranian Leishmania species originating from patients and animal reservoir hosts. Genomic nucleic acids were extracted from 50 cultured isolates belonging to the species Leishmania major, Leishmania tropica, and Leishmania infantum. A partial sequence of the viral RNA-dependent RNA polymerase (RdRp) gene was amplified, sequenced and compared with appropriate sequences from the GenBank database. We detected the virus in two parasite specimens: an isolate of L. infantum derived from a visceral leishmaniasis (VL) patient who was unresponsive to meglumine antimoniate treatment, and an L. major isolate originating from a great gerbil, Rhombomys opimus. The Iranian LRV sequences showed the highest similarities to an Old World L. major LRV2 and were genetically distant from LRV1 isolates detected in New World Leishmania parasites. We could not attribute treatment failure in VL patient to the presence of LRV due to the limited number of specimens analyzed. Further studies with inclusion of more clinical samples are required to elucidate the potential role of LRVs in pathogenesis or treatment failure of Old World leishmaniasis.

Introduction: Various methods are used for the diagnosis of visceral leishmaniasis (VL), such as microscopic examination, culture and inoculation of laboratory animals; however, serological assays are commonly used for the detection of antibodies in serum samples with a wide range of specificity and sensitivity., Methods: The purpose of this study was to compare three serological methods, including rA2-ELISA, the recombinant KE16 (rKE16) dipstick test and the direct agglutination test (DAT), for the detection of antibodies against VL antigens. The assays utilized 350 statistically based random serum samples from domestic dogs with clinical symptoms as well as samples from asymptomatic and healthy dogs from rural and urban areas of the Meshkinshahr district, northwestern Iran., Results: Samples were assessed, and the following positive rates were obtained: 11.5% by rKE16, 26.9% by DAT and 49.8% by ELISA. The sensitivity among symptomatic dogs was 32.4% with rKE16, 100% with DAT and 52.9% with ELISA. Conversely, rA2-ELISA was less specific for asymptomatic dogs, at 46.5%, compared with DAT, at 88.9%., Conclusions: This study recommends rA2-ELISA as a parallel assay combined with DAT to detect VL infection among dogs. Further evaluations should be performed to develop an inexpensive and reliable serologic test for the detection of Leishmania infantum among infected dogs.

Objective: To identify the vectors and reservoirs of cutaneous leishmaniasis in the endemic focus of Farashband, Fars Province, South of Iran., Methods: Sticky papers and Sherman trap were used for collection of sand flies and rodents, respectively. Polymerase chain reaction (PCR) of kDNA, ITS1-rDNA were used for identification of Leishmania parasite in sand flies as well as rodents., Results: Totally 2 010 sand flies were collected and the species of Phlebotomus papatasi Scopoli was the common specimen in outdoors and indoors places. PCR technique was employed on 130 females of Phlebotomus papatasi. One of them (0.76%) was positive to parasite Leishmania major (L. major) and one specimen (0.76%) was positive to Leishmania infantum. Microscopic investigation on blood smear of the animal reservoirs for amastigote parasites revealed 16 (44%) infected Tatera indica. Infection of them to L. major was confirmed by PCR against kDNA loci of the parasite., Conclusions: The results indicated that Phlebotomus papatasi was the dominant species circulating two species of parasites including L. major and Leishmania infantum among human and reservoirs. Furthermore, Tatera indica is the only main host reservoir for maintenance of the parasite source in the area., (Copyright © 2013 Asian Pacific Tropical Biomedical Magazine. Published by Elsevier B.V. All rights reserved.)

Visceral leishmaniasis (VL) is endemic in Northwest and southern Iran. Reports of cutaneous leishmaniasis (CL) in Northwest areas are rare, and its etiological agents are unknown. In the current study, we report six CL and two post kala-azar dermal leishmaniasis (PKDL) cases caused by Leishmania infantum from endemic areas of VL in the Northwest. Smears were made from skin lesions of 30 suspected patients in 2002-2011, and CL was determined by microscopy or culture. Leishmania spp. were identified by nested-PCR assay. The disease was confirmed in 20 out of 30 (66%) suspected patients by parasitological examinations. L. infantum was identified in eight and Leishmania major in 12 CL cases by nested-PCR. Cutaneous leishmaniasis patients infected with L. major had the history of travel to CL endemic areas. L. infantum antibodies were detected by direct agglutination test (DAT) at titers of 1:3200 in two cases with history of VL. Results of this study indicated that L. infantum is a causative agent of CL as well as PKDL in the VL endemic areas.

The diagnosis of visceral leishmaniasis (VL) in humans and animal reservoir hosts is difficult, particularly in rural areas where the disease is endemic and laboratory facilities are limited. This study aimed to develop a latex agglutination test (LAT) for the rapid detection of anti-Leishmania antibodies against the A2 antigen derived from the amastigote form as well as those against crude antigens derived from the promastigote form of an Iranian strain of Leishmania (Leishmania) infantum. The A2 antigen (42-100 kDa) was prepared from the amastigote form of L. infantum, purified with electroelution and compared with the crude antigen from the promastigote form of L. infantum. Both antigens showed appropriate intensity reactions, were selected using dot blotting of positive and negative pooled sera and used to sensitize 0.9-μm latex beads. The tests were carried out on sera from 43 symptomatic, human patients with VL confirmed by parasitological examination and direct agglutination test (DAT), 30 healthy controls and 32 patients with other infections but without VL. Canine sera were collected from 63 domestic dogs with VL confirmed using parasitological examinations and DAT and 31 healthy dogs from areas non-endemic for VL. Compared with the controls, human sera from DAT-confirmed patients yielded a sensitivity of 88.4% (95% CI, 82.1-94.5%) and specificity of 93.5% (95% CI, 87.0-99.7%) on A2-LAT (amastigote) when 1:3200 was used as the cut-off titre. A good degree of agreement was found between A2-LAT and DAT (0.914). LAT required 3-5 min to complete, versus the 12-18 h needed for DAT. Compared with the controls, A2-LAT of canine sera from DAT-confirmed cases yielded a sensitivity of 95.2% (95% CI, 95.0-95.4%) and specificity of 100% (95% CI 100%) when 1:320 was used as the cut-off titre. A good degree of agreement was found between A2-LAT and DAT (0.968). Similarly, the sensitivity and specificity of Pro.-LAT (promastigote) was calculated to be 88.4% and 91.9%, respectively for human sera and 96.8% and 90.3%, respectively for canine sera. No statistically significant differences were observed between A2-LAT and Pro.-LAT for the detection of human and canine L. infantum infections. In conclusion, A2-LAT and Pro.-LAT could be used in parallel to screen for L. infantum infections in humans and dogs in areas endemic for VL in Iran., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Canine visceral leishmaniasis (CVL) is endemic in northwestern Iran. This study aimed to compare real-time PCR, conventional PCR, and the direct agglutination test (DAT) for the diagnosis Leishmania infantum infection in 167 serum samples of domestic dog. Bone marrow was used for parasitological examination (smears and/or culture) in symptomatic visceral leishmaniasis, and serum was used for detection of L. infantum kinetoplast DNA (kDNA) by both conventional PCR and real-time PCR, while anti-L. infantum antibodies in sera were measured by DAT. The sera were collected from 37 symptomatic and 112 asymptomatic dogs during April to May 2011. Eighteen presumed negative samples were obtained from healthy dogs kept in non-endemic areas with no history of CVL and used as controls. All 18 samples were negative by DAT and Dipstick rK39. DAT confirmed previous exposure to L. infantum for all 149 serum samples collected from symptomatic and asymptomatic dogs in CVL endemic areas of Iran. Among the 37 symptomatic dogs, 20 (54%), 25 (67.6%), 36 (97.3%), and 37 (100%) showed L. infantum infection by parasitological methods, conventional PCR, real-time PCR, and DAT (≥ 1:80), respectively. Of 112 asymptomatic dogs, 79 (70.5%), 111 (99.1%), and 112 (100%) were shown to be positive by conventional PCR, and DAT (≥ 1:80), respectively. For ethical reasons, no asymptomatic or healthy control dogs were examined by parasitological methods. Three (16.7%) control dogs were positive by real-time PCR, but were negative by DAT, dipstick rK39, and conventional PCR methods. Parasitemia levels were measured by real-time PCR targeting kDNA using SYBR(®) green assay. This quantitative technique detected infection in 89.9% (150/167) of the domestic dogs that harbored L. infantum kDNA, ranging from 0.01 49 to 310.1 parasites/ml. The average was 16.60 parasites/ml. A good agreement (0.97) was found between real-time PCR and DAT at ≥ 1:80 titer, used as cut-off value by Kappa analysis. Thus, real-time PCR as a quantitative PCR assay on serum samples represents a valuable tool for initial diagnosis of CVL when whole blood is not available., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Zoonotic visceral leishmaniasis (VL) is endemic in northwestern Iran. Real-time PCR, conventional PCR, and the direct agglutination test (DAT) were used to diagnose Leishmania infantum infection in blood samples from 100 domestic dogs and 100 humans. Based on clinical evaluation, 82 humans and 72 dogs from the endemic area were categorized as having asymptomatic infection, DAT positive with no clinical signs of VL, or symptomatic infection, DAT positive with at least one sign of VL. Eighteen human samples containing no Leishmania antibodies (DAT(-)) and 28 dog DAT(-) sera from non-endemic areas with no history of VL constituted negative controls. All 46 DAT(-) samples were also negative by Dipstick rK39. Bone marrow material was used for parasitological examinations in symptomatic VL, and peripheral blood samples were used for detection of L. infantum infection using conventional PCR and real-time PCR in non-symptomatic subjects. Two DNA targets (ITS1 kDNA) were used for conventional PCR. L. infantum antibodies in sera were detected by DAT. Parasitemia was measured by real-time PCR targeting kDNA using Taqman Assay. All 72 (100%) symptomatic (38/38) and asymptomatic (34/34) dog DAT(+)samples, 45 of 48 (93.8%) symptomatic human DAT(+) samples, and 32 of 34 (94.1%) human asymptomatic cases were identified by real-time PCR. The mean (59.19 vs 12.38 parasite equivalents/mL of blood) and median (16.15 vs 1 parasite equivalents/mL of blood) ranges of parasitemia were higher in dogs than in humans (P<0.05). The highest agreement was obtained between real-time PCR and DAT (99% in dogs and 95% in humans). Sensitivity of 100% and 93.9%, specificity of 96.4% and 100%, positive predictive values of 98.6% and 100%, and negative predictive values of 100% and 78.3% were found by real-time PCR for dog and human samples, respectively., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Objective: To prepare and evaluate a glycerol-preserved antigen from an Iranian strain of Leishmania infantum (L. infantum) for use in glycerol-preserved direct agglutination tests (GP-DAT) as an alternative to freeze dried direct agglutination tests (FD-DAT) that use freeze-dried antigen., Methods: Glycerol-preserved DAT antigen was prepared and stored at different temperatures. We tested antigen stored at 4 °C, 22-37 °C and 50 °C over a period of 365 days. Seven hundred twenty-nine serum samples were collected from different geographical zones of Iran from 2007-2009, and 80 of these samples were pooled to produce sera. Each pooled serum contained 10 sera. All positive and negative pooled sera were separately tested for anti-L. infantum antibodies with GP-DAT, FD-DAT and formaldehyde-fixed direct agglutination test (FF-DAT) antigens; tests were performed on both human and dog sera over a period of 12 months., Results: There was strong agreement between the results obtained using GP-DAT and FD-DAT antigens stored at 22-37 °C for 12 months for both human (100%) and dog (100%) pooled sera. The direct agglutination test results were highly reproducible (weighted kappa: GP=0.833, FD=0.979 and FF=0.917)., Conclusions: Because GP-DAT antigen is highly stable over a range of temperatures and is easy to transport in the field, this type of antigen may be particularly useful in areas with endemic visceral leishmaniasis., (Copyright © 2012 Hainan Medical College. Published by Elsevier B.V. All rights reserved.)

Background: Visceral leishmaniasis (VL) is caused by Leishmania infantum in Mediterranean basin and is an endemic disease in some parts of Iran. Canines are the main reservoirs of VL in most of the endemic areas. Different serological methods have been introduced for diagnosis of canine visceral leishmaniasis (CVL)., Objective: In this survey a Fucose-Mannose Ligand (FML) ELISA, using native L. infantum antigen, was developed and its validity for detection of infected dogs in comparison with direct agglutination test (DAT) and PCR was evaluated., Methods: Blood samples of sixty ownership dogs (≤ 3 years old) were collected from Meshkin-shahr district in Ardabil province, North-west of Iran. Sera were separated for serological assays (DAT and FML-ELISA) and the buffy coats were collected for molecular evaluation., Results: Two out of the 60 (3.33%) samples were found to be positive (antibody titer of ≥ 1/320) in DAT while seven of the 60 (11.66%) samples were positive by FML-ELISA. Nine out of 60 (15%) buffy coat samples showed a band about 680 bp indicative of L. infantum in PCR. Three out of 60 dogs had Kala-azar symptoms and were positive by PCR and FML-ELISA, while two of these three dogs had antibody titers >1/320 in their serum samples. The sensitivity and specificity of FML-ELISA for the detection of CVL in both symptomatic and asymptomatic dogs were found to be 77.8% and 100%, respectively., Conclusion: Considering the acceptable sensitivity and high specificity of FML-ELISA, use of this serological method can be recommended for epidemiological surveys of CVL.

This study aimed to evaluate the performance of a fast agglutination screening test (FAST) for serodiagnosis of human Leishmania infantum infection in Iran. FAST is based on the direct agglutination test (DAT) but combines with a higher parasite concentration and is performed with only one serum dilution. The validity of FAST for the detection of L. infantum infection in the field was compared with the direct agglutination test on 110 confirmed or patients suspected of infection with leishmaniasis, 177 healthy individuals and 41 patients with other infectious diseases who were from northwestern and southern parts of Iran. In this study, we found a 1:1600 cut-off point empirically by seeking the best correlation (90.8) between sera confirmed with visceral leishmaniasis and healthy control sera. A sensitivity of 95.4% (95% CI, 91.4-99.4) and specificity of 88.5% (95% CI, 84.2-92.8) were found with 1:1600 as a cut-off titer when DAT-confirmed cases were compared with the control groups. A good degree of agreement was found between FAST and DAT (90.8%) by Kappa analysis. FAST requires 2 h for reading the results versus the 12-18 h needed for DAT. As FAST is simple, rapid, sensitive and non-invasive and does not require a higher volume of antigens or much expertise, it can be used for screening and serodiagnosis of human L. infantum infection., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Leishmania infantum is the causative agent of infantile visceral leishmaniasis (IVL) in the Mediterranean Basin and, based on isoenzyme typing of the parasite isolated from dogs; this parasite was considered to predominate in the all foci of IVL in Iran. However, based on PCR detection and sequencing of parasite Cysteine Protease B (CPB), only one out of seven sandfly infections in Phlebotomus perfiliewi transcaucasicus was found to be L. infantum in the current investigation. The six other infections were haplotypes of Leishmania donovani, the causative agent of anthroponotic visceral leishmaniasis (AVL) in West Africa and India. The deduced amino acid of the L. donovani haplotype was found to be novel and the shortest CPB protein reported within the Leishmania spp. Circulation of both L. donovani and L. infantum by P. perfiliewi transcaucasicus, in addition to previous data indicating its ability to circulate L. tropica, suggests that this species, like other vectors of VL, is a permissive vector. Finding L. donovani infecting P. perfiliewi transcaucasicus in the area demands extensive and intensive typing of natural Leishmania infections in epidemiological investigations in Iran and the Mediterranean Basin in general.

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.

Canine visceral leishmaniasis (CVL) is not only a veterinary problem but has also a serious public health importance. Rapid detection of CVL is highly important for control of human visceral leishmaniasis in Iran. This study was aimed to compare the fast agglutination screening test (FAST) with direct agglutination test (DAT) as a standard serological test for the detection of anti-Leishmania antibodies on dog serum samples. DAT and FAST antigens were prepared in the School of Public Health, Tehran University of Medical Science. Altogether, 73 serum samples from Leishmania infantum infection dogs and 74 sera from healthy controls were collected from human VL/CVL endemic and non-endemic areas of Iran, respectively. All the sera were evaluated with both FAST and DAT techniques. A sensitivity of 98.60% (95% CI, 98.57-98.62) and specificity of 78.70% (95 CI%, 69.20-88.20) were found at a 1:160--(cut-off) titer when DAT confirmed cases were compared with healthy control. A good degree of agreement was observed between FAST and DAT (86.8%) by kappa analysis (p < 0.01). In conclusion, this study showed that FAST is very practical and simple diagnostic tool for the sero-diagnosis of CVL in endemic areas of Iran.

Visceral leishmaniasis is caused by Leishmania infantum, which is transmitted to humans by bites of phlebotomine sand flies and is one of the most important public health problems in Iran. To identify the vector(s), an investigation was carried out in Germi district, an important focus of the disease in Ardebil province in northwestern Iran, during July-September 2004 and 2005. Using sticky papers, CDC light traps and aspirators, 3,560 sand flies were collected and identified to species. Host bloodmeal preference and Leishmania infections in female specimens were evaluated using enzyme-linked immunosorbent assay (ELISA) for the former and microscopic examination followed by polymerase chain reaction (PCR) assay using species-specific kinetoplast minicircle primers for the latter. Nine sand fly species are present in the district, including Phlebotomus kandelakii Shchurenkova, Phlebotomus perfiliewi transcaucasicus Perfil'ev, Phlebotomus major Annandale, Phlebotomus balcanicus Theodor, Phlebotomus halepensis Theodor, Phlebotomus brevis Theodor & Meshghali, Phlebotomus papatasi Scopoli, Sergentomyia dentata Sinton, and Sergentomyia sintoni Pringle, with P. p. transcaucasicus being the most prevalent representative of the genus Phlebotomus at 45%. The anthropophilic index for P. p. transcaucasicus was 36.3%, indicating a strong preference for humans. Of 905 female P. p. transcuacasicus dissected, 10 (1.1%) were found naturally infected with promastigotes. Species-specific amplification of promastigotes eluted from Giemsa-stained slides revealed specific PCR products of L. infantum DNA. Based on its high anthropophily and natural infections with L. infantum, and the fact that it was the only species found infected with L. infantum, we conclude that P. p. transcaucasicus is the principal vector of L. infantum in northwestern Iran.

Discrimination of Leishmania infantum and L. donovani, the members of the L. (L.) donovani complex, is important for diagnosis and epidemiological studies of visceral leishmaniasis (VL). We have developed two molecular tools including a restriction fragment length polymorphisms of amplified DNA (PCR-RFLP) and a PCR that are capable to discriminate L. donovani from L. infantum. Typing of the complex was performed by a simple PCR of cysteine protease B (cpb) gene followed by digestion with DraIII. The enzyme cuts the 741-bp amplicon of L. donovani into 400 and 341 bp fragments whereas the 702 bp of L. infantum remains intact. The designed PCR species-specific primer pair is specific for L. donovani and is capable of amplifying a 317 bp of 3' end of cpb gene of L. donovani whereas it does not generate an amplicon for L. infantum. The species-specific primers and the restriction enzyme were designed based on a 39 bp insertion/deletion (indel) in the middle of the cpb gene. Both assays could differentiate correctly the two species and are reliable and high-throughput alternatives for molecular diagnosis and epidemiological studies of VL in various foci.

Over the last decade, the incidence of visceral leishmaniasis (VL) has increased in many districts of the province of Fars, in southern Iran. Recent epidemiological reports indicate that asymptomatic human infections with Leishmania infantum (the causative agent of VL throughout the Mediterranean basin) occur more frequently in Iran than was previously believed. Between 2004 and 2006, blood samples were collected from 802 apparently healthy subjects from communities, in the north-west and south-east of Fars province, where VL cases had been recorded. Each of these samples was tested for anti-Leishmania antibodies, in direct agglutination tests (DAT), and for L. infantum kinetoplast DNA, in PCR-based assays. Of the 426 subjects from north-western Fars, eight (1.9%) were found seropositive and 68 (16.0%) PCR-positive. The corresponding values for the 376 subjects from south-eastern Fars were lower, with five (1.3%) seropositive and 32 (8.5%) PCR-positive. Of the 100 PCR-positive subjects, 18 (18.0%) each lived in a household in which there had been a case of VL, and six (6.0%) had had VL themselves (in each case, more than a year before the blood sampling for the present study). Although 21 of the PCR-positives have now been followed-up for at least 18 months, none has developed symptomatic VL. Since positivity in the PCR-based assay probably indicated the presence of L. infantum amastigotes in the peripheral blood of 12.5% of the subjects, it is clear that asymptomatic human carriers of L. infantum are quite common in the study areas and probably act as reservoirs in the transmission of the parasite, to humans and to dogs, by sandflies.

The incidence of human visceral leishmaniasis (VL) caused by Leishmania infantum, which is endemic in several parts of Iran, has recently increased in the Nourabad-Mamassani district of Fars province, in the south of the country. Between 2003 and 2005, 12,688 sandflies were caught in this focus, using CDC miniature light traps, sticky traps and aspirators, in an attempt to identify the main vector of L. infantum. When 120 of the parous, female Phlebotomus (Paraphlebotomus) alexandri caught were individually checked for Leishmania infection, in an assay based on PCR and kinetoplast minicircle primers (LINR4 and LIN17), five (4.2%) of the flies were found to be infected with L. infantum. Thirty-nine (32.5%) of the 120 blood-fed Ph. alexandri checked in an ELISA were found positive for human blood. Since Ph. alexandri was the third-most common species caught (representing over 17% of the total catch), was clearly anthropophilic, and was not infrequently infected with L. infantum, it is probably an important vector in the Nourabad-Mamassani focus. Although other sandfly species have been found naturally infected with promastigotes in Iran, Ph. alexandri is the first that has been proven to harbour L. infantum in the country.

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.

A rapid, sensitive and specific tool for detection of Leishmania infantum infection in dogs, would be highly desirable, because it would allow control interventions in endemic areas of Zoonotic visceral leishmaniosis (ZVL). In this study, we compared an immunochromatographic dipstick test with direct agglutination test (DAT) for detecting L. infantum infections in dogs from areas of ZVL endemic in Iran. The validity of the dipstick rk39 (Cypress Diagnostic Company, Belgium) for canine visceral leishmaniosis (CVL) was compared with a standard direct agglutination test on 116 clinically suspected dogs and 152 healthy controls from endemic areas of Ardabil and East Azerbaijan provinces, north-western of Iran for 1 year. A sensitivity of 70.9% and specificity of 84.9% were found at a 1:320 cut off titer when DAT confirmed cases were compared with healthy control. As the dipstick rk39 test is rapid, noninvasive and does not require much expertise or elaborate equipment, it can be used for screening and diagnosis of canine visceral leishmaniosis in remote endemic areas.

Canids are natural reservoirs of Leishmania infantum and have been promoted as experimental hosts to decipher the pathogenesis of human visceral leishmaniasis (VL). In this study, the presence of IgG antibodies as well as the presence of mononuclear leukocytes reactive to different cysteine proteinases (CPs) were examined in 13 L. infantum-infected dogs (six with symptoms, seven asymptomatic). Cysteine proteinases which belong to papain-like enzymes known as clan CA are the most studied CPs of parasite protozoa. These molecules are expressed by the intracellular stages of the parasite and could be immunogenic. We studied Type II CP (CPA) and Type I CP (CPB) with its long C-terminal extension (CTE) which could be highly immunogenic. We showed that the level of antibodies reactive to rCPA is low in both symptomatic and asymptomatic dogs. In contrast, when CPB and CTE were used as antigens, the level of total IgG (with IgG2 superior to IgG1) reached higher values in asymptomatic dogs than in dogs with VL. While the peripheral blood mononuclear cell (PBMC) reactivity was significant when cultured in the presence of freezed/thawed (F/T) lysate, it remained low in presence of CP although always higher for PBMC recovered from asymptomatic dogs. We showed the importance of CPB and CTE in particular as a target of immune response and their potential use for serodiagnosis in asymptomatic dogs.

In this study, the mature domains of type I (CPB) and type II (CPA) cysteine proteinases (CPs) of Leishmania infantum were expressed and their immunogenic properties defined using sera from active and recovered cases of human visceral leishmaniasis and sera from infected dogs. Immunoblotting and ELISA analysis indicated that a freeze/thaw extract of parasite antigens showed similar and intensive recognition in both active cases of human and dog sera but lower recognition in recovered human individuals. The total IgG of actively infected human sera was higher than in recovered cases when rCPs were used as antigen. In contrast to dog sera, both active and recovered human cases have higher recognition toward rCPB than rCPA. Furthermore, the asymptomatic dogs in contrast to the symptomatic cases exhibited specific lymphocyte proliferation to both crude antigens and rCPs.

One explanation proposed for the widespread failure to control zoonotic visceral leishmaniasis by culling infected domestic dogs is that wild canids or humans play significant roles in transmission. The aim of this study was to determine the importance of domestic dogs as the reservoir hosts of visceral leishmaniasis in northwest Iran. A random sample of 3,872 children and 199 dogs in 38 villages was surveyed by the direct agglutination test. Dog ownership details among these households were collected by questionnaire. Parasites isolated from 16 patients and 12 dogs were characterized as Leishmania infantum MON-1. Average seroprevalence in dogs (21.6%) was much higher than in children (7%). Child seropositivity increased significantly with village dog density in absolute terms (P < 0.001) and in relation to dog/human ratios (P = 0.028). Dog ownership within villages also was a significant risk factor for child seropositivity (P = 0.003).

Background: Mediterranean visceral leishmaniasis (MVL) is an infectious disease that affects both human and animals. Domestic dogs (Canis familiaris) are principal reservoir hosts of MVL caused by Leishmania infantum. Dogs are definitive hosts for Neospora caninum and a risk factor for infecting intermediate hosts. The immunosuppression caused by visceral leishmaniasis (VL) can promote the occurrence of co-infections with other agents such as neosporosis. This study aimed to determine the frequency of co-infection of the both protozoan parasites in the endemic areas of VL from Meshkin-Shahr District, north-west of Iran. Methods: Altogether, 171 serum samples were collected from domestic dogs of Meshkin- Shahr District by multistage cluster sampling from October 2008 to August 2009. The collected serum samples were tested for the detection of simultaneous infection of L. infantum and N. caninum using direct agglutination test (DAT) and indirect ELISA, respectively. Results: Of the 171 domestic dogs, 27 (15.8%) and 52 (30.4%) were showed antibodies against L. infantum and N. caninum, respectively. Simultaneous infections of N. caninum and L. infantum was found in 16 (9.4%) of the dogs. In VL-positive and VL-negative dogs, N. caninum infection was found in 59.3% and 25.0%, respectively. A statistically significant difference was found between VL-positive and VL-negative dogs with N. caninum infection (P= 0.001). Conclusion: These findings indicate that Meshkin-Shahr District in northwestern Iran is an active focus of canine visceral leishmaniasis (CVL). Neospora caninum and L. infantum co-infection is prevalent in the area and infection by L. infantum seems to enhance susceptibility to N. caninum infection in domestic dogs. [ABSTRACT FROM AUTHOR]

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 fortynine 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. [ABSTRACT FROM AUTHOR]