Background: The aim of this review was to describe the application of molecular methods in epidemiological aspects of malaria vectors, parasites, and human hosts in Iran and their critical role in malaria control and elimination programs.

Methods: Medline, EMBASE, Web of Science, Scopus, and Google Scholar databases were searched systematically for original published papers on PCR, the molecular identification of malaria vectors, the molecular epidemiology of malaria, insecticide resistance, and drug-resistant parasites, in Iran. In total, 51 studies on molecular entomology and 36 studies on molecular parasitology of malaria and three on human host were selected.

Results: Molecular methods are essential for improving the detection of malaria infection and monitoring antimalarial drugs and insecticide resistance in malaria elimination settings such as Iran.

Conclusion: The application of molecular methods may be of particular interest for malaria control/elimination programs, for monitoring progress towards malaria elimination, and for optimal orientation of program activities.

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.

Background: This study aimed to investigate the anti-Acanthamoeba effects of the most used marketed disinfecting solutions in Iran. Moreover, the efficacy of some nano-compounds was tested against pathogenic Acanthamoeba.

Methods: The present study was conducted in the School of Public Health, Tehran University of Medical sciences, Tehran, Iran during 2015-2016.  Cysts of Acanthamoeba T4 genotype (7 x 10⁴ /ml) mixed at the equal volume with contact lens solutions including Opti-free, Ginza, ReNu, Maxima, Light, and Cyclean for the recommended time by the manufacturers. Nano-silver and nano-gold compounds were also treated with the amoebae. Chlorhexidine 0.02% and normal saline were used as positive and negative controls, respectively. Dead and alive amoebae were determined using vital stain and suspension was cultured in non-nutrient agar. The entire process was repeated at least three times.

Results: In none of the solutions in the manufacturer's brochure recommended time, full cytotoxic effect was observed on the cysts of Acanthamoeba. Opti free express solution destroyed the cysts after 6 days. Nanosilver and nano-gold compounds showed no cytotoxic effect on the cysts of Acanthamoeba.

Conclusion: None of the Nanoparticles compounds as well as contact lenses disinfecting solutions which studied was effective on Acanthamoeba cysts in the manufacturer's brochure recommended time. However, continuing study on Nano-silver and Nano-gold compounds to find effective ingredients against Acanthamoeba are highly recommended.

Background: Cutaneous leishmaniasis (CL) is one of the world health problems. Leishmania major is the etiological agent of zoonotic cutaneous leishmaniasis (ZCL). Promastigote and amastigote are two morphological forms of Leishmania parasites that express different proteins and p27 is an important gene encoding cytochrome c oxidase (COX) component. P27 gene expresses a 27 kDa protein that essential in ATP synthesis. This study aimed to compare p27 gene expression in promastigote and amastigote forms in Iranian strain of L. major (MRHO/IR/75/ER).

Methods: This study was conducted in 2015. Clinical isolates of CL patients from north, center, west and south parts of Iran were collected and identified by PCR-RFLP. After RNA extraction of promastigotes and amastigotes and cDNA synthesis, the expression level of p27 gene was compared by real-time RT-PCR.

Results: By comparison of expression level between amastigote and promastigote forms of Iranian strain of L. major, up-regulation of p27 gene (2.73 fold) was observed in amastigotes. Moreover, there was no significant difference in p27 gene expression between L. major isolates.

Conclusion: p27 gene and protein can be considered as a target in recombinant vaccine production and treatment process.

Background: Despite the global efforts to control schistosomiasis, still prevalence in endemic regions unchanged. The present study was conducted to investigate the possible role of artesunate (AS) and praziquantel (PZQ) combination in enhancing cure in pre-patent and patent Schistosoma mansoni infection, and study the role of apoptosis in evaluation of the drugs efficacy.

Methods: Eighty laboratory-bred Swiss albino male mice were classified into four groups (20 mice each); control, PZQ treated (500 mg/kg), AS treated (400 mg/kg) and combined AS (400 mg/kg) + PZQ (500 mg/g) groups. Efficacy of the drugs was assessed by parasitological (egg count/gram stool, worm burden, tissue egg load, oogram pattern), histopathological (haematoxylin and eosin –for detection of type of hepatic granulomas, number & diameter) and immunohistochemical studies (P53 and Bcl-2 markers for determination of inflammatory cells and the degree of apoptosis).

Results: Significant reduction was recorded in stool egg count, tissue egg count (liver and intestine), worm burden, granuloma number and size and changed oogram patterns in artesunate -praziquantel combined group followed by artesunate monotherapy group. There was a significant increase in the apoptotic proteins P53 and slight increase in anti-apoptotic proteins Bcl-2 in the infected group compared to the control healthy group. A significant decrease and increase in P53 & Bcl-2 expressions respectively were observed in artesunate – praziquantel combined group compared to control infected group.

Conclusion: artesunate-praziquantel combination is a potential upcoming chemotherapy for schistosomiasis mansoni. Both Bcl-2 and P53 are good markers assessing S. mansoni apoptosis, morbidity and chemotherapy efficacy.

Background: Currently, most of the available serological diagnostic kits for strongyloidiasis are based on the use of the crude antigens of Strongyloides ratti, which are good, but with less sensitivity towards the infection. Hence, this study aimed to produce and evaluate monoclonal antibody for detecting soluble parasite antigen in animal sera.

Methods: The study was conducted in the Department of Medical Microbiology and Parasitology, University Putra Malaysia in 2014-2017. Saline extract protein from the infective larvae of S. ratti was used to immunize BALB/c mice and subsequent fusion of the B-cells with myeloma cells (SP2/0) using 50% PEG. The hybridomas were cultured in HAT medium and cloned by limiting dilutions. Positive hybrids were screened by indirect ELISA. The ascites fluid from the antibody-secreting hybridoma was purified and the MAb was characterized by western-blots and evaluated in sandwich ELISA for reactivity against the homologous and heterologous antigens.

Results: An IgG1 that recognizes a 30 and 34 kDa protein bands was obtained. The MAb was recognized by all S. ratti-related antigens and cross-reacted with only Toxocara canis antigens in both assays. The minimum antigen detection limit was found to be 5 ng/ml. All antibody-positive rat and dog sera evaluated have shown antigen-positive reactions in Sandwich-ELISA.

Conclusion: The MAb produced, was able to detect antigens in strongyloidiasis and toxocariasis in animal models and may also be useful for the serological detection of active strongyloidiasis and visceral toxocariasis in human sera.

Background: Toxoplasma gondii is a widely prevalent intracellular protozoan parasite which causes serious clinical and veterinary problems. Development of an effective vaccine for controlling toxoplasmosis is an extremely important aim. In the present study, the protective efficacy of recombinant multiepitope antigen (USM.TOXO1) expressing nine potential epitopes identified from SAG1, GRA2, and GRA7 of Toxoplasma gondii was evaluated in BALB/c mice.

Methods: Mice were immunized subcutaneously with three doses of USM.TOXO1 antigen (10 µg/ml). Following the immunization, the IgG antibody, IgG subclass, IFN-γ and IL-4 production were evaluated using ELISA, the study was conducted at Animal Research and Service Center (ARASC), USM Health Campus in 2016.

Results: Mice immunized with USM.TOXO1 significantly induced a mixed Th1/Th2 response polarized toward the IgG1 antibody isotype. While the cytokine analysis revealed a significant release of IFN-γ cytokines.

Conclusion: USM.TOXO1 is a potential vaccine candidate that elicits strong immunity in BALB/c mice. The proven immunogenicity of the generated antigen can serve as a premise for further use of epitope-based vaccine in the immunoprevention of human and animal toxoplasmosis.

Background: Theileria annulata is an economically important cattle disease in North Africa that occurs in subtropical and tropical areas. Accurate and rapid, molecular diagnosis of tropical theileriosis is an important issue that allows early treatment and, prevents transmission. We developed and validated a Theileria annulata specific LAMP assay targeting the cytochrome b multicopy gene, in order to increase the DNA detection sensitivity.

Methods: The methodology was used to evaluate the occurrences of T. annulata in 88 field samples collected in Northern Tunisia during 2013-2014. The specificity and sensitivity of the LAMP assays were compared to conventional cytochrome b PCR and routine microscopy commonly used on naturally infected cattle blood samples.

Results: The PCR assay showed a sensitivity of 70% and specificity around 75%. Our LAMP assay showed a suitable sensitivity 78.7% and specificity 87.5%, with, however, positive (98.4%) and negative (29.1%) predictive values.

Conclusion: The LAMP assay is a simple and convenient diagnostic tool for tropical theileriosis. Moreover, LAMP does not require experienced staff and specialized equipment for sampling procedures and it is practical outside laboratories and can be used for field diagnosis.

Background: We aimed to construct an adenovirus expressing a cross-reactive fragment of the apical membrane antigen 1 (AMA1) antigen and evaluated the concomitant immune response in BABL/c mice, allowing protection against N. caninum and T. gondii infection.

Methods: The study was conducted in Agricultural College of Yanbian University, Yanji, Jilin, China In 2015-2016. Primers were designed using the AMA1 gene sequences of N. caninum (AB265823.1) and T. gondii (AF010264.1). After linearization of the plasmid ADV4-NcAMA1 and the framework plasmid pacAd5, a total of 293T cells were co-transfected and Ad5-NcAMA1 recombinant adenovirus were packed. BALB/c mice were inoculated. Simultaneously serum IgG antibody levels and IFN-γand IL-4 cytokine levels were determined by ELISA. After immunization three times in two weeks, each group of BABL/c mice were divided into two groups, respectively given intraperitoneal inoculation by the Neospora tachyzoite and Toxoplasma tachyzoite. Then we observed the clinical symptoms and statistical survival rate of mice.

Results: The level of IgG in BABL/c mice immunized with Ad5-NcAMA1 was significantly increased when compared with that of pVAX1-NcAMA1 and PBS groups (P<0.01). At the same time, the cytokine levels of IFN-γ and IL-4 were also higher in the Ad4-NcAMA1 group than in the control groups (P<0.01). Moreover, BABL/c mice immunized with Ad5-NcAMA1, pVAX1-NcAMA1, and PBS showed survival rates of 75%, 45% and 20% after N. caninum infection, and 45%, 10% and 0% after T. gondii infection, respectively.

Conclusion: The adenovirus vaccineAd5-NcAMA1 could provide protective immunity against N. caninum and T. gondii infection.

Background: Chrysomela (=Melasoma) populi is one of the most serious pests on poplar plantations. In the present study, a microsporidian pathogen, Nosema melasomae infecting Crysomela populi is re-recorded from a new geographical locality and its spore ultrastructure is given for the first time.

Methods: Larvae and adults of C. populi were dissected in Ringer’s solution and prepared wet smears were examined under a microscope. Detected fresh and stained spores were measured and photographed using an Olympus BX51 microscope with a DP-25 digital camera and a DP2-BSW Soft Imaging System. The ultrastructure of the pathogen was studied with a Philips EM 208 transmission electron microscope using standard preparation techniques as previously described

Results: Fresh spores of the microsporidian pathogen are elongate, 4.86 ± 0.71 µm in length and 1.64 ± 0.19 µm in width. The spore wall is considerable thin, measured 60 to 100 nm and consists of a clear endospore (40 to 80 nm) and an electron-dense, uniform exospore (15 to 30 nm). The polar filament is isofilar and has only 6-8 coils. Nuclei in the cell are 400-560 nm in diameter. The polaroplast has a thin lamellated structure.

Conclusion: The pathogen from C. populi is N. melasomae Sidor & Jodal, 1986 and its systematic position given by Sidor and Jodal. The spore ultrastructure of N. melasomae differs from those of other microsporidia infecting chrysomelids.

Background: Intestinal parasites are the most common causes of gastrointestinal disease in canine. Stray dogs and wild candies can represent potential reservoirs of enteropathogens to other hosts. Therefore, present study determined the prevalence of intestinal parasites in canine in Mazandaran Province, northern, Iran.

Methods: Overall, 58 small intestinal samples of animals (42 stray dogs and 16 jackals) were collected from Oct 2012 to Dec 2013. The intestine contents were studied to detect and identify helminth infections. Then, the helminths were collected and their morphological traits were identified.

Results: Overall among infected stray dogs and jackals, 11 species were found. Three species of nematodes, seven species of cestodes and one trematode were observed. The prevalence of gastrointestinal helminths of stray dogs and jackals were 59.5% and 50%, respectively. Among registered zoonotic helminths A. caninum was the predominant parasite both stray dogs and jackals. Interestingly, Spirometra spp. was reported in these animals. Moreover, A. caninum showed a higher percentage rate in center region of province.

Background: Avian haemosporidians are able to parasitize numerous bird species all over the world. The extensive range of blood parasites infection rate is between 50% and 100% or less percentage. Haemoparasites with major effects on physiology, ecology, health, population dynamics, sexual selection and production success of avian hosts may promote species extinction.

Methods: To evaluate haemosporidians infection rate in Iranian birds, 136 individuals were examined by microscopic observation of stained blood smears under light microscope. These samples belonged to 10 different families of Songbirds from the east of Iran from April to August 2014-2016.

Results: Fifty-one passerine birds were detected as harboring Haemoproteus spp. Furthermore, we recorded Haemoproteus spp. infection of Granativora bruniceps, Oenanthe pleschanka for the first time in the world and eight more species for Iran.

Conclusion: Age and sampling localities do not influence the infection rate of Haemoproteus spp. from the eastern provinces of Iran. The relative high infection of avian haematozoa revealed this region might provide suitable sites for future studies on these parasites and the relationship with their hosts and vectors.

Background: Climate condition is expected to have significant in rodents’ diversity and in the seasonal pattern of diseases carried by different rodents. In an effort to aid in the study of the biodiversity of parasites of rodents in different climate zoon we examined climate patterns in the parasite assemblages of different rodents from Mar 2015 to Feb 2016.

Methods: Of 253 captured rodents in three climate zone of Iran, thirteen species of rodents were recognized. Rodents included Mus musculus, Microtus, Apodemus witherbyi, Calomyscus elburzensis, Meriones libycus, Tatera indica, Alactaga elater, and Arvicola amphibius. Trapped rodents humanely sacrificed and the gastrointestinal and respiratory tracts were removed and examined to identify parasitic helminths. Parasites were identified using key morphological characteristics.

Results: Of 253 rodents examined, 109 (43.08%) were positive for helminth infection including Syphacia obvelata (20.1%), Aspicularis tetraptera (9.9%), Trichuris muris (0.3%), Capillaria sp. (0.3%), Physaloptera sp. (0.7%), Gongylonema sp. (1.1%),  Nippostrongylus brasiliensis (6.7%) Heligmosomoides polygyrus (4.3%) Hymenolepis diminuta (3.1%), H. nana (0.8%), Cysticercus fasciolaris, (2.7%), Mesocestoides sp. larva (0.3%) and Moniliformis moniliformis (0.3%). Notocotylus neyrai was the only species of Trematoda isolated from water vole (Arvicola amphibius) for the first time in Iran.

Background: “Cerithidea cingulata” is reported from south of Iran, may act as intermediate host of Heterophyes heterophyes and cercaria dermatitis. As parasitological aspects of this brackish snail were not studied in Iran, this study was conducted in 2016, in Hormoz Strait, south of Iran.

Methods: Totally 402 snails were collected from 36 locations of three main regions in Hormozgan Province, Iran in 2015. In each location, one square meter was checked, and snails were collected for parasitological study. Snails were crashed and cercariae were studied using light microscope, in some cases natural red staining was used for better resolution.

Results: Mean length of snails was about 20.33 mm, width 5.57 mm. The aperture length was 5.10 and spire was 15.22. Important founded cercaria was cysticercus cercariae, echinostoma cercaria, furcocercus cercaria, furcocystocercous cercaria, gastrostomy cercaria, gymnocephalus cercaria, monostome cercaria, pelurolophocercus cercaria and xiphidiocercaria.

Background: Although carmine, as a synthetic dye, is the major substance for staining helminths, it may impose some adverse effects on human health. In the present study, we evaluated the aqueous extracts of onion (Allium cepa) skin, walnut (Juglans regia) husk, and madder (Rubia tinctorum) roots as potential herbal dyes for staining parasitic helminths.

Methods: Aqueous solutions (5%, 10% and 20%, w/v) of each herbal prepared from dried and powdered husk of walnut, skins of red onion, and madder roots in distilled water. Parasitic helminths including Fasciola spp., Dicrocoelium denderiticum, Echinococcus gronulosus protoscolices, Moniezia spp., and Haemonchus contortus were stained by different concentrations of herbal dyes according to carmine staining method. The structural clarity and quality of stained internal organs of the helminths such as suckers, intestine, and reproductive systems were scored by semi-quantitative evaluation in comparison with carmine stained samples.

Results: The optimum concentrations of extracts for helminths staining were 10% (w/v) of A. cepa and J. regia, and 20% (w/v) of R. tinctorum with final scores of 3.1, 3 and 2.8, respectively. In general, A. cepa and J. regia extracts showed higher quality in staining Platyhelminthes, while R. tinctorum extract presented relatively higher quality in staining Nematoda.

Conclusion: Considering proper quality of A. cepa, J. regia and R. tinctorum extracts in staining the helminths, they may be safe, eco-friendly, and inexpensive alternatives to carmine dye.

Background: Encephalitozoon cuniculi is an opportunistic microsporidian parasite that can affect a number of different species of mammalian animals and humans. The parasite can pose also threat for rabbits even though it causes several sporadic and asymptomatic infections. Infection of eyes is common and clinical symptom of ocular infection may include uveitis and cataracts. We found out subacute findings in naturally infected animals and show here a first described eye lesions as well as central nervous system and kidneys in Turkey.

Methods: The rabbits (n:171) of breeding units were observed to daily clinical examination for infection of E. cuniculi during three years. The eyes of five rabbits (2.9%) showed white intraocular masses or cataracts in the breeding units during daily examinations. The infection was described clinicopathologically in collected organ samples in the animals. During observation, macroscopically, corneal lesions and opacity and impaired lens were taken into attention as well as hyperemia in central nervous system and kidney. Histopathologically, parasitophorous vacuoles pertaining to E. cuniculi were detected in all three tissues during different routine Haematoxylin-Eosin and Gram stainings.

Results: Degenerative and necrotic changes in epithelium of cornea and lens and also neurons and tubules were predominantly observed in addition to nonpurulent interstitiel nephritis and encephalitis.

Conclusion: The results from study lead to subacute findings especially in eye during natural E. cuniculi infections following asymptomatic and latent changes among breeding colony. The lesions indicated sub-acute stage of E. cuniculi infection in eye lesions of rabbit in Turkey.

Background: Human hydatid disease imposes significant impacts on public health by producing substantial morbidity and mortality in involved communities. This study aimed to evaluate the epidemiology and clinical features of hydatid cyst in northern Iran as a breeding center for the infection.

Methods: In this cross-sectional study, the hospital records of all hydatidosis-affected patients admitted in three teaching hospitals of Mazandaran Province between Mar 2005-2015 were reviewed. Hydatidosis-relevant demographic characteristics, clinical findings, and laboratory data were collected. The descriptive statistical analysis was performed by SPSS software.

Results: Totally, 79 patients with the mean age of 42.00±23.82 yr were admitted with cystic echinococcosis (CE) diagnosis. Moreover, the highest and the lowest prevalence of CE cases were in age ranges of 50-59 (19.0%) and more than 80 (5.0%) yr, respectively. Male/female ratio was 0.88 (47.0% vs. 53.0%). Majority of the cases were urban residents (54.0%) and had no close contact with animals (58.0%). Nearly, two third of the patients (n=54), the affected organ was liver. The diameter of the cysts was variable from 2 to 15 cm. Most of the patients had a single hydatid cyst. Four patients were diagnosed as secondary hydatid cyst. Medical treatment with antiparasitic agents was done for 47 individuals and in 7 cases; it was the only treatment approach. Percutaneous puncture-aspiration-injection reinjection (PAIR) technique was applied for 6 cases. Sixty-six patients underwent radical surgery. No data was available on eosinophil count or serological tests.

Conclusion: CE is approximately prevalent in Iranian population. Development of new diagnostic methods and therapeutic procedures is worthy. Moreover, it is necessary to design and develop a registry and surveillance system by a multidisciplinary team.

Background: Opisthorchis viverrini is a serious health problem in Southeast Asia. The infection is associated with cholangiocarcinoma. Therefore, this study was aimed to detect O. viverrini infections among the ASEAN population in Thailand.

Methods: A cross-sectional study was conducted among 249 individuals from ASEAN populations in Thailand including Thai, Laotian, Cambodian, and Myanmar. Participants were screened using the O. viverrini verbal screening test (OvVST). Fecal samples were processed by the mini-parasep sf parasite fecal concentrator.

Results: The infection rate of O. viverrini was 27.21%. The majority of infections was detected in females, in the age group 31-40 yr old, in the primary school education level, and in the occupation of labor. By country, O. viverrini infection was detected more often in the Lao PDR (30.77%). In screening for O. viverrini infection, OvVST had a high sensitivity (93.48%), specificity (86.70%), NPV (98.32%), and accuracy (87.95%). The PPV was 61.43% for OvVTS. The observed agreement was substantial for OvVST (k-value = 0.64).