Identification and differentiation of Fasciola hepatica and F. gigantica using multiplex PCR technique.

We aimed to present an alternate method instead of PCR-RFLP and also develop an optimized method for rapid, time-saving and affordable molecular-based approach to discriminate species of liver fluke, Fasciola hepatica and F. gigantica. Seventy-six samples of F. hepatica and 28 F. gigantica were collected from the slaughterhouses of endemic regions in Iran. Following a comprehensive analysis of the mitochondrial complete sequences of both F. hepatica and F. gigantica, the extracted DNAs from all samples were used as templates in multiplex PCR reactions containing two sets of primers specific for cytochrome c oxidase I (cox I) gene of both species. In a parallel experiment, PCR-RFLP was performed for each sample using internal transcribed spacer (ITS1) sequence. Furthermore, following a PCR amplification for cox I gene, the amplicons were purified for sequencing. To assess the validity of the multiplex PCR approach, the obtained data from the multiplex PCR and PCR-RFLP experiments were compared with each other. By sequence analysis of 104 samples, 76 and 28 samples were identified as F. hepatica and F. gigantica, respectively. Results revealed 100% and 92% of accuracy as for multiplex PCR and PCR-RFLP. The designed multiplex PCR strategy offers a valid alternative approach to the conventional methods with distinctive features including convenience, cost-effectiveness, time-saving (3 hours from sampling to obtain final results) and high efficacy.

In Vitro Study on Four Types of Commercial Lectins on Leishmania infantum, L. major and L. tropica with Stage-Specific Binding and Leishmania Species Identification.

Background: We aimed to verify the susceptibility of Leishmania infantum, L. major and L. tropica, to commercial lectins in order to identify the three Leishmania species. Methods: The degree of agglutination was determined both macroscopically and microscopically and was scored negative (-) to positive (from 1+- 4+) based on their percentage of agglutination. Results: Jacalin and UEA-1 were capable of agglutination of L. infantum isolates in both logarithmic and stationary phases at a concentration of 1000 µg/ml (100%). L. tropica isolates showed agglutination with the lectin UEA-1 in both logarithmic and stationary phases (62.5% and 87.5%). L. major and L. tropica showed 75% agglutination with lectin Jacalin in both logarithmic and stationary phases. L. tropica isolates showed 25% agglutination with the lectin WGA in the logarithmic phase. L. infantum, L. major and L. tropica isolates showed 25, 12.5 and 37.5% agglutination in the stationary phase, however, did not show agglutination in logarithmic phases. L. major isolates showed 12.5% agglutination with the lectin PHA in the stationary phase, however, were incapable of agglutination with the L. tropica and L. infantum in both logarithmic and stationary phases. Conclusion: Despite the fact, that JCA and I-UEA lectins were not able to completely separate L. infantum, L. major and L. tropica. WGA lectin and PHA lectin can help in separating the species of Leishmania parasites.

In vivo anti-leishmanial activity of concocted herbal topical preparation against Leishmania major (MRHO/IR/75/ER).

Cutaneous leishmaniosis (CL) is considered as one of the most important tropical diseases. Herbal therapy is the ideal treatment for CL because of the reduced injection pain, availability, lower cost and non-toxicity effects. The present study aimed to evaluate the in vivo antileishmanial activity of concocted herbal topical preparation (Aloe vera, Perovskia abrotanoides, Nigella sativa, propolis, lavender and olive oil) to evaluate its efficacy against Leishmania major (MRHO/IR/75/ER) in comparison to the gold standard treatment. Following the cause of cutaneous leishmaniosis, the BALB/c mice were divided into three groups, test group (ointment formulation), positive control (Glucantime) and negative control (untreated), respectively, which were treated twice a day for 28 consecutive days. The lesion size and parasite burden were evaluated for in vivo evaluation. The herbal topical ointment was able to significantly decline the lesion progression and reduce parasite burden in mice inoculated with L. major promastigotes in the test group compared with the negative control group (P=<0.001). In mice treated with the formulation, the number of amastigotes significantly decreased (P=<0.001), compared with that in the negative control group. Moreover, comparative features of both treatments showed there was no difference between the herbal-treated and glucantime-treat mice (P=0.63). The herbal topical ointment displayed significant in vivo antileishmanial activities. It may be that using ointment formulation beside other skin repair compounds can be used as an alternative medicine in the treatment and healing of human CL lesions. Further investigations are needed to study the pharmacologic and pharmacokinetics aspects of ointment formulation in the treatment and healing of human CL lesions.

First Report of Passerilepis parina (Cestoda: Hymenolepididae) from Parus major in Iran: A Probable Fatal Case Due to Hyperinfection.

Cestodes are important parasites that can affect the health of humans and wildlife. Among these, the genus Passerilepis is an important endoparasite of Passeriform birds while poorly studied in Iran. During a parasitological field survey in central parts of Iran in 2018, thirty-two cestodes, as an obstructive intertwined mass, recovered from the intestine of a recently dead Parus major (great tit). Morphological characteristics of recovered cestodes were drawn carefully by a camera lucida equipped microscope and identification was carried out using standard keys. All of the collected cestodes were identified as P. parina. In the current study, we recorded P. parina from great tit for the first time in Iran.

A comparative study of nested-PCR and direct agglutination test (DAT) for the detection of Leishmania infantum infection in symptomatic and asymptomatic domestic dogs.

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.