Molecular and morphological characterization of Parabronema skrjabini of sheep and goats at three different geographical zones in Iran

Parabronema skrjabini is a spirurid nematode of the family Habronematidae that lives in the abomasum of ruminants such as sheep and goats. The purpose of this study was to investigate the molecular and morphological aspects of Parabronema skrjabini in sheep and goats in Iran. The worms were collected from these animal species from three different regions. An internal transcribed spacer 2 ribosomal DNA (ITS2-rDNA) fragment of Parabronema skrjabini was amplified by polymerase chain reaction (PCR) using a pair of specific primers (Para-Ir-R and Para-Ir-F). Morphological studies based on the body length, the frontal shield, spicules of male and egg dimensions were performed. ITS2-rDNA sequences were between 167 and 299bp in different isolates. ITS2 homology in different isolates was between 68 % and 77% compared with the sequence data in GenBank. Morphological results showed that the average length of male and female worms in sheep were 16.5mm and 36mm and in goats 16mm and 35.5mm, respectively. The average length of the small and large spicules in sheep were 657.5μm and 304.07μm and in goats 653.08μm and 302.66μm, respectively. To our knowledge, this is the first study in the world exploring the genetic diversity of Parabronema in sheep and goats. Add this sentence in discussion: the low ITS2-rDNA identity in different isolates from Iran as compared to the reference sequence in GenBank (68–77%) raise questions regarding the species identity of the parasites isolated in Iran

Design and construction of a new recombinant fusion protein (2b2t+EPC1) and its assessment for serodiagnosis of cystic echinococcosis

The immunodiagnostic tests for cystic echinococcosis (CE) are mostly serological tests based on ELISA that use hydatid cyst antigens for primary screening because of its simple preparation and availability. The challenge to develop new serological methods (as compared to those based on the hydatid cyst fluid antigens) to meet the gold standard remains. Appropriate sources of antigenic material are necessary for application to improve the efficacy of immunodiagnostic tests at a population level. In the current study, a fusion protein containing the coding sequence of antigen B2t and two sequences of EPC1 antigen with some modifications was reconstructed. Using bioinformatics tools, these sequences were joined together by applying the sequence of a rigid α-helix-forming linker to obtain an appropriate structure of a fusion protein. Synthetic recombinant fusion protein was expressed using pET28a as a vector and evaluated by indirect ELISA test for sera from patients with hepatic CE and other parasitic infections. The sensitivity of the fusion protein was lower (88.46) than the available ELISA kit (96.15). However, the differences in sensitivity were not statistically significant as compared to the recombinant fusion peptide with the commercial kit (p = 0.269). The specificity of the recombinant fusion protein (95.45) was not significantly lower than the commercial kit (96.59; p = 1.000). Moreover, surprisingly there was no difference in the cross-reactivity values of performance between the recombinant-ELISA and commercial kit. The positive and negative predictive values of the recombinant antigen were achieved as 92 and 93.33, respectively, while for the commercial kit, they were obtained as 94.33 and 97.70, respectively. In conclusion, as an early evaluation of these antigens the performance of our recombinant fusion protein in ELISA is relatively promising. Although, it seemed that this peptide with specific antigenic epitopes might be more appropriate for the serological evaluation of CE by use of bioinformatics tools, our findings showed that cross-reactions and a negative reaction could occur in clinical performance. This fusion protein may have utility for diagnosis in humans, but further evaluation is needed using the WHO ultrasound classification for CE. © 2018 APMIS. Published by John Wiley & Sons Lt

Canine microfilaraemia in some regions of Iran

Background: Dirofilaria immitis and Dirofilaria repens are vector-borne zoonotic parasites which affect mainly dogs and humans worldwide. In Iran, information about the distribution of those nematodes is scant in several regions. Therefore, we investigated the prevalence of these filarial parasites in stray dogs from five Iranian provinces where no information about these parasites is available. Methods: Blood samples were collected from 344 stray dogs in five provinces of Iran (i.e. Mazandaran, Gilan, Esfahan, Qazvin and Loresan). The presence of microfilariae was assessed using direct smear, modified Knott’s test, molecular detection of filarial DNA (cox1 gene) and Wolbachia endosymbiont of parasitic nematodes (ftsZ gene) by conventional PCR (cPCR). All of the PCR products were sequenced and phylogenetic analysis was performed. Results: In total, 75 dogs (21.8%) were found to be positive for D. immitis by cPCR. Infection was detected in all provinces, with the highest prevalence in Gilan province (22/28; 78.6%). Acanthocheilonema reconditum was diagnosed in five dogs (1.4%) from three provinces (i.e. Esfahan, Mazandaran, Gilan). Two dogs were infected with both parasites and three were only infected with A. reconditum. Dirofilaria repens infection was not found in the examined population. Representative sequences of the D. immitis cox1 gene from dogs from the northern provinces (Mazandaran, Gilan, Qazvin) were grouped together and distinctly separate from the ones from western and central provinces (Lorestan and Esfahan), suggesting that different nematode populations are present in the country. Conclusion: The data reported herein fill existing gaps in knowledge about canine filarial infection in two Iranian provinces and record the highest prevalence of D. immitis ever reported in the country (i.e. 78.6%). A geographical review of the literature about Dirofilaria spp. and A. reconditum infections in dogs and humans has also been summarized, indicating that D. immitis and D. repens are distributed in 22 of 31 provinces in Iran, whereas A. reconditum is present in fewer regions. Effective control strategies are advocated for owned dogs, and a national program for the management of stray dogs is needed to minimize the risk of infection in animals and humans. Graphical Abstract: [Figure not available: see fulltext.