Loop mediated isothermal amplification (LAMP) of Theileria annulata DNA

In the past three decades, as an alternative to PCR (polymerase chain reaction) new diagnostic techniques like LAMP (loop mediated isothermal amplification) whereby target DNA can be amplified under isothermal conditions without using thermocycler have been developed. The LAMP method allows the synthesis of large amounts of DNA in a short time with high specificity and rapid and easy detection of generated products. In this study, specificity and sensitivity of LAMP method was evaluated for the detection of T. annulata in acute infected and/or carriers cattle using primer pair specifically designed to amplify merozoite surface antigen gene (Mero1), 30 kDa major merozoite surface antigen gene (Tams-1) and cytochrome b gene of T.annulata. Primer pairs with highest sensitivity were used to evaluate the applicability of LAMP to the field samples. Two LAMP primers (CYTOB1 and CYTOB341) targeting cytochrome b gene specifically amplified DNA of different T. annulata isolates successfully while no amplification was seen in other species DNAs and BL20. CYTOB1 primers detected T. annulata Ankara / D7 DNA up to 2 fg, however the detection limit of CYTOB341 was 10 fold lower. The sensitivity of CYTOB1 LAMP assay was same with F3/B3 PCR, however when compared with that of cytob1 PCR a 10 fold lower sensitivity was found. The LAMP product was confirmed by restriction digestion and sequencing. Results obtained from this study indicated that none of the designed primer pairs specific to target genes (Tams-1 and Mero1), except cytochrome b gene was able to specifically and sensitively detect different isolates of T. annulata. Consequently, it was shown that LAMP method using CYTOB1 primers is less effective than the cytob1 PCR in terms of detecting T. annulata in the field sample

High genetic diversity and differentiation of the babesia ovis population in Turkey

Babesia ovis is a tick‐transmitted protozoan haemoparasite causing ovine babesiosis in sheep and goats leading to considerable economic loss in Turkey and neighbouring countries. There are no vaccines available, therapeutic drugs leave toxic residues in meat and milk, and tick vector control entails environmental risks. A panel of eight mini‐ and micro‐satellite marker loci was developed and applied to study genetic diversity and substructuring of B. ovis from western, central and eastern Turkey. A high genetic diversity (He = 0.799) was found for the sample of overall B. ovis population (n = 107) analyzed. Principle component analysis (PCoA) revealed the existence of three parasite subpopulations: (a) a small subpopulation of isolates from Aydin, western Turkey; (b) a second cluster predominantly generated by isolates from western Turkey; and (c) a third cluster predominantly formed by isolates from central and eastern Turkey. Two B. ovis isolates from Israel included in the analysis clustered with isolates from central and eastern Turkey. This finding strongly suggests substructuring of a major Turkish population into western versus central–eastern subpopulations, while the additional smaller B. ovis population found in Aydin could have been introduced, more recently, to Turkey. STRUCTURE analysis suggests a limited exchange of parasite strains between the western and the central–eastern regions and vice versa, possibly due to limited trading of sheep. Importantly, evidence for recombinant genotypes was obtained in regionally interchanged parasite isolates. Important climatic differences between the western and the central/eastern region, with average yearly temperatures of 21°C versus 15°C, correspond with the identified geographical substructuring. We hypothesize that the different climatic conditions may result in variation in the activity of subpopulations of Rhipicephalus spp. tick vectors, which, in turn, could selectively maintain and transmit different parasite populations. These findings may have important implications for vaccine development and the spread of drug resistance.Instituto de PatobiologíaFil: Mira, Anabela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Unlu, Ahmet Hakan. Van Yuzuncu Yil University. Vocational School of Gevas; TurquíaFil: Bilgic, Huseyin Bilgin. Aydin Adnan Menderes University. Faculty of Veterinary Medicine. Department of Parasitology; TurquíaFil: Bakirci, Serkan. Aydin Adnan Menderes University. Faculty of Veterinary Medicine. Department of Parasitology; TurquíaFil: Hacilarlioglu, Selin. Aydin Adnan Menderes University. Faculty of Veterinary Medicine. Department of Parasitology; TurquíaFil: Karagenc, Tulin. Aydin Adnan Menderes University. Faculty of Veterinary Medicine. Department of Parasitology; TurquíaFil: Carletti, Tamara. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; ArgentinaFil: Weir, William. Universityof Glasgow. College of Medical, Veterinary and Life Sciences; Reino UnidoFil: Shiels, Brian. Universityof Glasgow. College of Medical, Veterinary and Life Sciences; Reino UnidoFil: Shkap, Varda. Kimron Veterinary Institute. Division of Parasitology; IsraelFil: Aktas, Munir. Firat University. Faculty of Veterinary Medicine. Department of Parasitology; TurquíaFil: Florin-Christensen, Monica. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiologia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Schnittger, Leonhard. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiologia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Multifunctional 3D printing of heterogeneous hydrogel structures

Multimaterial additive manufacturing or three-dimensional (3D) printing of hydrogel structures provides the opportunity to engineer geometrically dependent functionalities. However, current fabrication methods are mostly limited to one type of material or only provide one type of functionality. In this paper, we report a novel method of multimaterial deposition of hydrogel structures based on an aspiration-on-demand protocol, in which the constitutive multimaterial segments of extruded filaments were first assembled in liquid state by sequential aspiration of inks into a glass capillary, followed by in situ gel formation. We printed different patterned objects with varying chemical, electrical, mechanical, and biological properties by tuning process and material related parameters, to demonstrate the abilities of this method in producing heterogeneous and multi-functional hydrogel structures. Our results show the potential of proposed method in producing heterogeneous objects with spatially controlled functionalities while preserving structural integrity at the switching interface between different segments. We anticipate that this method would introduce new opportunities in multimaterial additive manufacturing of hydrogels for diverse applications such as biosensors, flexible electronics, tissue engineering and organ printing

Identification of candidate transmission-blocking antigen genes in Theileria annulata and related vector-borne apicomplexan parasites

Background: Vector-borne apicomplexan parasites are a major cause of mortality and morbidity to humans and livestock globally. The most important disease syndromes caused by these parasites are malaria, babesiosis and theileriosis. Strategies for control often target parasite stages in the mammalian host that cause disease, but this can result in reservoir infections that promote pathogen transmission and generate economic loss. Optimal control strategies should protect against clinical disease, block transmission and be applicable across related genera of parasites. We have used bioinformatics and transcriptomics to screen for transmission-blocking candidate antigens in the tick-borne apicomplexan parasite, Theileria annulata. Results: A number of candidate antigen genes were identified which encoded amino acid domains that are conserved across vector-borne Apicomplexa (Babesia, Plasmodium and Theileria), including the Pfs48/45 6-cys domain and a novel cysteine-rich domain. Expression profiling confirmed that selected candidate genes are expressed by life cycle stages within infected ticks. Additionally, putative B cell epitopes were identified in the T. annulata gene sequences encoding the 6-cys and cysteine rich domains, in a gene encoding a putative papain-family cysteine peptidase, with similarity to the Plasmodium SERA family, and the gene encoding the T. annulata major merozoite/piroplasm surface antigen, Tams1. Conclusions: Candidate genes were identified that encode proteins with similarity to known transmission blocking candidates in related parasites, while one is a novel candidate conserved across vector-borne apicomplexans and has a potential role in the sexual phase of the life cycle. The results indicate that a ‘One Health’ approach could be utilised to develop a transmission-blocking strategy effective against vector-borne apicomplexan parasites of animals and humans

Gerbils, As Experimental Animals (Meriones unguiculatus): Is A Good Role Model for Leishmania major?

Amaç: Bu çalışma, deneysel olarak enfekte edilen gerbillerde (Meriones unguiculatus), klasik tanıdaki smear örnekleri yanında zenginleştirilmiş Novy-MacNeal-Nicolle (NNN) besiyerine ekim ve Polimeraz Zincir Reaksiyonu (PZR) testi kullanılarak, Leishmania major amastigotlarının yayılabilme ve visseralize olabilme olasılıklarının gözlenmesi amacı ile gerçekleştirilmiştir.Yöntemler: Çalışmada kullanılan L.major suşu Bitlis ilimiz kırsalında yaşayan ve 18 yaşında olan erkek hastadan izole edilmiştir. Gerbillerin enfeksiyonu için de bu suştan faydalanılmıştır. 10 adet gerbile toplam 1×108/mL dozda promastigot inokule edilmiştir. Enfeksiyonun visseralize olup olmadığı yönünden incelenmesi amacıyla hayvanlara ötenazi uygulanarak nekropsileri yapılmıştır. Nekropside tüm iç organlardan preparatlar hazırlanarak Giemsa ile boyanmış ve promastigot gelişimi için NNN besiyerine ekimler yapılmıştır. Bununla birlikte enfeksiyonu doğrulamak için gerçek zamanlı PZR testi uygulanmıştır.Bulgular: Enfeksiyondan sonraki ilk ayda, L. major ile enfekte edilmiş hayvanlardan alınan ve Giemsa ile boyanan doku örneklerinin muayenesinde, tüm dokularda (karaciğer, dalak, akciğer, böbrek, kalp, testis), Leishmania amastigotlarına rastlanılmış ve yapılan NNN besiyeri ekimlerinde 26°C'de Leishmania promastigotlarının ürediği görülmüştür. Leishmania parazitlerinin ribozomal internal transcribed spacer 1 (ITS1) bölgesine özgü tasarlanan primer ve problar ile gerçek zamanlı PZR testi yardımıyla gerbillerden alınan örneklerin L. major parazitine uygun erime eğrisi gösterdiği görülmüştür.Sonuç: Çalışma neticesinde, Zoonotik Kutanöz Leishmaniasis etkeni olan L.major'un gerbillerde visseralize olduğu hem giemsa ile boyalı preparatlarda, hem de PZR ile doğrulanmıştır.Objective: This study aimed to observation the possible visceralization tendency and dissemination of L. major amastigotes in gerbils (Mer-iones unguiculatus) using a classic smear technique, inoculated into enriched Novy-MacNeal-Nicolle (NNN) culture and polymerase chain reaction (PCR) assay for diagnosis of infection.Methods: In this study, L. major isolated from a man who 18 years old, living in Bitlis province of Turkey. This strain also was utilized to infect gerbils. A total of 1×108/mL promastigotes were inoculated to 10 gerbils. Necropsy was performed on infected gerbils for monitoring the visceralization tendency of the parasites. Tissue samples were prepared from each animal and stained by Giemsa and inoculated into NNN culture. However, a real-time PCR assay was performed to confirm the infection the clinical material. Results: Examination of Giemsa-stained tissue smears showed that infected animals with L.major were positive for Leishmania amastigotes in all tissues at the first month post infection and Leishmania promastigotes were cultured at 26°C in culture flasks containing NNN. Melting curve analyses of ribozomal internal transcribed spacer 1 (ITS-1) PCR showed the peak concordant with L. major.Conclusion: As a result, the present study confirmed by both Giemsa-stained smears and PCR, visceralization and dissemination of L. major amastigotes, the principal cause of zoonotic cutaneous leishmaniasis in gerbils

Parasites Detected by Examination of Fecal Samples in Wrestling Camels

First historical findings on camel wrestling, which is now practiced as a festival in Turkey, particularly in certain regions (Marmara, Aegean, Mediterranean) date back to the 15th century. In terms of animal husbandry, parasitic diseases may result in negative outcomes ranging from loss of performance to death for camels. In the present study, annual camel wrestling arenas were visited between December and March (2010-2011), and stool samples were collected from camels from different cities for parasitological analysis. Stool samples of 109 camels from 7 different cities (Aydin, Izmir, Manisa, Denizli, Mugla, Balikesir, and Canakkale) were examined using Baermann-Wetzel stool culture, flotation, and sedimentation techniques for the parasites that live in gastrointestinal tract. The analyses revealed that 74% of the camels (81 of 109) were infected with one or more parasites: Trichostrongylus spp. (47.7%), Ostertagia spp. (27.5%), Dicrocoelium spp. (24.7%), Trichuris spp. (11.9%), Eimeria cameli (11.9%), Capillaria spp. (6.4%), Fasciola spp. (6.4%), Dictyocaulus viviparous (5.5%), Haemonchus spp. (4.5%), Oesophagostomum spp. (4.5%), Cooperia spp. (4.5%), Cooperia oncophora (3.6%), Nematodirus spp. (3.6%), Chabertia ovina (2.7%), Eimeria spp. (1.8%), and Paramphistomum spp. (0.9%). 16 different parasites, at the level of species and genus, were found, of which 14 were helminth (11 nematodes, 3 trematodes), and 2 were protozoans. The present study was the first to report Ostertagia spp., Fasciola spp. Dictyocaulus viviparus, Haemonchus spp., Oesophagostomum spp., Cooperia spp., Cooperia oncophora, Chabertia ovina and Paramphistomum spp. in camels in Turkey. As high as 74 percent of the incidence of parasitic diseases and the wide variety of parasites found in the present study suggest that parasitic infections may be overlooked entity in wrestling camels that are meticulously brought up