Exposure to Neospora spp. and Besnoitia spp. in wildlife from Israel

[EN] Neosporosis and besnoitiosis, caused by cyst-forming protozoa Neospora caninum and Besnoitia besnoiti, respectively, are parasitic infestations of livestock in Israel. These parasites cause significant economic losses in cattle due to reproductive and productive disorders. Both parasites have been detected in several wild ruminant species throughout other regions of the world, while the existence of a sylvatic life cycle in Israel remains uncertain. Thus, a wide panel of 871 sera from two wild carnivores and nine wild ruminant species were tested. All sera were first analysed by MAT for an initial screening and positive samples were confirmed a posteriori by Western blot. Additionally, a complementary IFAT was used for the detection of antibodies against N. caninum. Neospora antibodies were present in six out of the 11 species investigated, whereas Besnoitia antibodies were undetected. Golden jackal, red fox, addax, Arabian oryx, Persian fallow deer, mouflon, mountain gazelle, Nubian ibex, scimitar horned oryx and water buffalo were seropositive against N. caninum infection by IFAT and/or MAT. Moreover, the presence of Neospora spp.-specific antibodies was confirmed by Western blot in golden jackal (6/189; 3.2%), red fox (1/75; 1.3%), Persian fallow deer (13/232; 5.6%), mouflon (1/15; 16.7%), Nubian ibex (22/55; 40%) and water buffalo (12/18; 66.7%). Addax (1/49) and water buffalo (1/18) were MAT-seropositive against B. besnoiti but were seronegative by Western blot. Hence, Neospora sylvatic cycle is present in Israel and may cross over to a domestic life cycle. In contrast, wildlife species investigated are unlikely to present a risk of transmitting Besnoitia to livestock in IsraelSIPart of this work was supported by a research project from the Spanish Ministry of Science and Innovation (AGL 2010-20561) and by The Israeli Services and Animal Health (Ministry of Agriculture and Rural Development). Daniel Gutiérrez Expósito was financially supported by the Ministry of Science and Innovation (grant no. BES-2011- 043753). The authors would like to thank the rangers of the Israel Nature & Parks Authority for their cooperation. Finally, we also acknowledge Elena Blinder, Ricardo Wolkomirsky and Noam Sugar for their excellent technical assistanc

Chemotherapy of experimental Besnoitia besnoiti infection in rabbits

Rabbits were infected with a bovine strain of Besnoitia besnoiti parasites derived from VERO cell cultures. Oxytetracycline*, given at 30 mg/kg i.m. simultaneously with infection, prevented the development of orchitis. The controls received no treatment. All infected animals showed a transient febrile reaction. It is concluded that oxytetracycline has some therapeutic potential against Besnoitia besnoiti and that rabbits are suitable models for therapeutic trials.The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. Adobe Acrobat XI Pro was used to OCR the text and also for the merging and conversion to the final presentation PDF-format

Bovine besnoitiosis : transfer of colostral antibodies with observations possibly relating to natural transmission of the infection

Colostral antibodies to B. besnoiti were detected by immunofluorescence in four calves born to two Besnoitia-infected dams, with titres ranging from 1:64 to 1:1024. A specific antibody titre of 1:1024 was found in colostrum collected from one of the dams. Two of the newborn calves, when sampled immediately after birth, were serologically negative to B. besnoiti, but became positive on the next day. In all the calves, antibodies were detectable up to the age of 4 months. Observations concerning passive transfer of antibodies from Besnoitia-infected dams to offspring, and transmission of the infection among infected and non-infected closely kept cows, are discussed.The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. Adobe Acrobat XI Pro was used to OCR the text and also for the merging and conversion to the final presentation PDF-format.mn201

Functional and Immunological Relevance of Anaplasma marginale Major Surface Protein 1a Sequence and Structural Analysis.

Bovine anaplasmosis is caused by cattle infection with the tick-borne bacterium, Anaplasma marginale. The major surface protein 1a (MSP1a) has been used as a genetic marker for identifying A. marginale strains based on N-terminal tandem repeats and a 5'-UTR microsatellite located in the msp1a gene. The MSP1a tandem repeats contain immune relevant elements and functional domains that bind to bovine erythrocytes and tick cells, thus providing information about the evolution of host-pathogen and vector-pathogen interactions. Here we propose one nomenclature for A. marginale strain classification based on MSP1a. All tandem repeats among A. marginale strains were classified and the amino acid variability/frequency in each position was determined. The sequence variation at immunodominant B cell epitopes was determined and the secondary (2D) structure of the tandem repeats was modeled. A total of 224 different strains of A. marginale were classified, showing 11 genotypes based on the 5'-UTR microsatellite and 193 different tandem repeats with high amino acid variability per position. Our results showed phylogenetic correlation between MSP1a sequence, secondary structure, B-cell epitope composition and tick transmissibility of A. marginale strains. The analysis of MSP1a sequences provides relevant information about the biology of A. marginale to design vaccines with a cross-protective capacity based on MSP1a B-cell epitopes

Isolation of Besnoitia besnoiti from infected cattle in Portugal

Abstract Besnoitia besnoiti, an obligate intracellular protozoan parasite belonging to the phylum apicomplexa, is the causative agent of bovine besnoitiosis. Besnoitiosis is responsible for significant losses in the cattle industry of Africa and Mediterranean countries due to the high morbidity rate, abortion and infertility in males. The acute stage of disease is associated with the proliferative forms (tachyzoites) and is characterized by fever, whimpery, general weakness and swelling of the superficial lymph nodes. During the following chronic stage, a huge number of cysts are formed mainly in the subcutaneous tissues. This process is non-reversible, and chronic besnoitiosis is characterized by hyper-sclerodermia, hyperkeratosis, alopecia and, in bulls, atrophy, sclerosis and focal necrosis that cause irreversible lesions in the testis. In this paper we report on the identification of large cysts in the skin of a cow and a bull in Portugal, which presented loss of hair and enlargement and pachydermis all over the body. The observation of a two-layered cyst wall within the host cell, the encapsulation of the host cell by a large outer cyst wall, and the subcutaneous localization of the cysts within the host, were characteristic for B. besnoiti. The parasites were isolated from the infected animals and successfully propagated in Vero cells without prior passages in laboratory animals. Morphological characterization of B. besnoiti tachyzoites and the amplification of the 149 bp segment from the internal transcribed spacer 1 (ITS1), aided with specific primers, confirmed the identification of B. besnoiti. Keywords: Besnoitia besnoiti; Bovine besnoitiosis; Portugal; Cyst; In vitro cell cultur

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

Transcriptome and proteome response of Rhipicephalus annulatus tick vector to Babesia bigemina infection

Funding Information: RHIBAB - PTDC/CVT/112050/2009 “Differential expression and functional characterization of tick (Rhipicephalus annulatus) genes in response to pathogen infection (B. bigemina).” SA is the recipient of a post-doctoral grant supported by FCT Funding Information: The authors would like to acknowledge Fundação para a Ciência e Tecnologia (FCT) for funds to GHTM – UID/Multi/04413/2013. Publisher Copyright: Copyright © 2019 Antunes, Couto, Ferrolho, Sanches, Merino Charrez, De la Cruz Hernández, Mazuz, Villar, Shkap, de la Fuente and Domingos. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.A system biology approach was used to gain insight into tick biology and interactions between vector and pathogen. Rhipicephalus annulatus is one of the main vectors of Babesia bigemina which has a massive impact on animal health. It is vital to obtain more information about this relationship, to better understand tick and pathogen biology, pathogen transmission dynamics, and new potential control approaches. In ticks, salivary glands (SGs) play a key role during pathogen infection and transmission. RNA sequencing obtained from uninfected and B. bigemina infected SGs obtained from fed female ticks resulted in 6823 and 6475 unigenes, respectively. From these, 360 unigenes were found to be differentially expressed (p < 0.05). Reversed phase liquid chromatography-mass spectrometry identified a total of 3679 tick proteins. Among them 406 were differently represented in response to Babesia infection. The omics data obtained suggested that Babesia infection lead to a reduction in the levels of mRNA and proteins (n = 237 transcripts, n = 212 proteins) when compared to uninfected controls. Integrated transcriptomics and proteomics datasets suggested a key role for stress response and apoptosis pathways in response to infection. Thus, six genes coding for GP80, death-associated protein kinase (DAPK-1), bax inhibitor-1 related (BI-1), heat shock protein (HSP), heat shock transcription factor (PHSTF), and queuine trna-ribosyltransferase (QtRibosyl) were selected and RNA interference (RNAi) performed. Gene silencing was obtained for all genes except phstf. Knockdown of gp80, dapk-1, and bi-1 led to a significant increase in Babesia infection levels while hsp and QtRibosyl knockdown resulted in a non-significant decrease of infection levels when compared to the respective controls. Gene knockdown did not affect tick survival, but engorged female weight and egg production were affected in the gp80, dapk-1, and QtRibosyl-silenced groups in comparison to controls. These results advanced our understanding of tick-Babesia molecular interactions, and suggested new tick antigens as putative targets for vaccination to control tick infestations and pathogen infection/transmission.publishersversionpublishe

Besnoitia besnoiti lytic cycle in vitro and differences in invasion and intracellular proliferation among isolates

© 2016 Frey et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.[EN]Background: Bovine besnoitiosis, caused by the protozoan Besnoitia besnoiti, reduces productivity and fertility of affected herds. Besnoitiosis continues to expand in Europe and no effective control tools are currently available. Experimental models are urgently needed. Herein, we describe for the first time the kinetics of standardised in vitro models for the B. besnoiti lytic cycle. This will aid to study the pathogenesis of the disease, in the screening for vaccine targets and drugs potentially useful for the treatment of besnoitiosis. Methods: We compared invasion and proliferation of one B. tarandi (from Finland) and seven B. besnoiti isolates (Bb-Spain1, Bb-Spain2, Bb-Israel, Bb-Evora03, Bb-Ger1, Bb-France, Bb-Italy2) in MARC-145 cell culture. Host cell invasion was studied at 4, 6, 8 and 24 h post infection (hpi), and proliferation characteristics were compared at 24, 48, 72, 96, 120, and 144 hpi. Results: In Besnoitia spp., the key parameters that determine the sequential adhesion-invasion, proliferation and egress steps are clearly distinct from those in the related apicomplexans Toxoplasma gondii and Neospora caninum. Besnoitia spp. host cell invasion is a rather slow process, since only 50 % of parasites were found intracellular after 3-6 h of exposure to host cells, and invasion still took place after 24 h. Invasion efficacy was significantly higher for Bb-France, Bb-Evora03 and Bb-Israel. In addition, the time span for endodyogeny to take place was as long as 18-35 h. Bb-Israel and B. tarandi isolates were most prolific, as determined by the tachyzoite yield at 72 hpi. The total tachyzoite yield could not be predicted neither by invasion-related parameters (velocity and half time invasion) nor by proliferation parameters (lag phase and doubling time (dT)). The lytic cycle of Besnoitia was asynchronous as evidenced by the presence of three different plaque-forming tachyzoite categories (lysis plaques, large and small parasitophorous vacuoles). Conclusions: This study provides first insights into the lytic cycle of B. besnoiti isolates and a standardised in vitro model that allows screening of drug candidates for the treatment of besnoitiosis.SIThis study was funded by the Spanish Ministry of Economy and Competitiveness (AGL2013-46442-R) and CYTED (Thematic Network 113RT0469 Protozoovac). CFF and NM are supported by the Swiss National Science Foundation (grants no. PBBEP3_141435 and PBBEP3_139398, respectively). We gratefully acknowledge Vanessa Navarro for her excellent technical assistance, and Andrew Hemphill for critical reading of the manuscript

Graphene-Assisted Integrated Nonlinear Optics

‎The unique linear and massless band structure of graphene in a purely two-dimensional Dirac fermionic structure has ignited intense research since the first monolayer graphene was isolated in the laboratory‎. ‎Not only does it offer new inroads into low-dimensional physics; graphene exhibits several peculiar properties that promise to widen the realm of opportunities for integrated optics and photonics‎. ‎This thesis is an attempt to shed light on the exceptional nonlinear optical properties of graphene and their potential applications in integrated photonics‎. ‎Following a theoretical exploration of light-graphene interaction‎, ‎disruptive new insight into the nonlinear optics of graphene was generated‎. ‎It now appears that graphene can efficiently enable photon-photon interaction in a fully integrated fashion‎. ‎This property‎, ‎taken together with ultrawideband tunability and ultrafast carrier dynamics could be fully exploited within integrated photonics for a variety of applications including harmonic generation and all-optical signal processing‎. ‎The multidisciplinary work described herein combines theoretical modeling and experimentation to proceed one step further toward this goal‎. ‎This thesis begins by presenting a semiclassical theory of light-graphene interaction‎. ‎The emphasis is placed on the nonlinear optical response of graphene from the standpoint of its underlying chiral symmetry‎. ‎The peculiar energy‎- ‎momentum dispersion of the quasiparticles in graphene entails a diverging field-induced interband coupling‎. ‎Following a many-body study of the carrier relaxations dynamics in graphene‎, ‎it will be shown that the charged carriers in the vicinity of the Dirac point undergo an unconventional saturation effect that can be induced by an arbitrarily weak electromagnetic field‎. ‎The perturbative treatment of the optical response of graphene is revisited and a theoretical model is developed to estimate the nonlinear optical coefficients including the Kerr coefficient of graphene‎. ‎The theoretical models are complimented by the experimental results‎. ‎The peculiar nonlinear optical properties of graphene together with its ablity to being integrated with optical platforms would render it possible to perform nonlinear optics in graphene integrated nanophotonic structures‎. ‎Here‎, ‎the suitability of graphene for nonlinear optical applications is investigated both theoretically and experimentally‎. ‎The emphasis is placed on an on-chip platform for ultrafast all-optical amplitude modulation‎. ‎The experimental results indicate strong all-optical modulation in a graphene-cladded planar photonic crystal nanocavity‎. ‎This development relies heavily on the unique properties of graphene‎, ‎including its fast carrier dynamics and the special phonon induced relaxation mechanism‎. ‎Finally‎, ‎the potential application of graphene based all-optical modulation in time resolved nonlinear spectroscopy is also discussed‎