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Baeta B.A.,Federal Rural University of Rio de Janeiro | Ribeiro C.C.D.U.,Federal Rural University of Rio de Janeiro | Teixeira R.C.,Federal Rural University of Rio de Janeiro | Cabezas-Cruz A.,University of Lille Nord de France | And 5 more authors.
Ticks and Tick-borne Diseases | Year: 2015

IDE8 tick cell cultures have been used for the isolation and propagation of several isolates of Anaplasma marginale. The genetic heterogeneity of A. marginale strains in cattle is diverse in endemic regions worldwide and the analyses of msp1α (major surface protein 1 alpha) gene sequences have allowed the identification of different strains. This study reports the isolation and propagation of two new isolates of A. marginale in IDE8 cells from blood of two cattle and their morphological and molecular characterization using light microscopy and the msp1α gene, respectively. Small colonies were observed in cytospin smears of each of the isolates 60 days after culture initiation. Based on msp1α sequence variation, the two isolates were found to be separate strains and were named AmRio1 and AmRio2. Analysis of msp1α microsatellite in both strains resulted in a single genotype, genotype E. The amino acid sequence of one MSP1α tandem repeat from the strain AmRio1 resulted in a new sequence (named 162) with one amino acid change. The results of these phylogenetic analyses demonstrated that A. marginale strains from Brazil and Argentina formed two large clusters of which one was less divergent that the other. © 2014 Elsevier GmbH. Source


Cruz A.C.,University of South Bohemia | Zweygarth E.,Ludwig Maximilians University of Munich | Ribeiro M.F.B.,Federal University of Minas Gerais | Da Silveira J.A.G.,Federal University of Minas Gerais | And 6 more authors.
Parasites and Vectors | Year: 2012

Background: Ehrlichia species are the etiological agents of emerging and life-threatening tick-borne human zoonoses that inflict serious and fatal infections in companion animals and livestock. The aim of this paper was to phylogeneticaly characterise a new species of Ehrlichia isolated from Rhipicephalus (Boophilus) microplus from Minas Gerais, Brazil. Methods. The agent was isolated from the hemolymph of Rhipicephalus (B.) microplus engorged females that had been collected from naturally infested cattle in a farm in the state of Minas Gerais, Brazil. This agent was then established and cultured in IDE8 tick cells. The molecular and phylogenetic analysis was based on 16S rRNA, groEL, dsb, gltA and gp36 genes. We used the maximum likelihood method to construct the phylogenetic trees. Results: The phylogenetic trees based on 16S rRNA, groEL, dsb and gltA showed that the Ehrlichia spp isolated in this study falls in a clade separated from any previously reported Ehrlichia spp. The molecular analysis of the ortholog of gp36, the major immunoreactive glycoproteins in E. canis and ortholog of the E. chaffeensis gp47, showed a unique tandem repeat of 9 amino acids (VPAASGDAQ) when compared with those reported for E. canis, E. chaffeensis and the related mucin-like protein in E. ruminantium. Conclusions: Based on the molecular and phylogenetic analysis of the 16S rRNA, groEL, dsb and gltA genes we concluded that this tick-derived microorganism isolated in Brazil is a new species, named E. mineirensis (UFMG-EV), with predicted novel antigenic properties in the gp36 ortholog glycoprotein. Further studies on this new Ehrlichia spp should address questions about its transmissibility by ticks and its pathogenicity for mammalian hosts. © 2012 Cruz et al.; licensee BioMed Central Ltd. Source


Lis K.,Ludwig Maximilians University of Munich | Najm N.,Ludwig Maximilians University of Munich | de la Fuente J.,Institute Investigacion Of Recursos Cinegeticos | de la Fuente J.,Oklahoma State University | And 5 more authors.
Ticks and Tick-borne Diseases | Year: 2014

Anaplasma marginale (Rickettsiales: Anaplasmataceae) is an obligate intracellular bacterium that multiplies exclusively within membrane-bound vacuoles in the cytoplasm of host cells. A number of A. marginale isolates can be propagated in the Ixodes scapularis IDE8 tick cell line, which provides a reliable source of antigens for a wide variety of studies. However, because of its intracellular nature, separation of bacteria from host cell materials remains an important constraint for researchers. In the present study, we evaluated the use of Percoll gradients for purification of two Brazilian strains of A. marginale grown in IDE8 tick cells. The purified A. marginale monitored in Giemsa-stained smears contained only minimal amounts of IDE8 cell stroma. The total protein yields were 1.2mg and 1.7mg, while the DNA titers quantified with real-time PCR were 6.4×109 for UFMG1 and 4.87×109 for UFMG2 copies in the purified material, respectively. Additionally, we confirmed the viability of purified bacteria by infecting tick cells after being freshly purified and after retrieval from long-term storage. Importantly, the viability of the organisms is preserved after use of this separation method, and therefore the purified organisms can be used in enzymatic assays and other research approaches where live organisms would be preferred. © 2014 Elsevier GmbH. Source


Tonk M.,Academy of Sciences of the Czech Republic | Tonk M.,University of South Bohemia | Cabezas-Cruz A.,Academy of Sciences of the Czech Republic | Cabezas-Cruz A.,University of South Bohemia | And 9 more authors.
Gene | Year: 2014

The hard-bodied tick Ixodes ricinus (castor bean tick) is the most common tick species in Europe. I. ricinus is a vector of the causative agents of diseases that affect humans and animals including tick-borne encephalitis, borreliosis, tick-borne fever and babesiosis. The innate immune system provides ticks with quite an efficient defence against some pathogenic microorganisms in the event of their penetration into the tick body or through the blood meal. Antimicrobial peptides (AMPs) constitute an important feature of the tick immune system. Defensins are a well-known class of AMPs. Members of the defensin family of proteins have been reported in several tick species. So far, only two defensins had been identified from I. ricinus. In this study, we report the identification of six novel putative defensins from I. ricinus at the genomic and transcriptional levels. At the genomic level they show differences with one being intronless, while others contain two introns. The expression pattern of these molecules in the salivary glands, midgut, ovary, Malpighian tubules, haemolymph and the tick cell line IRE/CTVM19 was determined. Some of them are tissue specific while others seem to be ubiquitous. Molecular and phylogenetic analyses show that these novel members of the I. ricinus defensin family differ phylogenetically and structurally; nevertheless, the cysteine pattern is highly conserved among the family members. Finally, antimicrobial-peptide prediction tools were used to predict putative antimicrobial activity of our defensins. They show putative antimicrobial activity mainly against Gram-positive bacteria. This study displays the diversity of the defensin family in the tick I. ricinus. © 2014 Elsevier B.V. Source


Cabezas-Cruz A.,Academy of Sciences of the Czech Republic | Passos L.M.F.,Ludwig Maximilians University of Munich | Passos L.M.F.,Federal University of Minas Gerais | Lis K.,Ludwig Maximilians University of Munich | And 13 more authors.
PLoS ONE | Year: 2013

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. © 2013 Cabezas-Cruz et al. Source

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