Differential expression and functional characterization of cattle tick genes in response to pathogen infection (Babesia bigemina)

O conceito “One health” reconhece a necessidade do trabalho conjunto de veterinários, profissionais de saúde e cientistas, dada a interface dinâmica entre pessoas, animais e ambiente. Este conceito é muito importante em zoonoses, tais como doenças associadas a carraças (DAC´s) que dependem de animais como reservatório. Os protozoários do género Babesia são agentes patogénicos transmitidos por carraças que causam a doença denominada babesiose num variado número de animais incluindo o Homem. Particularmente a B. bovis e B. bigemina são transmitidas por carraças, relacionadas com gado, Rhipicephalus (Boophilus) annulatus e R. microplus sendo estas consideradas os ectoparasitas de maior importância, com largo impacto económico na produção animal. O uso tradicional de acaricidas no controlo de carraças apresenta diversas desvantagens incluindo a seleção de carraças acaricido-resistentes e contaminação ambiental com resíduos químicos. As vacinas destacam-se como uma alternativa ao uso de acaricidas. O objetivo destas vacinas é a proteção contra DAC´s através do controlo das infestações pelos vectores e redução de transmissão de doença. As proteínas envolvidas nas interações carraça-agente patogénico podem ser bons candidatos para essas novas vacinas mas a sua identificação e validação continuam a ser obstáculos. Os objetivos do presente estudo foram, primeiro, a identificação de genes de R. annulatus diferenciadamente expressos em resposta à infeção por B. bigemina, segundo, a validação da influência destes genes no processo de infeção e finalmente a caracterização dos antigénios identificados, a fim de selecionar os melhores candidatos, para o desenvolvimento de uma potencial nova vacina. A fim de alcançar os objectivos propostos, clones de uma biblioteca de hibridização subtrativa por supressão (SSH) foram sequenciados e analisados. Os genes diferenciadamente expressos com prováveis funções relacionadas com a interface carraça- agente patogénico, foram selecionadas para validação dos resultados de SSH por real time RT-PCR. A análise funcional conduzida por RNA de interferência mostra que, nas condições do presente estudo, o silenciamento dos genes que codificam para as proteínas, sérica amilóide A e TROSPA levam á redução de níveis de infecção em R. annulatus e em R. microplus em comparação com o grupo controlo. Em R. microplus é demonstrada a influência também da calreticulina (CRT). As proteínas TROSPA e CRT foram selecionadas e obtidas usando um sistema de expressão em Escherichia coli e anticorpos poli/monoclonais foram produzidos. O reconhecimento das proteínas nativas foi confirmado por Western blotting e imunofluorescência em tecidos de carraça. O efeito dos anticorpos específicos, suplementados à refeição de sangue, foi demonstrado pela avaliação do peso final e/ou ovoposição em carraças alimentadas artificialmente. Não foi observado efeito significativo na aquisição de B. bigemina. Os resultados mostram as vantagens e desvantagens do sistema in vitro de alimentação artificial de carraças por tubos capilares na caracterização de antigénios protetores de carraça. Diferentes estudos caracterizaram a interface carraça- agente patogénico a nível molecular no entanto, o presente estudo apresenta a primeira análise funcional de genes em carraças infectadas com B. bigemina. Os resultados apresentados contribuem para um maior conhecimento do papel de genes de carraça no processo de infeção/multiplicação por Babesia sp., bem como para o desenvolvimento de novas vacinas.The “One Health” approach recognizes the need for veterinarians, human health professionals, and environmental scientists to work together given the dynamic interface among people, animals, and the environment.This approach is increasingly important for zoonotic diseases, suchas tick borne diseases (TBD ́s) which rely on animals as reservoirs. Babesiaspp. are tick-borne pathogens that cause a disease called babesiosis in a wide range of animals and also humans. Particularity, B. bovisand B. bigeminaare transmitted by cattle ticks, Rhipicephalus (Boophilus) annulatus andR. microplusbeing considered the most important cattle ectoparasites with major economic impact on cattle production. The traditional use ofchemicals to control ticks has serious drawbacks, including the selection of acaricide-resistant ticks and contamination of the environment with chemical residues. Research on alternatives to the use of acaricides is strongly represented by tick vaccines considered a more cost-effective and environmentally safe strategy. The ultimate goal of tick vaccines is to protect against TBD ́s through the control of vector infestations and reducing pathogen infection and transmission. Tick proteins involved in tick-pathogen interactions may provide good candidate protective antigens for these new vaccines but their identification andvalidation are still limiting steps. The objectives of this study were first to identify R. annulatusgenes differentially expressed in response to infection with B. bigemina, second to validate the influence of these genes in the infection in both R. annulatusand R. microplusticks and finallyto characterize identified antigens to select the best candidates for future vaccine development. In order to achieve these goals, suppression-subtractive hybridization (SSH) library clones were sequenced and analyzed. After molecular function gene ontology assignment differentially expressed genes with putative functions in tick-pathogen interactions were selected for validation of SSH results by real-time RT-PCR. Functional analysis by RNA interference showed that under the conditions of the present study,knockdown of trospaand serum amyloid A significantly reduced B. bigeminainfection levels in R. annulatusand R. micropluswhen compared to controls. In R. microplusalso calreticulinshowed infection reduction. TROSPA and CRT were selected, recombinant proteins were obtained using Escherichiacoliexpression system and poly/monoclonal antibodies were generated. Their specificity against tick recombinant proteins was confirmed by Western blottingand against native proteins in tick tissuesusing immunofluorescence. Capillary-fed ticks ingested antibodies added to the blood meal and the effect ofthese antibodies on tick weight and/or oviposition was shown. No significant effect was observed on pathogen acquisition. The results highlighted the advantages and disadvantages of in vitrotick capillary feeding for the characterization of candidate tick protective antigens. Several studies have characterized the tick-pathogen interface at the molecular level. However, to our knowledge this is the first report of functional genomics studies in ticks infected with B. bigemina. The results reported here increased our understanding of the role of tick genes in Babesiainfection/multiplication and contribute to the development of a vaccine with impact on both tick infestation and pathogen infection

Unraveling the genetic background of individuals with a clinical familial hypercholesterolemia phenotype

Familial hypercholesterolemia (FH) is a common genetic disorder of lipid metabolism caused by pathogenic/likely pathogenic variants in LDLR, APOB, and PCSK9 genes. Variants in FH-phenocopy genes (LDLRAP1, APOE, LIPA, ABCG5, and ABCG8), polygenic hypercholesterolemia, and hyperlipoprotein (a) [Lp(a)] can also mimic a clinical FH phenotype. We aim to present a new diagnostic tool to unravel the genetic background of clinical FH phenotype. Biochemical and genetic study was performed in 1,005 individuals with clinical diagnosis of FH, referred to the Portuguese FH Study. A next-generation sequencing panel, covering eight genes and eight SNPs to determine LDL-C polygenic risk score and LPA genetic score, was validated, and used in this study. FH was genetically confirmed in 417 index cases: 408 heterozygotes and 9 homozygotes. Cascade screening increased the identification to 1,000 FH individuals, including 11 homozygotes. FH-negative individuals (phenotype positive and genotype negative) have Lp(a) >50 mg/dl (30%), high polygenic risk score (16%), other monogenic lipid metabolism disorders (1%), and heterozygous pathogenic variants in FH-phenocopy genes (2%). Heterozygous variants of uncertain significance were identified in primary genes (12%) and phenocopy genes (7%). Overall, 42% of our cohort was genetically confirmed with FH. In the remaining individuals, other causes for high LDL-C were identified in 68%. Hyper-Lp(a) or polygenic hypercholesterolemia may be the cause of the clinical FH phenotype in almost half of FH-negative individuals. A small part has pathogenic variants in ABCG5/ABCG8 in heterozygosity that can cause hypercholesterolemia and should be further investigated. This extended next-generation sequencing panel identifies individuals with FH and FH-phenocopies, allowing to personalize each person’s treatment according to the affected pathway

Characterisation of microbial attack on archaeological bone

As part of an EU funded project to investigate the factors influencing bone preservation in the archaeological record, more than 250 bones from 41 archaeological sites in five countries spanning four climatic regions were studied for diagenetic alteration. Sites were selected to cover a range of environmental conditions and archaeological contexts. Microscopic and physical (mercury intrusion porosimetry) analyses of these bones revealed that the majority (68%) had suffered microbial attack. Furthermore, significant differences were found between animal and human bone in both the state of preservation and the type of microbial attack present. These differences in preservation might result from differences in early taphonomy of the bones. © 2003 Elsevier Science Ltd. All rights reserved

NEOTROPICAL ALIEN MAMMALS: a data set of occurrence and abundance of alien mammals in the Neotropics

Biological invasion is one of the main threats to native biodiversity. For a species to become invasive, it must be voluntarily or involuntarily introduced by humans into a nonnative habitat. Mammals were among first taxa to be introduced worldwide for game, meat, and labor, yet the number of species introduced in the Neotropics remains unknown. In this data set, we make available occurrence and abundance data on mammal species that (1) transposed a geographical barrier and (2) were voluntarily or involuntarily introduced by humans into the Neotropics. Our data set is composed of 73,738 historical and current georeferenced records on alien mammal species of which around 96% correspond to occurrence data on 77 species belonging to eight orders and 26 families. Data cover 26 continental countries in the Neotropics, ranging from Mexico and its frontier regions (southern Florida and coastal-central Florida in the southeast United States) to Argentina, Paraguay, Chile, and Uruguay, and the 13 countries of Caribbean islands. Our data set also includes neotropical species (e.g., Callithrix sp., Myocastor coypus, Nasua nasua) considered alien in particular areas of Neotropics. The most numerous species in terms of records are from Bos sp. (n = 37,782), Sus scrofa (n = 6,730), and Canis familiaris (n = 10,084); 17 species were represented by only one record (e.g., Syncerus caffer, Cervus timorensis, Cervus unicolor, Canis latrans). Primates have the highest number of species in the data set (n = 20 species), partly because of uncertainties regarding taxonomic identification of the genera Callithrix, which includes the species Callithrix aurita, Callithrix flaviceps, Callithrix geoffroyi, Callithrix jacchus, Callithrix kuhlii, Callithrix penicillata, and their hybrids. This unique data set will be a valuable source of information on invasion risk assessments, biodiversity redistribution and conservation-related research. There are no copyright restrictions. Please cite this data paper when using the data in publications. We also request that researchers and teachers inform us on how they are using the data