Mitochondrial diversity and the origins of African and European cattle.

The nature of domestic cattle origins in Africa are unclear as archaeological data are relatively sparse. The earliest domesticates were humpless, or Bos taurus, in morphology and may have shared a common origin with the ancestors of European cattle in the Near East. Alternatively, local strains of the wild ox, the aurochs, may have been adopted by peoples in either continent either before or after cultural influence from the Levant. This study examines mitochondrial DNA displacement loop sequence variation in 90 extant bovines drawn from Africa, Europe, and India. Phylogeny estimation and analysis of molecular variance verify that sequences cluster significantly into continental groups. The Indian Bos indicus samples are most markedly distinct from the others, which is indicative of a B. taurus nature for both European and African ancestors. When a calibration of sequence divergence is performed using comparisons with bison sequences and an estimate of 1 Myr since the Bison/Bos Leptobos common ancestor, estimates of 117-275,000 B.P. and 22-26,000 B.P. are obtained for the separation between Indians and others and between African and European ancestors, respectively. As cattle domestication is thought to have occurred approximately 10,000 B.P., these estimates suggest the domestication of genetically discrete aurochsen strains as the origins of each continental population. Additionally, patterns of variation that are indicative of population expansions (probably associated with the domestication process) are discernible in Africa and Europe. Notably, the genetic signatures of these expansions are clearly younger than the corresponding signature of African/European divergence.

The standard of perfection for American domestic rabbits illustrated : the origin, history and full descriptions of all recognized varieties of domestic rabbits, with revised American and European standards /

"Compiled for the American Rabbit Association."

Italian wild hop (Humulus lupulus L.): from biodiversity evaluation to varietal selection

Hop (Humulus lupulus L.) is a dioecius perennial plant. The cultivation is specific for female plants, used mainly for brewing and pharmacology. Female inflorescence, known as cone or strobili, contains bitter acids, essential oil and polyphenols. Commercial hop cultivation provides better results in regions between 45 and 55 degrees north or south in latitude, an area that also includes the northern part of Italy, where hop is endemic. Despite several studies have been conducted on the characterization of wild hops biodiversity in the U.S.A. and Europe, a lack in literature concerning the description of Italian wild hops genetic variability is still present. The increasing request of hop varieties improved in important traits, like diseases, resistance and valuable aroma profile, is bringing the hop industry. Moreover, Italian agricultural sector needs new impulse to be competitive in the market. In this view, Italian wild hop biodiversity is a resource, useful for the obtaining of Italian hop varieties, characterized by peculiar aromatic traits and more adaptable to Mediterranean climate, making their cultivation more sustainable. Based on this consideration, the present Ph.D. thesis deals with the evaluation of the Italian hop biodiversity, through the characterization of the wild samples under different point of view. The project started with the recovery of wild hop samples in different areas of north of Italy to consitue a collection field, where 11 commercial cultivars of US and European origin were grown, to have a complete vision of the hop panorama. Ph.D. project followed different research lines, the results of each one contributed to completly characterize the northern Italian hop wild biodiversity: • the morphological description showed a high phenological variability (Study 1); • the genetic characterization confirmed the rich biodiversity of the Italian population and showed a significant genetic distance between Italian genotypes and the commercial cultivars, taken in consideration (Study 2); • the need of an early sex discrimination method leads to an improvement of a genetic marker, developing a more efficient marker (Study 3); • a complete morphologic, genetic and chemical analysis of plants gave results to select the most promising genotypes (Study 4); • the comparison between the performance of wild hops and commercial cultivars in the same collection field indicated that some wild genotypes had a higher environment adaptability (Study 5); • the evaluation of the terroir, obtained comparing commercial cultivars in the collection field and the same genotypes purchased in the market, showed the influence of the northern Italian environment on the aromatic profile (Study 5); • a new analytical method for the revelation of bioactive metabolites and a simple extraction procedure were developed (Study 6). In conclusion, the Ph.D. thesis, contains the first characterization of Italian wild hop, made under field condition. The present study: i) permits to obtain a complete and significative description of the genotypes; ii) allows the identification of the most promising wild Italian genotypes; iii) allows the identification of commercial cultivars more adaptable the northern Italian climate.

Regional climate change impacts on wild animals' living territory in Central Europe

In this paper the projected future impact of climate change has been analyzed for the quality of living conditions of the European terrestrial vertebrates (amphibians, reptiles, birds, mammals) in the Carpathian Basin. According to the climate scenarios, warmer and drier climatic conditions are likely to occur in the Carpathian Basin by end of this century. Simultaneous analysis of climate parameters, climate simulations and animal range datasets enables us to evaluate the vulnerability of different European species to regional warming and climate change. The spatial climate analogy technique is used to analyze the estimated rapid change of the wild animals’ habitats and their northward migration. For the reference climate data of Debrecen is considered, and three spatial analogue regions are compared. The results suggest that generally a significant decline in habitats is very likely for most of the analyzed animal groups by the end of the 21st century. The largest rate of decline is estimated for birds. However, living conditions for reptiles may improve in the future due to the warmer and drier climatic conditions, which are favourable for these species.

Enhancing public speaking skills - an evaluation of the Presentation Trainer in the wild

Full paper presented at EC-TEL 2016

Study of antioxidant, antiproliferative and apoptosis-inducing properties of wild mushrooms from the Northeast of Portugal

Mushrooms are known as a powerful source of bioactive compounds including antioxidants, inhibitors of human tumour cell lines growth, inducers of apoptosis and enhancers of immunity. Indeed, many pre-clinical studies have been conducted in human tumour cell lines and in some cases a number of compounds isolated from mushrooms have followed to clinical trials. The Northeast of Portugal is one of the European regions with higher wild mushrooms diversity. However, to our knowledge, no studies had been conducted so far to verify their bioactivities. The main aim of this work was the evaluation of the bioactive properties (antioxidant properties and growth inhibitory potential on human tumour cell lines) of wild edible mushrooms collected in the Northeast of Portugal. Once properly identified, methanolic, ethanolic and boiling water extracts were prepared from thirty eight wild mushroom species collected in that region. Chemical characterization was obtained by high performance liquid chromatography (HPLC) coupled to a photodiode array detector (DAD) or to a refraction index detector (RI). Antioxidant activity assays were carried out in those extracts, including evaluation of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals scavenging capacity, reducing power and inhibition of β-carotene bleaching. Extract-induced cell growth inhibition was assessed with the sulforhodamine B assay in four human tumour cell lines (NCI-H460 - lung cancer, MCF-7 -breast cancer, HCT-15 -colon cancer and AGS - gastric cancer). The effects on cell cycle profile and apoptosis were evaluated by flow cytometry and the effect on the expression levels of proteins related to cell cycle and apoptosis was further investigated by Western blotting. Three wild edible mushroom species revealed growth inhibitory activity in the studied human tumour cell lines: Clitocybe alexandri ethanolic extract, Lepista inversa methanolic extract and Suillus collinitus methanolic extract. C. alexandri ethanolic extract induced an S-phase cell cycle arrest and increased the percentage of apoptotic cells, in the NCI-H460 cell line. The analysed mushroom species also provided interesting antioxidant potential, mainly the boiling water extract of L. inversa which showed the highest DPPH radical scavenging activity, reducing power and β-carotene bleaching inhibition. S. collinitus methanolic extract induced a slight increase in the number of cells in G1, with a concomitant decrease in the percentage of cells in the S phase of the cell cycle and an increase in the percentage of apoptotic cells, in the MCF-7 cell line. The combined use of the S. collinitus methanolic extract and etoposide caused a greater decrease in the percentage of cell growth, when compared to either of them used individually, indicating the potential benefit of this combination. The tested extracts were chemically characterized and protocatechuic, p-hydroxybenzoic, p-coumaric and cinnamic acids were the main compounds identified on the phenolic (methanolic and ethanolic) extracts, while mannitol, trehalose and arabinose were the main sugars found in the polysaccharidic (boiling water) extracts after hydrolysis. The individual compounds identified in the extracts were submitted to a screening of tumour cells growth inhibitory activity, but only the phenolic acids and a related compound, cinnamic acid, presented activity. This compound was found to be the most potent one regarding cell growth inhibition in the NCI-H460 cell line. The effect of the individual and combined treatment with the identified compounds was also evaluated. Cinnamic and protochatequic acids caused a statistically significantly reduction in the number of viable cells. In addition, p-hydroxybenzoic acid did not show any significantly reduction in the viable cell number. Nevertheless, it was verified that the concomitant use of the three compounds provided the strongest decrease in the viable cell number, suggesting a possible concomitant effect of those compounds. Overall, the present work has contributed to further understand the bioactive potential of wild edible mushrooms from the Northeast of Portugal. This study allowed to identify some species with antioxidant or tumour cell growth inhibitory potential.

What can exploratory modelling tell us about the ecobiology of European sea bass ( Dicentrarchus labrax ): a comprehensive overview

European sea bass, Dicentrarchus labrax, is a highly valuable species in Europe, both for aquaculture in the Mediterranean Sea and for commercial and recreational fisheries in the North East Atlantic Ocean. Subjected to increasing fishing pressure, the wild population has recently experienced significant recruitment fluctuation as well as a northward extension of its distribution area in the North Sea. While the nature of the ecological and/or physiological processes involved remains unresolved, ontogenetic habitat shifts and adult site fidelity could increase the species’ vulnerability to climate change and overfishing. As managers look for expert information to propose management scenarios leading to sustainable exploitation, exploratory modelling appears to be a cost-efficient approach to enhance the understanding of recruitment dynamics and the spatio-temporal scales over which fish populations function. A conceptual modelling framework and its specific data requirements are discussed to tackle some sound ecological questions regarding this species. We consequently provide an updated review of current knowledge on bass population structure, biology and ecology. This paper will hence be particularly valuable to develop spatially-explicit models of European sea bass dynamics under environmental and anthropogenic forcing. Knowledge gaps requiring further research efforts are also reported.

African swine fever virus transmission cycles in Central Europe: evaluation of wild boar-soft tick contacts through detection of antibodies against Ornithodoros erraticus saliva antigen.

BACKGROUND African swine fever (ASF) is one of the most complex viral diseases affecting both domestic and wild pigs. It is caused by ASF virus (ASFV), the only DNA virus which can be efficiently transmitted by an arthropod vector, soft ticks of the genus Ornithodoros. These ticks can be part of ASFV-transmission cycles, and in Europe, O. erraticus was shown to be responsible for long-term maintenance of ASFV in Spain and Portugal. In 2014, the disease has been reintroduced into the European Union, affecting domestic pigs and, importantly, also the Eurasian wild boar population. In a first attempt to assess the risk of a tick-wild boar transmission cycle in Central Europe that would further complicate eradication of the disease, over 700 pre-existing serum samples from wild boar hunted in four representative German Federal States were investigated for the presence of antibodies directed against salivary antigen of Ornithodoros erraticus ticks using an indirect ELISA format. RESULTS Out of these samples, 16 reacted with moderate to high optical densities that could be indicative of tick bites in sampled wild boar. However, these samples did not show a spatial clustering (they were collected from distant geographical regions) and were of bad quality (hemolysis/impurities). Furthermore, all positive samples came from areas with suboptimal climate for soft ticks. For this reason, false positive reactions are likely. CONCLUSION In conclusion, the study did not provide stringent evidence for soft tick-wild boar contact in the investigated German Federal States and thus, a relevant involvement in the epidemiology of ASF in German wild boar is unlikely. This fact would facilitate the eradication of ASF in the area, although other complex relations (wild boar biology and interactions with domestic pigs) need to be considered.

Modular framework to assess the risk of African swine fever virus entry into the European Union

BACKGROUND The recent occurrence and spread of African swine fever (ASF) in Eastern Europe is perceived as a serious risk for the pig industry in the European Union (EU). In order to estimate the potential risk of ASF virus (ASFV) entering the EU, several pathways of introduction were previously assessed separately. The present work aimed to integrate five of these assessments (legal imports of pigs, legal imports of products, illegal imports of products, fomites associated with transport and wild boar movements) into a modular tool that facilitates the visualization and comprehension of the relative risk of ASFV introduction into the EU by each analyzed pathway. RESULTS The framework's results indicate that 48% of EU countries are at relatively high risk (risk score 4 or 5 out of 5) for ASFV entry for at least one analyzed pathway. Four of these countries obtained the maximum risk score for one pathway: Bulgaria for legally imported products during the high risk period (HRP); Finland for wild boar; Slovenia and Sweden for legally imported pigs during the HRP. Distribution of risk considerably differed from one pathway to another; for some pathways, the risk was concentrated in a few countries (e.g., transport fomites), whereas other pathways incurred a high risk for 4 or 5 countries (legal pigs, illegal imports and wild boar). CONCLUSIONS The modular framework, developed to estimate the risk of ASFV entry into the EU, is available in a public domain, and is a transparent, easy-to-interpret tool that can be updated and adapted if required. The model's results determine the EU countries at higher risk for each ASFV introduction route, and provide a useful basis to develop a global coordinated program to improve ASFV prevention in the EU.

A Bayesian approach to study the risk variables for tuberculosis occurrence in domestic and wild ungulates in South Central Spain

BACKGROUND Bovine tuberculosis (bTB) is a chronic infectious disease mainly caused by Mycobacterium bovis. Although eradication is a priority for the European authorities, bTB remains active or even increasing in many countries, causing significant economic losses. The integral consideration of epidemiological factors is crucial to more cost-effectively allocate control measures. The aim of this study was to identify the nature and extent of the association between TB distribution and a list of potential risk factors regarding cattle, wild ungulates and environmental aspects in Ciudad Real, a Spanish province with one of the highest TB herd prevalences. RESULTS We used a Bayesian mixed effects multivariable logistic regression model to predict TB occurrence in either domestic or wild mammals per municipality in 2007 by using information from the previous year. The municipal TB distribution and endemicity was clustered in the western part of the region and clearly overlapped with the explanatory variables identified in the final model: (1) incident cattle farms, (2) number of years of veterinary inspection of big game hunting events, (3) prevalence in wild boar, (4) number of sampled cattle, (5) persistent bTB-infected cattle farms, (6) prevalence in red deer, (7) proportion of beef farms, and (8) farms devoted to bullfighting cattle. CONCLUSIONS The combination of these eight variables in the final model highlights the importance of the persistence of the infection in the hosts, surveillance efforts and some cattle management choices in the circulation of M. bovis in the region. The spatial distribution of these variables, together with particular Mediterranean features that favour the wildlife-livestock interface may explain the M. bovis persistence in this region. Sanitary authorities should allocate efforts towards specific areas and epidemiological situations where the wildlife-livestock interface seems to critically hamper the definitive bTB eradication success.

Identification of Hotspots in the European Union for the Introduction of Four Zoonotic Arboviroses by Live Animal Trade

Live animal trade is considered a major mode of introduction of viruses from enzootic foci into disease-free areas. Due to societal and behavioural changes, some wild animal species may nowadays be considered as pet species. The species diversity of animals involved in international trade is thus increasing. This could benefit pathogens that have a broad host range such as arboviruses. The objective of this study was to analyze the risk posed by live animal imports for the introduction, in the European Union (EU), of four arboviruses that affect human and horses: Eastern and Western equine encephalomyelitis, Venezuelan equine encephalitis and Japanese encephalitis. Importation data for a five-years period (2005-2009, extracted from the EU TRACES database), environmental data (used as a proxy for the presence of vectors) and horses and human population density data (impacting the occurrence of clinical cases) were combined to derive spatially explicit risk indicators for virus introduction and for the potential consequences of such introductions. Results showed the existence of hotspots where the introduction risk was the highest in Belgium, in the Netherlands and in the north of Italy. This risk was higher for Eastern equine encephalomyelitis (EEE) than for the three other diseases. It was mainly attributed to exotic pet species such as rodents, reptiles or cage birds, imported in small-sized containments from a wide variety of geographic origins. The increasing species and origin diversity of these animals may have in the future a strong impact on the risk of introduction of arboviruses in the EU.

A real time Taqman RT-PCR for the detection of rabbit hemorrhagic disease virus 2 (RHDV2)

As key prey, the wild rabbit downsize constitutes a major drawback on the endangered Iberian lynx (Lynx pardinus) re-introduction in the Iberia. Several captive breeding units mostly located in Alentejo, endeavour the wild rabbit repopulation of depleted areas assigned for the lynx re-introduction. Here we report an RHDV2 outbreak that occurred in early 2016 in a wild rabbit captive breeding unit located in Barrancos municipality. The estimated mortality rate between March and April 2016 was approximately 8.67%. Anatomopathologic examination was carried out for 13 victimized rabbits. Molecular characterization was based on the complete vp60 capsid gene. The 13 rabbit carcasses investigated showed typical macroscopic RHD lesions testing positive to RHDV2- RNA. Comparison of the vp60 nucleotide sequences obtained from two specimens with others publically available disclosed similarities below 98.22% with RHDV2 strains originated in the Iberia and Azores and revealed that the two identical strains from Barrancos-2016 contain six unique single synonymous nucleotide polymorphisms. In the phylogenetic analysis performed, the Barrancos-2016 strains clustered apart from other known strains, meaning they may represent new evolutionary RHDV2 lineages. No clear epidemiological link could be traced for this outbreak where the mortalities were lower compared with previous years. Yet, network analysis suggested a possible connection between the missing intermediates from which the strains from Barrancos 2013, 2014 and 2016 have derived. It is therefore possible that RHDV2 has circulated endemically in the region since 2012, with periodic epizootic occurrences. Still, six years after its emergence in wild rabbits, RHDV2 continues to pose difficulties to the establishment of natural wild rabbit populations that are crucial for the self-sustainability of the local ecosystems.

Wild carrot differentiation in Europe and positive selection at DcAOX1 gene

By definition, the domestication process leads to an overall reduction of crop genetic diversity. This lead to the current search of genomic regions in wild crop relatives (CWR), an important task for modern carrot breeding. Nowadays massive sequencing possibilities can allow for discovery of novel genetic resources in wild populations, but this quest could be aided by the use of a surrogate gene (to first identify and prioritize novel wild populations for increased sequencing effort). Alternative oxidase (AOX) gene family seems to be linked to all kinds of abiotic and biotic stress reactions in various organisms and thus have the potential to be used in the identification of CWR hotspots of environment-adapted diversity. High variability of DcAOX1 was found in populations of wild carrot sampled across a West-European environmental gradient. Even though no direct relation was found with the analyzed climatic conditions or with physical distance, population differentiation exists and results mainly from the polymorphisms associated with DcAOX1 exon 1 and intron 1. The relatively high number of amino acid changes and the identification of several unusually variable positions (through a likelihood ratio test), suggests that DcAOX1 gene might be under positive selection. However, if positive selection is considered, it only acts on some specific populations (i.e. is in the form of adaptive differences in different population locations) given the observed high genetic diversity. We were able to identify two populations with higher levels of differentiation which are promising as hot spots of specific functional diversity.

Challenges in the rabbit haemorrhagic disease 2 (RHDV2) molecular diagnosis of vaccinated rabbits

Molecular methods are fundamental tools for the diagnosis of viral infections. While interpretation of results is straightforward for unvaccinated animals, where positivity represents ongoing or past infections, the presence of vaccine virus in the tissues of recently vaccinated animals may mislead diagnosis. In this study, we investigated the interference of RHDV2 vaccination in the results of a RT-qPCR for RHDV2 detection, and possible associations between mean Cq values of five animal groups differing in age, vaccination status and origin (domestic/wild). Viral sequences from vaccinated rabbits that died of RHDV2 infection (n = 14) were compared with the sequences from the commercial vaccines used in those animals. Group Cq means were compared through Independent t-test and One-way ANOVA. We proved that RHDV2 vaccine-RNA is not detected by the RT-qPCR as early as 15 days post- vaccination, an important fact in assisting results interpretation for diagnosis. Cq values of vaccinated and non-vaccinated infected domestic adults showed a statistically significant difference (p < 0.05), demonstrating that vaccination-induced immunity reduces viral loads and delays disease progression. Contrarily, in vaccinated young rabbits higher viral loads were registered compared to non-vaccinated kittens. No significant variation (p = 0.3824) was observed between viral loads of non- vaccinated domestic and wild RHDV2-victimised rabbits. Although the reduced number of vaccinated young animals analysed hampered a robust statistical analysis, this occurrence suggests that passively acquired maternal antibodies may inhibit the active immune response to vaccination, delaying protection and favouring disease progression. Our finding emphasises the importance of adapting kitten RHDV2 vaccination schedules to circumvent this interference phenomenon.