Hugo Fernández-Bellon

Histological and immunohistochemical study of clinically normal skin of Leishmania infantum-infected dogs

Skin lesions are the most usual manifestation of canine leishmaniosis. The aim of this study was to investigate the histological pattern and parasite load in clinically normal skin of Leishmania-infected dogs. Two groups of Leishmania-infected dogs were studied. Group A consisted of 15 symptomless animals which, although seronegative or only mildly seropositive, gave a positive polymerase chain reaction (PCR) for Leishmania in the skin. Group B consisted of 20 clinically affected dogs which were highly seropositive and PCR-positive. Biopsies of normal skin from all dogs were processed for routine histology and Leishmania immunohistochemistry. The study demonstrated microscopical lesions and the presence of parasites in the skin from dogs of group B, but not group A. The results cast doubt on the relevance of infected but symptomless dogs in the epidemiology of canine leishmaniosis. In contrast, however, the clinically normal skin of sick dogs harbours the parasite and probably plays a role in the transmission of leishmaniosis.

The interplay between DNA methylation and sequence divergence in recent human evolution

Despite the increasing knowledge about DNA methylation, the understanding of human epigenome evolution is in its infancy. Using whole genome bisulfite sequencing we identified hundreds of differentially methylated regions (DMRs) in humans compared to non-human primates and estimated that ∼25% of these regions were detectable throughout several human tissues. Human DMRs were enriched for specific histone modifications and the majority were located distal to transcription start sites, highlighting the importance of regions outside the direct regulatory context. We also found a significant excess of endogenous retrovirus elements in human-specific hypomethylated. We reported for the first time a close interplay between inter-species genetic and epigenetic variation in regions of incomplete lineage sorting, transcription factor binding sites and human differentially hypermethylated regions. Specifically, we observed an excess of human-specific substitutions in transcription factor binding sites located within human DMRs, suggesting that alteration of regulatory motifs underlies some human-specific methylation patterns. We also found that the acquisition of DNA hypermethylation in the human lineage is frequently coupled with a rapid evolution at nucleotide level in the neighborhood of these CpG sites. Taken together, our results reveal new insights into the mechanistic basis of human-specific DNA methylation patterns and the interpretation of inter-species non-coding variation.

Little evidence of seasonal variation of natural infection by Leishmania infantum in dogs in Spain.

Leishmania infantum, the etiological agent of canine leishmaniosis in the Mediterranean region, is vectored by Phlebotomus spp sandflies, which are active during the warmer months of the year. In order to determine whether seasonality in transmission induces seasonal changes in the prevalence of infection by L. infantum and of parasite-specific immune response, two groups of dogs, one in February (n=37) and another in October (n=42), were studied. Clinical signs compatible with leishmaniosis, as well as presence of microscopic skin lesions in the muzzle were recorded for all dogs. Assays were also performed for detection of L. infantum parasites in muzzle skin samples (PCR, immunohistochemistry and culture), specific serum antibodies (ELISA), and specific lymphocyte proliferation and interferon-gamma production. Although prevalence of non-specific clinical signs increased significantly after the sandfly season, this was not the case for Leishmania-specific markers: positivity by PCR (24% vs. 21%) or immunohistochemistry (3% vs. 2%) of muzzle skin samples, as well as lymphocyte proliferation (59% vs. 50%) or interferon-gamma production (21% vs. 27%) were similar in February and in October. Only prevalence of positive specific antibody titers increased noticeably in October (8% vs. 20%), although this was not statistically significant. Overall, the sandfly season did not have a marked impact on the prevalence L. infantum infection or parasite-specific immune responses analyzed in this study.

Aggressive behavior and hair cortisol levels in captive Dorcas gazelles (Gazella dorcas) as animal-based welfare indicators

Ensuring welfare in captive wild animal populations is important not only for ethical and legal reasons, but also to maintain healthy individuals and populations. An increased level of social behaviors such as aggression can reduce welfare by causing physical damage and chronic stress to animals. Recently, cortisol in hair has been advanced as a non-invasive indicator to quantify long-lasting stress in many species. The sensitivity of social behavior and hair cortisol concentration was evaluated in several groups of dorcas gazelles (Gazella dorcas). Four different groups of gazelles from three different zoos were observed and the expression of intra-specific affiliative and negative social behaviors was assessed across the different groups. Hair samples were taken from sub-groups of animals and analyzed for cortisol concentrations. Significant differences between groups of dorcas gazelles were found in frequency of negative social behavior and hair cortisol concentration. Despite the low sample size, these two parameters had a positive Spearman correlation coefficient (rs  = +0.80, P = 0.20). These results suggest that hair cortisol levels are sensitive to differences in the social structure of dorcas gazelles. Zoo Biol. 35:467-473, 2016.

Functional Implications of Human-Specific Changes in Great Ape microRNAs.

microRNAs are crucial post-transcriptional regulators of gene expression involved in a wide range of biological processes. Although microRNAs are highly conserved among species, the functional implications of existing lineage-specific changes and their role in determining differences between humans and other great apes have not been specifically addressed. We analyzed the recent evolutionary history of 1,595 human microRNAs by looking at their intra- and inter-species variation in great apes using high-coverage sequenced genomes of 82 individuals including gorillas, orangutans, bonobos, chimpanzees and humans. We explored the strength of purifying selection among microRNA regions and found that the seed and mature regions are under similar and stronger constraint than the precursor region. We further constructed a comprehensive catalogue of microRNA species-specific nucleotide substitutions among great apes and, for the first time, investigated the biological relevance that human-specific changes in microRNAs may have had in great ape evolution. Expression and functional analyses of four microRNAs (miR-299-3p, miR-503-3p, miR-508-3p and miR-541-3p) revealed that lineage-specific nucleotide substitutions and changes in the length of these microRNAs alter their expression as well as the repertoires of target genes and regulatory networks. We suggest that the studied molecular changes could have modified crucial microRNA functions shaping phenotypes that, ultimately, became human-specific. Our work provides a frame to study the impact that regulatory changes may have in the recent evolution of our species.

Vaccination against H5 avian influenza virus induces long-term humoral immune responses in flamingoes (Phoenicopterus spp.)

Avian influenza (AI) can represent a threat to endangered wild birds, as demonstrated with the H5N1 highly pathogenic AI (HPAI) outbreaks. Vaccination against AI using inactivated H5-vaccines has been shown to induce humoral immune response in zoo bird species. In this study, the long-term efficacy of H5-vaccination was evaluated in flamingoes from Barcelona Zoo. Specific H5-antibody titres were maintained at high levels (geometric mean titres ≥32) for over 7 years after vaccination, both against the H5N9 and H5N3 vaccine strains, as well as H5N3 and H5N1 reference strains. In addition the breadth of the immune response was also studied by testing antibody production against H1-, H3-, H4-, H7-, and H10-subtypes. It was observed that most flamingoes presented specific antibodies against H1 virus subtypes, but titres to the other HA-subtypes were rarely detected. We show that AI-vaccines can induce immunity lasting seven years in flamingoes, which suggests that vaccination can provide long term protection from HPAI outbreaks in zoo birds.

RNA editing independently occurs at three mir-376a-1 sites and may compromise the stability of the microRNA hairpin

RNA editing is being recognized as an important post-transcriptional mechanism that may have crucial roles in introducing genetic variation and phenotypic diversity. Despite microRNA editing recurrence, defining its biological relevance is still under extended debate. To better understand microRNA editing function and regulation we performed an exhaustive characterization of the A-to-I site-specific patterns in mir-376a-1, a mammalian microRNA which RNA editing is involved in the regulation of development and in disease. Thorough an integrative approach based on high-throughput small RNA sequencing, Sanger sequencing and computer simulations we explored mir-376a-1 editing in samples from various individuals and primate species including human placenta and macaque, gorilla, chimpanzee and human brain cortex. We observed that mir-376a-1 editing is a common phenomenon in the mature and primary microRNA molecules and it is more frequently detected in brain than in placenta. Primary mir-376a-1 is edited at three positions, -1, +4 and +44. Editing frequency estimations and in silico simulations indicated that editing was not equally recurrent along the three mir-376a-1 sites, nevertheless no epistatic interactions among them were observed. Particularly, the +4 site, located in the seed region of the mature miR-376a-5p, reached the highest editing frequency in all samples. Secondary structure predictions revealed that the +4 position was the one that conferred the highest stability to the mir-376a-1 hairpin. We suggest that molecular stability might partially explain the editing recurrence observed in certain microRNAs and that editing events conferring new functional regulatory roles in particular tissues and species could have been conserved along evolution, as it might be the case of mir-376a-1 in primate brain cortex.

Social organization and demography of reintroduced Dorcas gazelle (Gazella dorcas neglecta) in North Ferlo Fauna Reserve, Senegal

Abstract As part of a reintroduction project in Senegal, 23 (9.14) captive-born Dorcas gazelles were released into a 440-ha fenced-in area in Katané (North Ferlo Fauna Reserve, Senegal) in March 2009. After 4 years of seasonal monitoring, the gazelles showed progressive adaptation of their behavior to semi-wild living conditions. Breeding gradually became seasonal, and 53.8% of births occurred during the rainy season (July to September). Gazelle group size and composition varied seasonally. Groups were smallest during the dry season (2.29±1.72) and largest at the beginning of the rainy season (4.18±2.73). Social group composition also showed seasonal variation. There were always a larger proportion of solitary males than solitary females and mixed couples were observed throughout the year. All-male groups were found the least. The proportion of adult females with subadults and juveniles decreased during the early rainy season, while mixed adults, subadults and juveniles groups increased during this period. The mortality rate during the first weeks after release was 13%. Four years of monitoring after release, demographic traits of this released population reveal its adaptation from captive to natural-living conditions.

Using Farm Animal Welfare Protocols as a Base to Assess the Welfare of Wild Animals in Captivity—Case Study: Dorcas Gazelles (Gazella dorcas)

Simple Summary Animal welfare is gaining attention regarding the conservation of species not only due to ethical and legal reasons but also because optimal welfare can ensure stable and healthy populations. Currently, there is a lack of protocols that help to objectively assess welfare of wild animals in captivity. In this study, we have developed and applied a protocol for the assessment of welfare in captive dorcas gazelles (Gazella dorcas). We have gathered information from existing literature about the biology of this species in wild conditions, as well as in zoo husbandry, breeding, management and care guidelines developed for this species. We took a protocol developed for the on-farm welfare assessment in cattle as a reference and suggested 23 indicators that we considered useful to assess welfare in captive dorcas gazelles. To test the utility of this protocol, we then applied it in five groups of dorcas gazelles from three different zoos and we detected areas for improvement in all of the groups assessed. Abstract There is a lack of protocols specifically developed for the assessment of welfare of wild animals in captivity, even when it is known that providing good standards of welfare is important. The aim of this study was the development and the application of a protocol for the assessment of welfare in captive dorcas gazelles. The protocol was mainly developed taking into account the protocol for the assessment of welfare in cattle from the Welfare Quality® project, the available literature of the biology of this species and the Husbandry Guidelines developed for captive breeding and management of this species. The protocol was specifically developed for dorcas gazelles and included four principles, 10 criteria and 23 animal and environmental-based indicators. To test its utility, this protocol was applied to five different groups of gazelles from three different zoos. Its application made possible to detect areas for improvement in all groups assessed.

Daily salivary cortisol levels in response to stress factors in captive common bottlenose dolphins (Tursiops truncatus): a potential welfare indicator

DOLPHINS in a captive setting can be occasionally subjected to a variety of potentially stressful situations. The stress response comprises a variety of biological reactions to internal or external stimuli elicited when an individual perceives (real or not) a threat to its homoeostasis (Moberg and Mench 2000). The stress response is a complex interplay of behavioural and physiological strategies to cope with changes in the environment (Tsigos and Chrousos 2002, Sheriff and others 2011). Noise can act as a stressor to dolphins because cetaceans rely heavily on sound for many important life functions. This reliance on sound means it is quite likely that exposure to noise will have some detrimental effects on these life functions (Wright and others 2007). Observed effects of noise on marine mammals include: changes in vocalisations, respiration, swim speed, diving and foraging behaviour; displacement; avoidance; shifts in migration path; stress; hearing damage and strandings, but responses of marine mammals to noise can often be subtle and barely detectable (Weilgart 2007). If there is no obvious change in behaviour, stress is difficult to assess in dolphins, both individually and as a group. In order to detect the presence of stress responses in these situations, the endocrine response to stressors is assessed evaluating the hypothalamic–pituitary–adrenal (HPA) axis activity in dolphins and other cetaceans (Wright and others 2007, Amaral 2010). The HPA axis is a key element of the stress response and, when activated, the resultant hormonal cascade increases the levels of glucocorticoids (GCs) …