Detection of rare genetic variations in the promoter regions of the ATG16L gene in Parkinson's patients.

Mutations in the ATG genes have been related to impair autophagic function, contributing to the sporadic onset of Parkinsons Disease (PD). However, scarce studies have been performed about ins/del within the regulatory domains of the autophagy genes in sporadic PD patients. This study was aimed to found ins/del within part of the crucial core autophagy promotor gene region of the ATG16L1 in a groups of sporadic PD patients. After developing a genetic marker to find ins/del using fragment size analysis, a rare mutation by insertion (0.45%) was reported in the patients. This mutation was characterized by sequencing. No others ins/del were found. As a results, the frequency of this insertion should be considered as a rare genetic variant. An in silico analysis also highlighting the usefulness of a search GDV which revealed multiples ins/del within ATG16L1 promoter. Furthermore, these genetic insertions could be found in patients with sporadic PD in the ATG161L promoter gene. When a breakpoint as deletions, insertions or tandem duplication are located within a functional gene interruption of the gene and a loss of function was expected but removing or altering in the regulatory sequence can influence the expression or the regulation of a nearby gene which may impair healthy due to dosage effects in sporadic diseases.

Molecular survey of Besnoitia spp. (Apicomplexa) in faeces from European wild mesocarnivores in Spain.

Numerous studies have unsuccessfully tried to unravel the definitive host of the coccidian parasite Besnoitia besnoiti. Cattle infections by B. besnoiti cause a chronic and debilitating condition called bovine besnoitiosis that has emerged in Europe during the last two decades, mainly due to limitations in its control associated with the absence of vaccines and therapeutical tools. Although the exact transmission pathways of B. besnoiti is currently unknown, it is assumed that the parasite might have an indirect life cycle with a carnivore as definitive host. Current lack of studies in wildlife might underestimate the importance of free-living species in the epidemiology of B. besnoiti. Thus, the aim of the present study is to assess the presence of Besnoitia spp. in free-ranging mesocarnivores in Spain. DNA was searched by PCR on faeces collected from wild carnivores as a first approach to determine which species could be considered as potential definitive host candidates in further research. For this purpose, a total of 352 faecal samples from 12 free-living wild carnivore species belonging to the Canidae, Felidae, Herpestidae, Mustelidae, Procyonidae and Viverridae families were collected in seven Spanish regions. PCR testing showed that Besnoitia spp. DNA was present in four faecal samples from red foxes collected in western Spain, an area with the greatest density of extensively reared cattle and associated with high incidence of bovine besnoitiosis in the country. To date, this is the first report of a B. besnoiti-like sequence (99.57% homology) from carnivore faeces in a worldwide context. Red foxes might contribute to the epidemiology of B. besnoiti, although further studies, mostly based on bioassay, would be needed to elucidate the accuracy and extent of these interesting findings.

Blastocystis sp. Subtype Diversity in Wild Carnivore Species from Spain.

The occurrence and molecular diversity of the stramenopile eukaryote Blastocystis sp. was investigated by PCR and sequencing (Sanger and NGS) methods in 380 faecal specimens of free-living carnivores in Spain. Blastocystis sp. was confirmed in 1.6% (6/380) of the specimens analysed. Two samples from a common genet and a fox were successfully subtyped as ST7 by Sanger. Using NGS, ST14 was found in a fox and a European polecat, ST7 in a fox, and two additional foxes presented mixed infections of ST1/ST2/ST4 and ST1/ST2/ST7, respectively. Wild carnivore species could act as carriers of zoonotic Blastocystis subtypes.

Phaeocystis globosa Virus DNA Polymerase X: a "Swiss Army knife", Multifunctional DNA polymerase-lyase-ligase for Base Excision Repair.

Phaeocystis globosa virus 16T is a giant virus that belongs to the so-called nucleo-cytoplasmic large DNA virus (NCLDV) group. Its linear dsDNA genome contains an almost full complement of genes required to participate in viral base excision repair (BER). Among them is a gene coding for a bimodular protein consisting of an N-terminal Polß-like core fused to a C-terminal domain (PgVPolX), which shows homology with NAD+-dependent DNA ligases. Analysis of the biochemical features of the purified enzyme revealed that PgVPolX is a multifunctional protein that could act as a "Swiss army knife" enzyme during BER since it is endowed with: 1) a template-directed DNA polymerization activity, preferentially acting on DNA structures containing gaps; 2) 5'-deoxyribose-5-phosphate (dRP) and abasic (AP) site lyase activities; and 3) an NAD+-dependent DNA ligase activity. We show how the three activities act in concert to efficiently repair BER intermediates, leading us to suggest that PgVPolX may constitute, together with the viral AP-endonuclease, a BER pathway. This is the first time that this type of protein fusion has been demonstrated to be functional.

Occurrence and molecular genotyping of Giardia duodenalis and Cryptosporidium spp. in wild mesocarnivores in Spain.

There is a surprisingly scarce amount of epidemiological and molecular data on the prevalence, frequency, and diversity of the intestinal protozoan parasites Giardia duodenalis and Cryptosporidium spp. in wildlife in general and mesocarnivore species in particular. Consequently, the extent of the cyst/oocyst environmental contamination attributable to these wild host species and their potential implications for public veterinary health remain largely unknown. In this molecular epidemiological survey a total of 193 individual faecal samples from badgers (Meles meles, n=70), ferrets (Mustela putorius furo, n=2), genets (Genetta genetta, n=6), Iberian lynxes (Lynx pardinus, n=6), beech martens (Martes foina, n=8), mongooses (Herpestes ichneumon, n=2), otters (Lutra lutra, n=2), polecats (Mustela putorius, n=2), red foxes (Vulpes vulpes, n=87), wildcats (Felis silvestris, n=2), and wolves (Canis lupus, n=6) were obtained from road-killed, hunted, and accidentally found carcasses, and from camera-trap surveys or animals entering rescue shelters, during the period December 2003-April 2016. Investigated specimens were collected in five Spanish autonomous regions including Andalusia (n=1), Asturias (n=69), Basque Country (n=49), Castile-La Mancha (n=38), and Extremadura (n=36). The presence of cysts/oocysts was confirmed by PCR-based methods targeting the small subunit (ssu) ribosomal RNA gene of these parasite species. Genotyping of the obtained isolates were attempted at appropriate markers including the glutamate dehydrogenase (G. duodenalis) and the 60-kDa glycoprotein (C. parvum and C. ubiquitum) loci. Overall, G. duodenalis was detected in 8% (7/87) of red foxes, a single beech marten, and a single wolf, respectively. Cryptosporidium was identified in 3% (2/70) of badgers, 8% (7/87) of red foxes, a single genet, and a single mongoose, respectively. None of the nine G. duodenalis isolates generated could be genotyped at the assemblage/sub-assemblage level. Out of the nine Cryptosporidium isolates successfully characterized, three were identified as C. canis (one in a mongoose and two in red foxes), and three as C. parvum (one in a badger and three in red foxes). The remaining three isolates were assigned to C. felis (in a red fox), C. hominis (in a badger), and C. ubiquitum (in a red fox), respectively. Two additional Cryptosporidium isolates infecting a badger and a genet, respectively, were untypable. The red fox was confirmed as a suitable host of potentially zoonotic Cryptosporidium species, mainly C. parvum and C. ubiquitum. The high mobility and wide home range of red foxes, together with their increasing presence in urban and peri-urban settings, may led to the overlapping of sylvatic and domestic cycles of the parasite, and consequently, to an increased risk of cryptosporidiosis in production animals and humans. The detection of C. hominis oocysts in a badger raises the question of whether this finding represents a true infection or a sporadic event of mechanical passage of C. hominis oocyst of anthroponotic origin.

Low heterozygosity at microsatellite markers in Iberian red deer with small antlers.

Deer antlers are costly structures subjected to directional sexual selection that may be sensitive to heterozygosity. However, a relationship between heterozygosity and antler development has only been found for select protein-coding loci and MHC genes in one deer species (the white-tailed deer Odocoileus virginianus). Here, we study the relationship between multilocus heterozygosity at 11 microsatellite markers and antler size (AS) in a sample of 367 Iberian red deer males (Cervus elaphus hispanicus) from two study areas with different ecological and genetic conditions. We found that males with very small antlers (10% of the sampled individuals with the lowest values of AS) had lower levels of heterozygosity than those with bigger antlers (significant effect in an analysis of variance, P = 0.011). This relationship was noticeable mainly in situations of low genetic diversity, where the differences in heterozygosity between groups of males were greater. Finally, we conducted analyses to address the hypotheses proposed by the heterozygosity-fitness correlation, and we found the local effect as the most likely hypothesis. Our findings reveal an expected but not previously detected association between low heterozygosity and reduced AS, with implications for red deer evolution and management.