The wildcat (Felis s. silvestris) in the Mediterranean forest: sighting through photo-trapping and non-invasive hair collection for genetic purposes.

The European wildcat (Felis silvestris silvestris) is a mesocarnivore species widely distributed in Europe, from Eastern Europe to Portugal and from Scotland to Italy. Recent biogeographical studies of wildcat populations have endeavoured to assess in detail the various issues that pose a threat to this species, including hybridization with domestic cats. The use of non-invasive sampling methods supported by photo-trapping and some attractants has made it possible to gather genetic material for the detection of native wildcats in locally threatened populations, some of which live in the Iberian Peninsula. Testimonies of naturalists, hunters and farm workers led our team to choose specific areas in two large territories of Mediterranean forests where the presence of wildcats has been historically attested: the Almonte River basin and the Sierra de San Pedro Mountains. Between 2014 and 2018, non-invasive hair sampling was performed using valerian (Valeriana officinalis) as an attractant and supported by photo-trapping to guarantee the collection of genuine biological material (hair samples). The hair samples were genetically assessed by sequencing the nuclear gene IRBP (interphotoreceptor retinoid-binding protein) and the mtDNA gene ND4 (NADH dehydrogenase subunit 4). Despite the low density of wildcats, this combined protocol proved to be an applicable tool for detecting the presence of elusive wildcats and other mesocarnivore species in this remote region of southern Europe. In addition, non-invasive hair trapping contributes to the collection of genetic material from current wildcat populations. This procedure could enhance future management actions focused on collecting quality individualized biological material.

Application of real-time PCR for the identification of the endangered species Galemys pyrenaicus through faecal samples.

BACKGROUND: Currently, many micromammals are important targets for study. The endangered Galemys pyrenaicus is an outstanding example. Globally, their populations have suffered a substantial decline in last 20 years. In the surveyed area, the capture of desman is legally forbidden due to the high conservation concerns. Reason by non-invasive sampling through faeces is proposed for its monitoring. Furthermore, the confusion between faeces from desman and Mediterranean water shrews must be considered. Thus, the aim of this study was focused on developing RT-PCR assays to determine the presence of Galemys pyrenaicus and N. a. anomalus from non-invasive samples. METHODS AND RESULTS: The study was conducted in the mountains of the System Central of Extremadura (Spain). A total of 186 samples were collected from 2018 to 2021 by experts where historically reported and/or our previous studies confirmed their presence. RT-PCR assays using hydrolysis probes were designed to detect genetic material from both desman and Mediterranean water shrews and its specificity was confirmed. The reliability of the method was further assessed by PCR sequencing of mitochondrial Cyb and d-loop, resulting fully compatible with the RT-PCR approach. Intraspecific phylogenetic relationship was reported to improve knowledge about mtDNA variability in the desman from the Central System. CONCLUSIONS: We demonstrated that RT-PCR gives a gold opportunity to further map the species using faeces which minimizes disturbance and reports both population status and individual presence. Cost-effective RT-PCR combined with field-collected faeces allows us to better investigate the full range of occurrence of the species.

Comparative Genetic Analysis of the Promoters of the ATG16L1 and ATG5 Genes Associated with Sporadic Parkinson's Disease.

Sporadic Parkinson's disease, characterised by a decline in dopamine, usually manifests in people over 65 years of age. Although 10% of cases have a genetic (familial) basis, most PD is sporadic. Genome sequencing studies have associated several genetic variants with sporadic PD. Our aim was to analyse the promoter region of the ATG16L1 and ATG5 genes in sporadic PD patients and ethnically matched controls. Genotypes were obtained by using the Sanger method with primers designed by us. The number of haplotypes was estimated with DnaSP software, phylogeny was reconstructed in Network, and genetic divergence was explored with Fst. Seven and two haplotypes were obtained for ATG16L1 and ATG5, respectively. However, only ATG16L1 showed a significant contribution to PD and a significant excess of accumulated mutations that could influence sporadic PD disease. Of a total of seven haplotypes found, only four were unique to patients sharing the T allele (rs77820970). Recent studies using MAPT genes support the notion that the architecture of haplotypes is worthy of being considered genetically risky, as shown in our study, confirming that large-scale assessment in different populations could be relevant to understanding the role of population-specific heterogeneity. Finally, our data suggest that the architecture of certain haplotypes and ethnicity determine the risk of PD, linking haplotype variation and neurodegenerative processes.

Non-Invasive Wildlife Disease Surveillance Using Real Time PCR Assays: The Case of the Endangered Galemys pyrenaicus Populations from the Central System Mountains (Extremadura, Spain).

The Iberian desman (Galemys pyrenaicus) is a small semi-aquatic mammal that inhabits mountainous areas from the centre to the north of the Iberian Peninsula and the Pyrenees and is listed as endangered because it has suffered a serious decline. Since 1960, only three species of digeneans (Omphalometra flexuosa, Maritrema pyrenaica and Mathovius galemydis) and two nematodes (Aonchotheca galemydis and Paracuaria hispanica) have been reported from the desman, but no further information on health status and no data from Extremadura has been available. The aim of our study was to characterise the diversity and distribution of parasites and microbiomes of desmans in different areas of the Central System of Extremadura. Between 2019 and 2021 we collected 238 fecal samples and one tissue (intestine) sample that was obtained from a dead desman. DNA templates were processed by commercial or customised real-time PCR using TaqMan probes. Representative data were obtained for Cryptosporidium spp., Omphalometra spp., Eimeria spp., Salmonella spp., Staphylococcus spp. and Leptospira spp. Omphalometra spp. was studied using a newly developed PCR test. The screening of the dead desman allowed us to obtain, for the first time, a partial sequence of the 18SrDNA. This study is the most complete study of the desman, allowing us to identify parasites and the microbiome in populations of G. pyrenaicus using non-invasive sampling.

Detection of Myxoma Virus DNA in Ticks from Lagomorph Species in Spain Suggests Their Possible Role as Competent Vector in Viral Transmission.

Myxoma virus (MYXV) causes morbidity and mortality in European wild rabbits (Oryctolagus cuniculus) worldwide, and recently in Iberian hares (Lepus granatensis) in Spain. We aimed to assess the presence of MYXV-specific DNA in ixodid ticks collected from both hosts. A total of 417 ticks harvested from 30 wild lagomorphs, including wild rabbits and Iberian hares were collected from southern Spain. Enzyme-linked immunosorbent assay and PCR-sequencing were used to detect virus exposure and presence, respectively. Antibodies to MYXV were detected in 68% (17/25) of wild rabbits and in 67% (2/3) of Iberian hares. We detected MYXV DNA in 50.7% of pools of two different tick species (nymphs and adults of Rhipicephalus pusillus, and nymphs of Hyalomma lusitanicum) parasitizing rabbits and hares. The obtained partial sequence of the viral major envelope protein gene showed a mutation (G383A) within the MYXV_gp026 locus between the rabbit strain and Iberian hare strain (recently isolated in tissues of infected hares from Spain). However, in our study, the viral DNA presence was detected for the first time using tick DNA as the PCR-template, but the possible role of ticks as vectors of MYXV still needs to be elucidated.

Identification and genetic characterization of Sarcocystis arctica and Sarcocystis lutrae in red foxes (Vulpes vulpes) from Baltic States and Spain.

BACKGROUND: Typically, carnivores serve as definitive hosts for Sarcocystis spp. parasites; currently, their role as intermediate hosts is being elucidated. The present study aimed to identify and molecularly characterize Sarcocystis cysts detected in striated muscle of red foxes from different populations in Latvia, Lithuania and Spain. METHODS: Muscle samples from 411 red foxes (Vulpes vulpes) and 269 racoon dogs (Nyctereutes procyonoides) from Latvia, 41 red foxes from Lithuania and 22 red foxes from Spain were examined for the presence of Sarcocystis sarcocysts by light microscopy (LM). Sarcocystis spp. were identified by transmission electron microscopy (TEM) and molecular biology techniques. RESULTS: Sarcocystis cysts were detected in 11/411 (2.7%) Latvian, 3/41 (7.3%) Lithuanian, and 6/22 (27.3%) Spanish red foxes, however, cysts were not observed in the muscles of racoon dogs. Based on LM, TEM, 18S rDNA, 28S rDNA, ITS1, cox1 and rpoB sequences, Sarcocystis arctica and Sarcocystis lutrae cysts were identified in red fox muscles from Latvia and Lithuania, whereas only S. arctica was detected in Spain. The 18S rDNA, 28S rDNA and ITS1 sequences from the 21 isolates of S. arctica from Latvia, Lithuania and Spain were identical. By contrast, two and four haplotypes were determined based on mtDNA cox1 and apicoplast rpoB sequences, respectively. Polymorphisms were not detected between the two isolates of S. lutrae from Latvia and Lithuania. Based on phylogenetic results, S. arctica and S. lutrae were most closely related to Sarcocystis spp. using predatory mammals as intermediate hosts and to Sarcocystis species with a bird-bird life-cycle. CONCLUSIONS: Based on current knowledge, the red fox and Arctic fox (Vulpes lagopus) could act as intermediate host for the same two Sarcocystis species. Molecular results suggest the existence of two genetic lineages of S. arctica, and such divergence relies on its geographical distribution but not on their intermediate host species.

Seroprevalence of Theileria equi and Babesia caballi in horses in Spain.

Equine piroplasmoses are enzootic parasitic diseases distributed worldwide with high incidence in tropical and subtropical regions. In Spain, there is insufficient epidemiological data about equine piroplasmoses. The main aim of the present study was therefore to estimate the prevalence of Theileria equi and Babesia caballi in five regions and obtain information about the risk factors. This study was conducted in the central and south-western regions of Spain, using indirect fluorescence antibody testing (IFAT) in 3,100 sera samples from apparently healthy horses of different ages, breeds, coat colours, genders and geographical locations. The overall seroprevalence was 52%, consisting of 44% seropositive for T. equi and 21% for B. caballi. There was a significant association between age (p < 0.0001), breed (p < 0.004), geographical location (p < 0.0001) and the seroprevalence, but neither the coat colour nor the gender was significantly associated with prevalence. In addition, it was proved that most of the geographic areas showed a moderate to high prevalence. The statistical κ value was used to compare the results obtained by the IFAT and the competitive enzyme-linked immunosorbent assay (cELISA) utilised to test some samples (n = 108) and showed a higher concordance for T. equi (κ = 0.68) than for B. caballi (κ = 0.22). Consequently, this revealed the importance of developing an appropriate technique to detect each haemoparasite.

Genetic Variation of the ß-tubulin Gene of Babesia caballi Strains.

BACKGROUND: Equine piroplasmosis is caused by two haemoprotozoan parasites: Babesia caballi and Theileria equi. Negative economic impact on international trade has been associated to endemic sites. This is the reason why carrier detection requires reliable diagnostic methods. Various diagnostic modalities can be used alone or in combination including PCR. However, genetic variation of commonly used genes is still of debate. The aim of this research was to sequence the ß-tubulin gene of a B. caballi strain from Spain and to compare it with known ß-tubulin sequences. METHODS: DNA was isolated from a cryopreserved strain from Spain and acute and chronic carrier horses. Firstly, degenerated primer pairs were designed based on GenBank sequences of different Babesia and Theileria species for sequencing. The primers were redesigned to amplify both parasites, simultaneously. Finally, a species-specific primer pair for B. caballi was designed and a Restriction Fragment Length Polymorphism-PCR (PCR-RFLP) assay performed to know the difference of known B. caballi strains. RESULTS: We provided new insights of the ß-tubulin gene and a good molecular coverage of this gene, contributing with a number of useful primers to amplify T. equi and B. caballi. Moreover, PCR-RFLP assays based on the exon II of this gene confirmed the causative B. caballi strain in Spanish horses. CONCLUSION: We reported useful primer pairs for diagnostic and a new sequence of the ß-tubulin gene of B. caballi, which will facilitate the development of future assays and the detection of infected horses, preventing thus the spread of this disease worldwide.

Characterization of an outbreak of emerging bovine besnoitiosis in southwestern Spain.

Bovine besnoitiosis is an emerging disease in Europe, presenting quick spread toward central and southern Spain. Characterization of an outbreak in a free-ranging Limousin and Avileña beef cattle herd from southwestern Spain territories is attempted. Serological survey in the herd revealed increase of number of infected animals, from 34.3 % on first diagnoses/exams on December 2013 to 42.5 % in the second on April 2014. Blood analysis and serum biochemistry showed important alterations like leukocytosis (+33.2 % of mean value), with lymphocytosis (+205.3 %) and increase of LDH (+25.1 %), associated with tissue damage. Clinical cases were only observed in Limousin animals. Along with typical lesions of acute and chronic besnoitiosis, inflammatory and degenerative processes and parasitic cysts were present in the corpus cavernosum and the corpus spongiosum of penis. By using polymerase chain reaction (PCR) sequencing of 18S rDNA, Besnoitia besnoiti was confirmed as causative agent; microsatellite sequence analyses showed the homology of isolates with previously studied strains.

[Quality improvement program of nosocomial infection in colorectal cancer surgery]. / Programa de mejora de la infección nosocomial en cirugía por cáncer colorrectal.

BACKGROUND AND OBJECTIVE: The surgical-site infection (SSI) is a complication of colorectal neoplasia surgery. The objectives of the study were to identify the SSI risk factors associated with colon surgery and to describe a strategy of quality improvement using surgical-site rates. PATIENTS AND METHOD: Prospective cohort study of in-patients undergoing neoplasia colorectal surgery between 1st July 2002 to 30th June 2003. A descriptive analysis was implemented. Benchmarking was used as tool of quality improvement, and the outcomes were measured using the standardized infection ratio (SIR). To define the risk factors, the Chi square test and logistic regression test were used in univariate and multivariate analysis, respectively. RESULTS: 148 patients were included in the study. The SSI accumulative incidence rate (IA) was 10.14%, and the incidence rate was 6.47 SSI per 1000 days. The SIR was 1.53 the first semester and 1.02 the second one. The multivariate analysis identified two risk factors associated with SSI: unscheduled admission (odds ratio [OR] = 7.47, 95% confidence interval [CI] 2.03-27.48) and a risk index of American Society of Anaesthesiologists (ASA) > or = 3 (OR = 6.77, IC 95%, 1.15-39.84). CONCLUSIONS: An unscheduled admission and high risk ASA index were risk factors associated with SSI in patients undergoing colorectal surgery. The program of quality improvement based on benchmark achieved a reduction of SSI rates similar to the standard ones.

Programa de mejora de la infección nosocomial en cirugía por cáncer colorrectal / Quality improvement program of nosocomial infection in colorectal cancer surgery

Fundamento y objetivo: La infección nosocomial de la herida quirúrgica (INHQ) es una complicación de la cirugía colorrectal. Los objetivos del estudio son analizar los factores de riesgo asociados a la INHQ en cirugía de colon y establecer una estrategia de mejora basado en los indicadores de INHQ. Pacientes y método: Estudio longitudinal de cohortes prospectivo sobre pacientes intervenidos de neoplasia colorrectal entre el 1 de julio de 2002 y el 30 de junio de 2003. Se realizó un análisis descriptivo. Se utilizó la técnica de punto de referencia o comparación de resultados (benchmarking) como herramienta de estrategia de mejora y se evaluó con la razón de infección estandarizada (RIE). Se realizó un análisis univariante y multivariante de los factores de riesgo para desarrollar infección. Resultados: Se incluyó en el estudio a un total de 148 pacientes. La incidencia acumulada de INHQ fue de 10,14% y la densidad de incidencia de 6,47 infecciones por cada 1.000 días de estancia. La RIE del primer semestre fue de 1,53 y la del segundo semestre de 1,02. El análisis multivariante identificó 2 factores de riesgo independientes para el desarrollo de infección nosocomial: el ingreso urgente (odds ratio [OR] = 7,47; intervalo de confianza [IC] del 95%, 2,03-27,48) y el índice de riesgo ASA (American Society of Anesthesiologists) >= 3 (OR = 6,77; IC del 95%, 1,15-39,84). Conclusiones: El ingreso urgente y el riesgo de base elevado previo a la cirugía fueron los factores asociados a riesgo de INHQ tras cirugía colorrectal. El plan de mejora con benchmarking consiguió disminuir las tasas de INHQ e igualarlas al estándar

Background and objective: The surgical-site infection (SSI) is a complication of colorectal neoplasia surgery. The objectives of the study were to identify the SSI risk factors associated with colon surgery and to describe a strategy of quality improvement using surgical-site rates. Patients and method: Prospective cohort study of in-patients undergoing neoplasia colorectal surgery between 1st July 2002 to 30th June 2003. A descriptive analysis was implemented. Benchmarking was used as tool of quality improvement, and the outcomes were measured using the standardized infection ratio (SIR). To define the risk factors, the Chi square test and logistic regression test were used in univariate and multivariate analysis, respectively. Results: 148 patients were included in the study. The SSI accumulative incidence rate (IA) was 10.14%, and the incidence rate was 6.47 SSI per 1000 days. The SIR was 1.53 the first semester and 1.02 the second one. The multivariate analysis identified two risk factors associated with SSI: unscheduled admission (odds ratio [OR] = 7.47, 95% confidence interval [CI] 2.03-27.48) and a risk index of American Society of Anaesthesiologists (ASA) >= 3 (OR = 6.77, IC 95%, 1.15-39.84). Conclusions: An unscheduled admission and high risk ASA index were risk factors associated with SSI in patients undergoing colorectal surgery. The program of quality improvement based on benchmark achieved a reduction of SSI rates similar to the standard ones