Seroprevalence and Risk Factors Associated to Mycobacterium bovis in Wild Artiodactyl Species from Southern Spain, 2006–2010

The control of bovine tuberculosis (bTB) is at a critical point in the last stage of eradication in livestock. Wildlife species recently have emerged infected with TB in Europe, particularly ungulates in the Iberian Peninsula. Epidemiological information regarding TB in wild ungulates including affected species, prevalence, associated risk factors and appropriate diagnostic methods need to be obtained in these countries

Spatial distribution and risk factors of Brucellosis in Iberian wild ungulates

<p>Abstract</p> <p>Background</p> <p>The role of wildlife as a brucellosis reservoir for humans and domestic livestock remains to be properly established. The aim of this work was to determine the aetiology, apparent prevalence, spatial distribution and risk factors for brucellosis transmission in several Iberian wild ungulates.</p> <p>Methods</p> <p>A multi-species indirect immunosorbent assay (iELISA) using <it>Brucella </it>S-LPS antigen was developed. In several regions having brucellosis in livestock, individual serum samples were taken between 1999 and 2009 from 2,579 wild bovids, 6,448 wild cervids and4,454 Eurasian wild boar (<it>Sus scrofa</it>), and tested to assess brucellosis apparent prevalence. Strains isolated from wild boar were characterized to identify the presence of markers shared with the strains isolated from domestic pigs.</p> <p>Results</p> <p>Mean apparent prevalence below 0.5% was identified in chamois (<it>Rupicapra pyrenaica</it>), Iberian wild goat (<it>Capra pyrenaica</it>), and red deer (<it>Cervus elaphus</it>). Roe deer (<it>Capreolus capreolus</it>), fallow deer (<it>Dama dama</it>), mouflon (<it>Ovis aries</it>) and Barbary sheep (<it>Ammotragus lervia</it>) tested were seronegative. Only one red deer and one Iberian wild goat resulted positive in culture, isolating <it>B. abortus </it>biovar 1 and <it>B. melitensis </it>biovar 1, respectively. Apparent prevalence in wild boar ranged from 25% to 46% in the different regions studied, with the highest figures detected in South-Central Spain. The probability of wild boar being positive in the iELISA was also affected by age, age-by-sex interaction, sampling month, and the density of outdoor domestic pigs. A total of 104 bacterial isolates were obtained from wild boar, being all identified as <it>B. suis </it>biovar 2. DNA polymorphisms were similar to those found in domestic pigs.</p> <p>Conclusions</p> <p>In conclusion, brucellosis in wild boar is widespread in the Iberian Peninsula, thus representing an important threat for domestic pigs. By contrast, wild ruminants were not identified as a significant brucellosis reservoir for livestock.</p

Interactions between four species in a complex wildlife: livestock disease community : implications for Mycobacterium bovis maintenance and transmission

Livestock diseases such as bovine tuberculosis can have considerable negative effects on human health and economic activity. Wildlife reservoirs often hinder disease eradication in sympatric livestock populations. Therefore, quantifying interactions between wildlife and livestock is an important aspect of understanding disease persistence. This study was conducted on an extensive cattle farm in southwest Spain, where cattle, domestic pigs, wild boar and red deer are considered to be part of a tuberculosis host community. We tested the hypothesis that the frequency of both types of interactions would be greater at food and water sites, due to the aggregation of individuals from multiple species at these locations. We measured direct and indirect interactions between individuals using GPS and proximity loggers. Over 57,000 direct interactions were recorded over a 2-year period, of which 875 (1.5 %) occurred between different species and 216 (0.38 %) occurred between wildlife and livestock. Most direct and indirect interactions occurred at water sites. Over 90 % of indirect interactions between wildlife and livestock took place within the estimated 3-day environmental survival time of Mycobacterium bovis in this habitat. Red deer home ranges and daily activity patterns revealed significant spatial and temporal overlaps with cattle, particularly in autumn. Suids and red deer also cross the farm boundary regularly, introducing a between-farm interaction risk. The infrequent occurrence of direct interactions between individuals from different species suggests that they are unlikely to be the sole mode of disease transmission and that indirect interactions may play an important role

One-Tube-Only Standardized Site-Directed Mutagenesis: An Alternative Approach to Generate Amino Acid Substitution Collections

Contains fulltext : 171086.PDF (publisher's version ) (Open Access)Site-directed mutagenesis (SDM) is a powerful tool to create defined collections of protein variants for experimental and clinical purposes, but effectiveness is compromised when a large number of mutations is required. We present here a one-tube-only standardized SDM approach that generates comprehensive collections of amino acid substitution variants, including scanning- and single site-multiple mutations. The approach combines unified mutagenic primer design with the mixing of multiple distinct primer pairs and/or plasmid templates to increase the yield of a single inverse-PCR mutagenesis reaction. Also, a user-friendly program for automatic design of standardized primers for Ala-scanning mutagenesis is made available. Experimental results were compared with a modeling approach together with stochastic simulation data. For single site-multiple mutagenesis purposes and for simultaneous mutagenesis in different plasmid backgrounds, combination of primer sets and/or plasmid templates in a single reaction tube yielded the distinct mutations in a stochastic fashion. For scanning mutagenesis, we found that a combination of overlapping primer sets in a single PCR reaction allowed the yield of different individual mutations, although this yield did not necessarily follow a stochastic trend. Double mutants were generated when the overlap of primer pairs was below 60%. Our results illustrate that one-tube-only SDM effectively reduces the number of reactions required in large-scale mutagenesis strategies, facilitating the generation of comprehensive collections of protein variants suitable for functional analysis

Tailor-Made Protein Tyrosine Phosphatases: In Vitro Site-Directed Mutagenesis of PTEN and PTPRZ-B

In vitro site-directed mutagenesis (SDM) of protein tyrosine phosphatases (PTPs) is a commonly used approach to experimentally analyze PTP functions at the molecular and cellular level and to establish functional correlations with PTP alterations found in human disease. Here, using the tumor-suppressor PTEN and the receptor-type PTPRZ-B (short isoform from PTPRZ1 gene) phosphatases as examples, we provide a brief insight into the utility of specific mutations in the experimental analysis of PTP functions. We describe a standardized, rapid, and simple method of mutagenesis to perform single and multiple amino acid substitutions, as well as deletions of short nucleotide sequences, based on one-step inverse PCR and DpnI restriction enzyme treatment. This method of SDM is generally applicable to any other protein of interest