Dominga Soglia

Skin-scale genetic structure of Sarcoptes scabiei populations from individual hosts: Empirical evidence from Iberian ibex-derived mites

The objective of the present study was to examine the extent of genetic diversity among Sarcoptes scabiei individuals belonging to different skin subunits of the body from individual mangy hosts. Ten microsatellite primers were applied on 44 individual S. scabiei mites from three mangy Iberian ibexes from Sierra Nevada Mountain in Spain. Dendrograms of the mites from the individual Iberian ibexes, showing the proportion of shared alleles between pairs of individual mites representing three skin subpopulations (head, back, and abdomen subunits), allowed the clustering of some mite samples up to their skin subunits. This genetic diversity of S. scabiei at skin-scale did not have the same pattern in all considered hosts: for the first Iberian ibex (Cp1), only mites from the head subunit were grouped together; in the second individual (Cp2), the clustering was detected only for mites from the abdomen subunit; and for the third one (Cp3), only mites from the back subunit were clustered together. Our results suggest that the local colonization dynamics of S. scabiei would have influenced the nonrandom distribution of this ectoparasite, after a single infestation. Another presumable explanation to this skin-scale genetic structure could be the repeated infestations. To our knowledge, this is the first documentation of genetic structuring among S. scabiei at individual host skin-scale. Further studies are warranted to highlight determining factors of such trend, but the pattern underlined in the present study should be taken into account in diagnosis and monitoring protocols for studying the population genetic structure and life cycle of this neglected but important ectoparasite.

Sarcoptes mite from collection to DNA extraction : the lost realm of the neglected parasite

Sarcoptes mite from collection to DNA extraction forms the cornerstone for studies on Sarcoptes scabiei. Whilst the new science era took a shy leap into the different facets of mite studies, the cornerstone was almost entirely neglected. Mite collection, cleaning, storage and DNA extraction were, basically, humble attempts to extrapolate, adapt, modify or ‘pirate’ those existing methods to the peculiarities of Sarcoptes research. These aspects usually constituted few lines, bashfully mentioned, in the materials and methods section of some papers, which arose in unique problems concerning cost-effectiveness, time profitability, safety and even worse, the credibility of the results, creating contradictory conclusions in some cases. This ‘noisy’ situation encouraged us to collect, classify and review, for the first time to our knowledge, some aspects relating to studies on Sarcoptes mite from collection to DNA extraction, which will be useful for further studies on Sarcoptes, and have implications for the effective control of the diseases Sarcoptes mite causes. Further studies are needed, especially to compare the profitability, safety, sensibility and specificity of the different methods of this neglected realm of the ubiquitous ectoparasite.

HotSHOT Plus ThermalSHOCK, a new and efficient technique for preparation of PCR-quality mite genomic DNA

The present study adapted the HotSHOT method, a technique which has been successfully applied on different kinds of tissues, to studies of Sarcoptes. Some modifications of this technique were made which allowed the quick preparation of PCR-quality Sarcoptes genomic DNA (gDNA), namely applying sodium hydroxide as a substrate for three cycles of thermal shock, followed by a short incubation and pH adjustment with a Tris solution (HotSHOT Plus ThermalSHOCK). The performance of this technique was tested by amplifying a ~450-bp rDNA fragment of the second internal transcribed spacer (ITS-2) and by multi-locus genotyping using ten microsatellites on 520 individual Sarcoptes samples. No difference in performance was observed between gDNA samples prepared using the HotSHOT Plus ThermalSHOCK technique and those prepared using a commercial kit utilizing proteinase K digestion. The results demonstrated that the HotSHOT Plus ThermalSHOCK technique is time-saving, economic, and easily automatable for the preparation of PCR-quality mite gDNA, which has implications for studying the molecular biology of mites with human and animal health significance. Although tested in the present study using Sarcoptes mites as a model, this technique may find broad applicability in extraction of gDNA from other parasites with small sizes and hard bodies.

Microsatellites as markers for comparison among different populations of Sarcoptes scabiei

Abstract The aim of the present investigation was to analyse genetic variation and relationships of epizootic mange mites from sympatric Alpine chamois and red fox populations. The results of multi-locus genotyping using microsatellite marker loci support the hypothesis that gene flow between mite varieties on sympatric Alpine chamois and red fox is absent or extremely rare. Although the number of samples analysed until now is very small, the transmission of parasites seem to be more frequent when phylogenetically related host species are involved.

Development and validation of a microsatellite marker-based method for tracing infections by Microsporum canis

BACKGROUND Microsporum canis is a dermatophyte fungus harbored by cats and dogs and is frequently transmitted to humans. Molecular tools able to discriminate fungal isolates at the strain level would prove extremely useful for confirming the route of infection, thus contributing to optimization of prophylaxis and hygienic regimens. OBJECTIVE To develop and validate a microsatellite marker-based method for use in tracking infections by M. canis. METHODS Primers were designed against sequences flanking the microsatellites individuated by a BLAST search using the nucleotide sequence information assembled by the M. canis CBS 113480 genome project. The PCR conditions were standardized and fragment analysis was performed using a genetic analyzer. The resolving power of the markers was investigated on 26 unrelated M. canis strains while the reproducibility of the technique and the stability of the markers were evaluated on a single strain subcultured in time as well as on 36 strains isolated from nine outbreak episodes. RESULTS Eight markers were recognized as being the most polymorphic within the set of M. canis strains isolated from unrelated distant hosts, with a total of 22 multilocus genotypes, which corresponded to a genotypic diversity of 97%. Repeated tests on subcultures of M. canis reference strain CBS 113480 always yielded the same results. Identical multilocus genotypes were obtained for all the isolates from each outbreak episode. CONCLUSION The high resolving power and reproducibility of the markers that were identified support the potential of these tools to detect sources and routes of infection by M. canis.

Genetic variability of two Italian indigenous chicken breeds inferred from microsatellite marker analysis.

Abstract The objective of this study was to determine the genetic structure and variability of Bionda Piemontese and Bianca di Saluzzo (Piedmont, Northwest Italy) using an international set of microsatellite loci (AVIANDIV-FAO). Differences compared with commercial lines and other Italian breeds were verified to justify the implementation of conservation programmes. Flock contribution to genetic variability was assessed following the approach implemented in the MolKin software. Comparison was performed using the fixation index and the Reynolds genetic distance. The most likely number of different populations was estimated using the clustering procedure implemented in STRUCTURE. The molecular information suggests that management practices could have prevented random mating and produced inbreeding and heterogeneity across flocks. In this respect, Bionda and Bianca show substructuring and are more similar to British breeds than other continental European breeds. Bionda and Bianca fit into the European breeds provided with the highest number of alleles and expected heterozygosity. There is a clear distinction between the Piedmont breeds and the other populations. The Piedmont poultry differ from both commercial lines and other Italian breeds and retain a high level of genetic variability. As for other indigenous breeds, Bionda and Bianca could make an original contribution to the industry in the future. A collective planned approach to restoration is essential, because the flocks are managed with poor regulation. Enhancing connection between breeders with an efficient replacement interchange and mating plan is the right way of controlling inbreeding, preventing substructuring and increasing variability within the flocks.

Population genetic structure of Alpine chamois (Rupicapra r. rupicapra) in the Italian Alps

Analysis of the genetic diversity of the Alpine chamois in Italy was conducted using a pool of 26 microsatellite loci. A total of 209 animals were analyzed, representing six geographical populations from different location of the Southern slope of the Alps. Clear genetic differences have emerged between the sampled chamois groups. Some were consistent with an isolation-by-distance model. However, in parallel, other mechanisms intervened in areas that, in addition to being peripheral to the main alpine ridge, had suffered from recent bottlenecks. In such areas, genetic drift and a low rate of gene flow are likely explanations for the current genetic structure.

MUC1 gene polymorphism in three Nelore lines selected for growth and its association with growth and carcass traits

The objective of this study was to describe the VNTR polymorphism of the mucin 1 gene (MUC1) in three Nelore lines selected for yearling weight to determine whether allele and genotype frequencies of this polymorphism were affected by selection for growth. In addition, the effects of the polymorphism on growth and carcass traits were evaluated. Birth, weaning and yearling weights, rump height, Longissimus muscle area, backfat thickness, and rump fat thickness, were analyzed. A total of 295 Nelore heifers from the Beef Cattle Research Center, Instituto de Zootecnia de Sertãozinho, were used, including 41 of the control line, 102 of the selection line and 152 of the traditional. The selection and traditional lines comprise animals selected for higher yearling weight, whereas control line animals are selected for yearling weight close to the average. Five alleles were identified, with allele 1 being the most frequent in the three lines, especially in the lines selected for higher means for yearling weight. Heterozygosity was significantly higher in the control line. Association analyses showed significant effects of allele 1 on birth weight and weaning weight while the allele 3 exert significant effects on yearling weight and back fat thickness. Despite these findings, application of this marker to marker-assisted selection requires more consistent results based on the genotyping of a larger number of animals in order to increase the accuracy of the statistical analyses.

Polymorphism Analysis of Ch1 and Ch2 Genes in the Siberian Cat

Cats are usually spreaders of allergens that are critical for sensitive people; the Siberian cat is a breed supposed to be low level allergenic, according to some breeders’ statements. The sequence of the two genes, namely Ch1 and Ch2, that code for the allergen Fel d 1, the major allergen responsible for outbreaks of allergy symptoms, is not yet known in the Siberian cat, and finding this was the aim of our investigation. Notably, our work is the first survey of the genetic structure of these genes in Siberian cats. The comparison of the sequences of Siberian cats, non-Siberian cats, and sequences present in the National Center for Biotechnology Information database revealed a considerable number of mutations; some of those detected in the Siberian cat, due to their position in exon regions, could affect the Fel d 1 allergenic properties. Therefore, further investigations are recommended to assess if the identified mutations can be responsible for a reduced-allergen synthesis and can be used as markers for selection of low level allergenic cats.

Distinguishing industrial meat from that of indigenous chickens with molecular markers

&NA; The aim of investigation was to evaluate a traceability system to detect industrial chicken meat among indigenous products, considering issues that could affect assignment accuracy. The dataset included 2 Italian indigenous meat breeds, namely Bionda Piemontese (2 ecotypes) and Bianca di Saluzzo, one broiler line, and 3 layer lines. Assignment tests were performed using a standard panel of 28 microsatellite loci. To evaluate effects of inbreeding and substructure on assignment accuracy, a simulated dataset was prepared. Broilers and layers belong to homogeneous populations and never enter the clusters of indigenous breeds. Ambiguity or misallocation are expected between the Bionda ecotypes and between the 2 indigenous breeds, but it is unlikely that niche products provided by Bionda and Bianca will compete with one another. Non‐random mating reduces accuracy, but only populations having weak genetic differentiation are involved, namely those that are less interesting to discriminate. The dataset can be used as a reference population to distinguish commercial meat from indigenous meat with great accuracy. Misallocations increase as number of loci decreases, but only within or between the indigenous breeds. A subpanel of the most resolving 14 loci keeps sufficient informative content to provide accuracy and to correctly allocate additional test samples within the reference population. This analytical tool is economically sustainable as a method to detect fraud or mislabeling. Adoption of a monitoring system should increase the value of typical products because the additional burden of molecular analyses would improve commercial grade and perception of quality.