A mating scheme based on molecular parentage improves growth performance in slow-growing chickens... more A mating scheme based on molecular parentage improves growth performance in slow-growing chickens over three generations Dominga Soglia (1), Stefano Sartore (2), Sandra Maione (3), Cecilia Mugnai (4), Sihem Dabbou (5), Paola Sacchi (6), Roberto Rasero (7), Laura Gasco (8), Ivo Zoccarato (9), Francesco Gai (10) and Achille Schiavone (11)
Bovine serum amyloid A3: analysis of the genomic region / Soglia, Dominga; Sartore, Stefano; Maio... more Bovine serum amyloid A3: analysis of the genomic region / Soglia, Dominga; Sartore, Stefano; Maione, Sandra; Pepe, Elvira; Gallino, Alice; Rasero, Roberto; Sacchi, Paola. In: ITALIAN JOURNAL OF ANIMAL SCIENCE. ISSN 15944077. 14:Supplement 1(2015), pp. 124-124. ((Intervento presentato al convegno Animal Science and Production Association, 21st Congress tenutosi a Milano nel 9-12 giugno 2015. Original Citation: Bovine serum amyloid A3: analysis of the genomic region
further sub-structuring reflects the main post-domestication migration routes. The reconstruction... more further sub-structuring reflects the main post-domestication migration routes. The reconstruction of past migration events highlighted several exchanges mainly between African populations, which often involve admixed and cosmopolitan breeds. In addition, extensive gene flow was revealed within specific areas (e.g., southern Europe, Morocco and Mali-Burkina Faso-Nigeria), while isolation due to geographical causes (e.g. insularity) or human management has brought a decrease in local gene flow. Taken together, these results confirm that after domestication in the Fertile Crescent in the early Neolithic era (approx. 15,000 BP), domestic goats spread to Europe, Africa and Asia through divergent migration routes, which determined the major genomic background of the continental populations. During the following centuries, due to geographical and reproductive isolation, further sub-structuring of diversity occurred at the local level. This has been accompanied by additional migrations and/or importations, the traces of which are still detectable, such as the clear African signatures in the goat populations of the Canary Islands and Southern America.
The creation of genetic resource cryobanks provides a crucial link between in situ and ex situ te... more The creation of genetic resource cryobanks provides a crucial link between in situ and ex situ techniques to improve the efficiency of conservation programs. Aim of the present review is to describe all the activities developed for the implementation of the first Italian Semen Cryobank of Autochthonous Chicken and Turkey Breeds. These activities can be classified into three main topics: (1) identification of species-specific semen freezing/thawing reference procedures; (2) drafting Standard Operative Procedures (SOP) for the implementation of the semen cryobank; (3) storage of semen doses from Italian chicken and turkey breeds to establish the cryobank. Several trials have been developed to identify a specie-specific semen cryopreservation protocol for chickens and turkeys. The major results are reviewed and a final reference protocol described. Taking into consideration the FAO guidelines for cryoconservation of animal genetic resources, SOP were drafted with the aim to provide technical guidance and logistical support on the choice of priority breeds, selection of birds for semen production, infrastructures and storage sites, birds and semen management, cryopreservation process and doses traceability. Lastly, the Italian Semen Cryobank was created. A total of 112 semen doses from 22 cockerels of three breeds, and 74 doses from 12 turkey males of three breeds were stored in the Cryobank. Breed specific semen quality parameters assessed before and after cryopreservation are reported. The described activities provide information and tools useful for the implementation of semen cryobanking in avian species and might be transferred also to other species after appropriate adaptations. HIGHLIGHTS Implementation of the first Italian Semen Cryobank of Autochthonous Chicken and Turkey Breeds Drafting Standard Operative Procedures provides technical guidance and logistical support on the design and establishment of the cryobank Semen cryobank is a precious genetic reservoir and could be useful to safeguard genetic variability in small population in vivo conserved ARTICLE HISTORY
The purpose of this study was to investigate the effects of high levels of Tenebrio molitor dieta... more The purpose of this study was to investigate the effects of high levels of Tenebrio molitor dietary inclusion (15%) on molecular mechanisms that influence poultry health in a broiler chicken diet. The global gene expression of four tissues (breast, liver, jejunum, and caecum) was evaluated using the RNA-Seq approach. The analysis of differentially expressed genes suggested that the use of Tenebrio molitor leads to the overexpression of genes related to protein elongation required for tissue growth and development in the gut and liver. It would also appear to contain nutrients that reduce the expression of genes related to the immune system and inflammation of the mucosa. The dietary inclusion of Tenebrio molitor in poultry could also lead to a possible inactivation of the growth factor and a reduction of tissue free-radicals. No genes alterations have been detected in liver RNA expression that would discourage the use of larvae in feeding broilers.
Achille Schiavone, Alberto Brugiapaglia, Michele De Marco, Stefano Sartore, Dominga Soglia, Sandr... more Achille Schiavone, Alberto Brugiapaglia, Michele De Marco, Stefano Sartore, Dominga Soglia, Sandra Maione, Roberto Rasero, Paola Sacchi, Alessandra Dalmasso, Maria T. Bottero, Daniele Pattono, Martina Tarantola, Mario Giacobini, Luigi Bertolotti, Ivo Zoccarato, Laura Gasco, Cecilia Mugnai Dipartimento di Scienze Veterinarie, University of Torino, Italy Dipartimento di Scienze Agrarie Forestali e Alimentari, University of Torino, Italy Contact: [email protected]
A multiplex PCR for the simultaneous detection of some pathogenic genes of enteropathogenic, ente... more A multiplex PCR for the simultaneous detection of some pathogenic genes of enteropathogenic, enterotoxigenic and verocytotoxinproducing Escherichia coli was developed.
ABSTRACT In Italy, since 2005 a breeding plan to increase scrapie resistance has been adopted. Th... more ABSTRACT In Italy, since 2005 a breeding plan to increase scrapie resistance has been adopted. The impact of this selection on genetic diversity was assessed on Sambucana, an autochtonous sheep breed reared in southern Piedmont, by analysing the evolution of allele frequencies at different levels: PRNP (prion protein) gene, microsatellite loci on OAR13 (where PRNR maps), and microsatellite loci on other chromosomes, not subjected to selection for scrapie resistance. A total of 147 young rams, 80 born in 2004 and 67 in 2008-2009 were analysed. Evidence of diversity loss was observed for PRNP gene as a consequence of the directional selection. Diversity was affected in the immediate vicinity of PRNP but the effect on more distant loci on the same chromosome was trivial. With regard to neutral markers, lack of heterozygosis with no changeover of allele frequencies was observed suggesting an increase of inbreeding. Mating policies would be sufficient to solve these problems. A selection scheme based on genotyping rams and eliminating carriers of both susceptible and high susceptible alleles is the best way to improve natural resistance to scrapie with low costs and minimal problems in the current conservation programmes targeting rare breeds.
The present study adapted the HotSHOT method, a technique which has been successfully applied on ... more 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.
The objective of the present study was to examine the extent of genetic diversity among Sarcoptes... more 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 skinscale 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 forms the cornerstone for studies on Sarcoptes s... more 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.
Availability of mites is a recognized limiting factor of biological and genetic investigations of... more Availability of mites is a recognized limiting factor of biological and genetic investigations of the genus Sarcoptes. Current methods of deoxyribonucleic acid (DNA) extraction from individual mites also need substantial improvement in efficiency and operator friendliness. We have first developed a technique for efficient and safe extraction of living mites from scabietic skin samples (crusts or deep skin scrapings). Its core device is a large plastic syringe connected with a 1.5-ml Eppendorf tube. The source material is introduced in the syringe and the device in a shoe box with the tip half of the tube emerging. Mites migrate towards a heat source during a minimum of 36 h. Then, the tube is detached and the mites utilized without risks for the operators. A second technique allows operator-friendly manipulation of individual mites for DNA extraction. Fixed mites are isolated by adhesion to a small strip of polyvinyl chloride (PVC) adhesive tape operated with tweezers. Then, mite and strip are plunged in the lyses buffer and the sample twice submitted to thermal shock for disruption of the chitinous exoskeleton. Data show that the tape does not interfere with successive DNA extraction with a commercial kit. The corresponding protocol, that we briefly name "PVC adhesive tape + thermal shock + kit DNA extraction," compares favorably with the available ones.
The objective of this study was to describe the VNTR polymorphism of the mucin 1 gene (MUC1) in t... more 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.
Knowledge of the sex of individuals in natural populations greatly facilitates evolutionary ecolo... more Knowledge of the sex of individuals in natural populations greatly facilitates evolutionary ecology, breeding systems and genetics. Therefore, the development of a simple, not stressing and objective sexing test would facilitate conservation of the Short-toed Eagle (Circaetus gallicus), an endangered Accipitridae species living mainly in southern Europe and Asia. A PCR test was used employing primers that amplify two homologous fragments of both the CHD-W gene, unique of females, and the CHD-Z, occurring in the two sexes. The analysis of the PCR products obtained from blood DNA showed a band of about 380 bp, apparently unique in all individuals. The alignment of the sequences of the two fragments revealed that CHD-W is only 9 bp longer than CHD-Z (387 vs. 378 bp) while CHD-Z lacks the restriction site for Asp700I. After digestion male PCR products showed a unique band of 378 bp while fragments belonging to females resolved into three bands (378, 280 and 107 bp). Using feathers as DNA sources, the individual patterns obtained were identical with the corresponding blood DNA samples. This sexing technique is objective and non-invasive and could be useful for verifying the sex ratio theories and improving the management. q
Background: Microsporum canis is a dermatophyte fungus harbored by cats and dogs and is frequentl... more 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. ß
The aim of the present study was to assess whether individual Sarcoptes mites collected from froz... more The aim of the present study was to assess whether individual Sarcoptes mites collected from frozen skin ('postponed isolation' method) are suitable sources of PCRquality genomic DNA, and to test the effectiveness of this method in comparison with the 'direct isolation' method, often used through force of habit. Hundreds of single Sarcoptes scabiei samples, resulting from direct (live) or postponed (post-frozen) isolation, were tested using a *450 bp product (ITS-2) and multi-locus 109 genotyping with microsatellite markers. No statistical difference in yield of soluble DNA was found between the two isolation methods. Nevertheless, 19% of the reactions were classified as failed preparations in the direct isolation method, whereas the rate of unsuccessful reactions was 34% in the postponed isolation method. Consequently, post-frozen isolation is suitable and recommendable for Sarcoptes mite gDNA preparation, particularly when performing a balancing act among safety, practicability and profitability. These results have implications for mite collection for DNA extraction, and hence the needed wider leap of Sarcoptes into the genetic era.
A mating scheme based on molecular parentage improves growth performance in slow-growing chickens... more A mating scheme based on molecular parentage improves growth performance in slow-growing chickens over three generations Dominga Soglia (1), Stefano Sartore (2), Sandra Maione (3), Cecilia Mugnai (4), Sihem Dabbou (5), Paola Sacchi (6), Roberto Rasero (7), Laura Gasco (8), Ivo Zoccarato (9), Francesco Gai (10) and Achille Schiavone (11)
Bovine serum amyloid A3: analysis of the genomic region / Soglia, Dominga; Sartore, Stefano; Maio... more Bovine serum amyloid A3: analysis of the genomic region / Soglia, Dominga; Sartore, Stefano; Maione, Sandra; Pepe, Elvira; Gallino, Alice; Rasero, Roberto; Sacchi, Paola. In: ITALIAN JOURNAL OF ANIMAL SCIENCE. ISSN 15944077. 14:Supplement 1(2015), pp. 124-124. ((Intervento presentato al convegno Animal Science and Production Association, 21st Congress tenutosi a Milano nel 9-12 giugno 2015. Original Citation: Bovine serum amyloid A3: analysis of the genomic region
further sub-structuring reflects the main post-domestication migration routes. The reconstruction... more further sub-structuring reflects the main post-domestication migration routes. The reconstruction of past migration events highlighted several exchanges mainly between African populations, which often involve admixed and cosmopolitan breeds. In addition, extensive gene flow was revealed within specific areas (e.g., southern Europe, Morocco and Mali-Burkina Faso-Nigeria), while isolation due to geographical causes (e.g. insularity) or human management has brought a decrease in local gene flow. Taken together, these results confirm that after domestication in the Fertile Crescent in the early Neolithic era (approx. 15,000 BP), domestic goats spread to Europe, Africa and Asia through divergent migration routes, which determined the major genomic background of the continental populations. During the following centuries, due to geographical and reproductive isolation, further sub-structuring of diversity occurred at the local level. This has been accompanied by additional migrations and/or importations, the traces of which are still detectable, such as the clear African signatures in the goat populations of the Canary Islands and Southern America.
The creation of genetic resource cryobanks provides a crucial link between in situ and ex situ te... more The creation of genetic resource cryobanks provides a crucial link between in situ and ex situ techniques to improve the efficiency of conservation programs. Aim of the present review is to describe all the activities developed for the implementation of the first Italian Semen Cryobank of Autochthonous Chicken and Turkey Breeds. These activities can be classified into three main topics: (1) identification of species-specific semen freezing/thawing reference procedures; (2) drafting Standard Operative Procedures (SOP) for the implementation of the semen cryobank; (3) storage of semen doses from Italian chicken and turkey breeds to establish the cryobank. Several trials have been developed to identify a specie-specific semen cryopreservation protocol for chickens and turkeys. The major results are reviewed and a final reference protocol described. Taking into consideration the FAO guidelines for cryoconservation of animal genetic resources, SOP were drafted with the aim to provide technical guidance and logistical support on the choice of priority breeds, selection of birds for semen production, infrastructures and storage sites, birds and semen management, cryopreservation process and doses traceability. Lastly, the Italian Semen Cryobank was created. A total of 112 semen doses from 22 cockerels of three breeds, and 74 doses from 12 turkey males of three breeds were stored in the Cryobank. Breed specific semen quality parameters assessed before and after cryopreservation are reported. The described activities provide information and tools useful for the implementation of semen cryobanking in avian species and might be transferred also to other species after appropriate adaptations. HIGHLIGHTS Implementation of the first Italian Semen Cryobank of Autochthonous Chicken and Turkey Breeds Drafting Standard Operative Procedures provides technical guidance and logistical support on the design and establishment of the cryobank Semen cryobank is a precious genetic reservoir and could be useful to safeguard genetic variability in small population in vivo conserved ARTICLE HISTORY
The purpose of this study was to investigate the effects of high levels of Tenebrio molitor dieta... more The purpose of this study was to investigate the effects of high levels of Tenebrio molitor dietary inclusion (15%) on molecular mechanisms that influence poultry health in a broiler chicken diet. The global gene expression of four tissues (breast, liver, jejunum, and caecum) was evaluated using the RNA-Seq approach. The analysis of differentially expressed genes suggested that the use of Tenebrio molitor leads to the overexpression of genes related to protein elongation required for tissue growth and development in the gut and liver. It would also appear to contain nutrients that reduce the expression of genes related to the immune system and inflammation of the mucosa. The dietary inclusion of Tenebrio molitor in poultry could also lead to a possible inactivation of the growth factor and a reduction of tissue free-radicals. No genes alterations have been detected in liver RNA expression that would discourage the use of larvae in feeding broilers.
Achille Schiavone, Alberto Brugiapaglia, Michele De Marco, Stefano Sartore, Dominga Soglia, Sandr... more Achille Schiavone, Alberto Brugiapaglia, Michele De Marco, Stefano Sartore, Dominga Soglia, Sandra Maione, Roberto Rasero, Paola Sacchi, Alessandra Dalmasso, Maria T. Bottero, Daniele Pattono, Martina Tarantola, Mario Giacobini, Luigi Bertolotti, Ivo Zoccarato, Laura Gasco, Cecilia Mugnai Dipartimento di Scienze Veterinarie, University of Torino, Italy Dipartimento di Scienze Agrarie Forestali e Alimentari, University of Torino, Italy Contact: [email protected]
A multiplex PCR for the simultaneous detection of some pathogenic genes of enteropathogenic, ente... more A multiplex PCR for the simultaneous detection of some pathogenic genes of enteropathogenic, enterotoxigenic and verocytotoxinproducing Escherichia coli was developed.
ABSTRACT In Italy, since 2005 a breeding plan to increase scrapie resistance has been adopted. Th... more ABSTRACT In Italy, since 2005 a breeding plan to increase scrapie resistance has been adopted. The impact of this selection on genetic diversity was assessed on Sambucana, an autochtonous sheep breed reared in southern Piedmont, by analysing the evolution of allele frequencies at different levels: PRNP (prion protein) gene, microsatellite loci on OAR13 (where PRNR maps), and microsatellite loci on other chromosomes, not subjected to selection for scrapie resistance. A total of 147 young rams, 80 born in 2004 and 67 in 2008-2009 were analysed. Evidence of diversity loss was observed for PRNP gene as a consequence of the directional selection. Diversity was affected in the immediate vicinity of PRNP but the effect on more distant loci on the same chromosome was trivial. With regard to neutral markers, lack of heterozygosis with no changeover of allele frequencies was observed suggesting an increase of inbreeding. Mating policies would be sufficient to solve these problems. A selection scheme based on genotyping rams and eliminating carriers of both susceptible and high susceptible alleles is the best way to improve natural resistance to scrapie with low costs and minimal problems in the current conservation programmes targeting rare breeds.
The present study adapted the HotSHOT method, a technique which has been successfully applied on ... more 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.
The objective of the present study was to examine the extent of genetic diversity among Sarcoptes... more 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 skinscale 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 forms the cornerstone for studies on Sarcoptes s... more 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.
Availability of mites is a recognized limiting factor of biological and genetic investigations of... more Availability of mites is a recognized limiting factor of biological and genetic investigations of the genus Sarcoptes. Current methods of deoxyribonucleic acid (DNA) extraction from individual mites also need substantial improvement in efficiency and operator friendliness. We have first developed a technique for efficient and safe extraction of living mites from scabietic skin samples (crusts or deep skin scrapings). Its core device is a large plastic syringe connected with a 1.5-ml Eppendorf tube. The source material is introduced in the syringe and the device in a shoe box with the tip half of the tube emerging. Mites migrate towards a heat source during a minimum of 36 h. Then, the tube is detached and the mites utilized without risks for the operators. A second technique allows operator-friendly manipulation of individual mites for DNA extraction. Fixed mites are isolated by adhesion to a small strip of polyvinyl chloride (PVC) adhesive tape operated with tweezers. Then, mite and strip are plunged in the lyses buffer and the sample twice submitted to thermal shock for disruption of the chitinous exoskeleton. Data show that the tape does not interfere with successive DNA extraction with a commercial kit. The corresponding protocol, that we briefly name "PVC adhesive tape + thermal shock + kit DNA extraction," compares favorably with the available ones.
The objective of this study was to describe the VNTR polymorphism of the mucin 1 gene (MUC1) in t... more 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.
Knowledge of the sex of individuals in natural populations greatly facilitates evolutionary ecolo... more Knowledge of the sex of individuals in natural populations greatly facilitates evolutionary ecology, breeding systems and genetics. Therefore, the development of a simple, not stressing and objective sexing test would facilitate conservation of the Short-toed Eagle (Circaetus gallicus), an endangered Accipitridae species living mainly in southern Europe and Asia. A PCR test was used employing primers that amplify two homologous fragments of both the CHD-W gene, unique of females, and the CHD-Z, occurring in the two sexes. The analysis of the PCR products obtained from blood DNA showed a band of about 380 bp, apparently unique in all individuals. The alignment of the sequences of the two fragments revealed that CHD-W is only 9 bp longer than CHD-Z (387 vs. 378 bp) while CHD-Z lacks the restriction site for Asp700I. After digestion male PCR products showed a unique band of 378 bp while fragments belonging to females resolved into three bands (378, 280 and 107 bp). Using feathers as DNA sources, the individual patterns obtained were identical with the corresponding blood DNA samples. This sexing technique is objective and non-invasive and could be useful for verifying the sex ratio theories and improving the management. q
Background: Microsporum canis is a dermatophyte fungus harbored by cats and dogs and is frequentl... more 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. ß
The aim of the present study was to assess whether individual Sarcoptes mites collected from froz... more The aim of the present study was to assess whether individual Sarcoptes mites collected from frozen skin ('postponed isolation' method) are suitable sources of PCRquality genomic DNA, and to test the effectiveness of this method in comparison with the 'direct isolation' method, often used through force of habit. Hundreds of single Sarcoptes scabiei samples, resulting from direct (live) or postponed (post-frozen) isolation, were tested using a *450 bp product (ITS-2) and multi-locus 109 genotyping with microsatellite markers. No statistical difference in yield of soluble DNA was found between the two isolation methods. Nevertheless, 19% of the reactions were classified as failed preparations in the direct isolation method, whereas the rate of unsuccessful reactions was 34% in the postponed isolation method. Consequently, post-frozen isolation is suitable and recommendable for Sarcoptes mite gDNA preparation, particularly when performing a balancing act among safety, practicability and profitability. These results have implications for mite collection for DNA extraction, and hence the needed wider leap of Sarcoptes into the genetic era.
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Papers by Dominga Soglia