Emerging applications of machine learning in genomic medicine and healthcare.

The integration of artificial intelligence technologies has propelled the progress of clinical and genomic medicine in recent years. The significant increase in computing power has facilitated the ability of artificial intelligence models to analyze and extract features from extensive medical data and images, thereby contributing to the advancement of intelligent diagnostic tools. Artificial intelligence (AI) models have been utilized in the field of personalized medicine to integrate clinical data and genomic information of patients. This integration allows for the identification of customized treatment recommendations, ultimately leading to enhanced patient outcomes. Notwithstanding the notable advancements, the application of artificial intelligence (AI) in the field of medicine is impeded by various obstacles such as the limited availability of clinical and genomic data, the diversity of datasets, ethical implications, and the inconclusive interpretation of AI models' results. In this review, a comprehensive evaluation of multiple machine learning algorithms utilized in the fields of clinical and genomic medicine is conducted. Furthermore, we present an overview of the implementation of artificial intelligence (AI) in the fields of clinical medicine, drug discovery, and genomic medicine. Finally, a number of constraints pertaining to the implementation of artificial intelligence within the healthcare industry are examined.

Genetic diversity and breed-informative SNPs identification in domestic pig populations using coding SNPs.

Background: The use of breed-informative genetic markers, specifically coding Single Nucleotide Polymorphisms (SNPs), is crucial for breed traceability, authentication of meat and dairy products, and the preservation and improvement of pig breeds. By identifying breed informative markers, we aimed to gain insights into the genetic mechanisms that influence production traits, enabling informed decisions in animal management and promoting sustainable pig production to meet the growing demand for animal products. Methods: Our dataset consists of 300 coding SNPs genotyped from three Italian commercial pig populations: Landrace, Yorkshire, and Duroc. Firstly, we analyzed the genetic diversity among the populations. Then, we applied a discriminant analysis of principal components to identify the most informative SNPs for discriminating between these populations. Lastly, we conducted a functional enrichment analysis to identify the most enriched pathways related to the genetic variation observed in the pig populations. Results: The alpha diversity indexes revealed a high genetic diversity within the three breeds. The higher proportion of observed heterozygosity than expected revealed an excess of heterozygotes in the populations that was supported by negative values of the fixation index (FIS) and deviations from the Hardy-Weinberg equilibrium. The Euclidean distance, the pairwise FST, and the pairwise Nei's GST genetic distances revealed that Yorkshire and Landrace breeds are genetically the closest, with distance values of 2.242, 0.029, and 0.033, respectively. Conversely, Landrace and Duroc breeds showed the highest genetic divergence, with distance values of 2.815, 0.048, and 0.052, respectively. We identified 28 significant SNPs that are related to phenotypic traits and these SNPs were able to differentiate between the pig breeds with high accuracy. The Functional Enrichment Analysis of the informative SNPs highlighted biological functions related to DNA packaging, chromatin integrity, and the preparation of DNA into higher-order structures. Conclusion: Our study sheds light on the genetic underpinnings of phenotypic variation among three Italian pig breeds, offering potential insights into the mechanisms driving breed differentiation. By prioritizing breed-specific coding SNPs, our approach enables a more focused analysis of specific genomic regions relevant to the research question compared to analyzing the entire genome.

Oral ecological environment modifications by hard-cheese: from pH to microbiome: a prospective cohort study based on 16S rRNA metabarcoding approach.

BACKGROUND: The oral ecosystem conditions dental health, and is known to be positively modified by oral hygiene which cannot always be performed between meals, especially outside home. It is therefore important to identify the practices to be adopted to influence the oral environment in an anticariogenic direction. Milk and cheese are considered functional foods and have a role on oral health. There are several mechanisms by which cheese exerts its beneficial effects on teeth. The aim of the present study was to examine whether short term consumption of hard cheese would affect the oral pH and microbial flora of healthy adults modifying ecological oral environment. The Next Generation Sequencing (NGS) approach was applied to study the effect of Italian Grana Padano (GP), as a prototype of typical hard cheese, on the oral microbiota composition. Finally, we explored Streptococcus mutans/sanguinis ratio as a marker of protective biofilm composition. METHODS: Nine oral-healthy adults were instructed to eat 25 gr of GP cheese for 5 consecutive days. Three time points were chosen for supragingival samples collection and pH measurement. 16S rRNA-gene sequences were obtained both from oral samples and GP cheese using the MiSeq platform and analyzed against the expanded Human Oral Microbiome Database (eHOMD). ProgPerm was used to perform statistical analyses to investigate strain differential representation after cheese consumption. RESULTS: Taxonomic analyses of the oral microbiota revealed that Firmicutes was the most abundant phylum, followed by Proteobacteria and Actinobacteria. GP cheese significantly modifies oral pH, causing a shift toward basic conditions which are kept for a few hours. The Streptococcus mutans/Streptococcus sanguinis ratio lowers in the last observed timepoint. CONCLUSION: Our results reveal that a portion of GP cheese eaten after dinner provides important micronutrients (i.e. calcium, vitamins and some aminoacids such as arginine) and changes oral pH toward basic conditions, resulting in a light modification of the oral microbiome towards the reduction of the overall amount of acidophilic bacteria. Furthermore, the S. mutans/S. sanguinis ratio is reduced, contributing to obtain a more protecting environment towards caries establishment and evolution.

Comparison of three statistical approaches for feature selection for fine-scale genetic population assignment in four pig breeds.

BACKGROUND: Assigning animals to their corresponding breeds through breed informative single-nucleotide polymorphisms (SNPs) is required in many fields. For instance, it is used in the traceability and the authentication of meat and other livestock products. SNPs' information for several pork breeds are now accessible thanks to the availability of dense SNP chips. These SNP chips cover a large number of molecular markers distributed across the entire genome. To identify the pork breed from a sample of industrial meat, one must analyze a large panel of genetic markers depending on the SNP chip used. The analysis of such large datasets requires intensive work. This leads to the idea of creating less dense chips of breed informative markers based on a reduced number of SNPs. Therefore, the analysis of the data emanating from the genotyping of these reduced chips will require less time and effort. AIM: The objective of this study is to find the most informative SNPs for the discrimination between four pig breeds, namely Duroc, Landrace, Large White, and Pietrain. METHOD: The Illumina Porcine 60 k SNP chip was used to genotype SNPs distributed all over the individuals' genomes. Firstly, we used three different statistical approaches for feature selection: (i) principal component analysis (PCA), (ii) least absolute shrinkage and selection operator (LASSO), and (iii) random forest (RF). These three approaches identified three sets of SNPs; each set corresponds to one approach. Then, we combined the results of the three methods by setting up a final panel containing the SNPs which appear on the three sets altogether. RESULTS: Separately, each method resulted in a panel with the corresponding most discriminating SNPs. The PCA, the LASSO, and the random forest with Boruta algorithm highlighted 28,816, 50, and 286 SNPs, respectively. The number of SNPs selected by PCA is high compared to Boruta and LASSO because PCA chooses the variables while preserving as much information about the data as possible. The only downside of LASSO regression is that among a group of correlated variables, LASSO tends to select only one variable and ignore the others regardless of their importance. Contrarily to LASSO, the Boruta algorithm considers the interdependence between SNPs and selects informative variables even if they are correlated and have the same effect. The three panels shared 23 SNPs; the distribution of the individuals according to these SNPs showed a grouping of individuals of each breed in well-defined clusters without any overlapping. CONCLUSIONS: The biological pathways represented by 23 breed informative SNPs resulted by the combination of PCA, LASSO, and Boruta should be explored in further analysis. The results provided by our study are promising for further applications of this method in other livestock animals.

Prevalence and concordance of oral and genital HPV in women positive for cervical HPV infection and in their sexual stable partners: An Italian screening study.

OBJECTIVES: This cross-sectional study aimed to evaluate the prevalence and type of oral HPV-infection in women with a cervical HPV-lesion and in the oral and genital mucosa of their male partners. METHODS: The study group comprised 44 sexually-active women, 20-45 years with abnormal PAP smear, not more than 6 months prior to referral together with the male partners cohabiting in stable partnerships. A detailed questionnaire was administered concerning the HPV-related risk factors. Oral swabs, oral rinses, cervical swabs and urine samples were collected. HPV DNA was detected using two different polymerase chain reactions (PCRs): MY09-11 and FAP59-64. Positive samples were genotyped by Sanger sequencing and the INNO-LiPA HPV Genotyping Extra II probe assay. The association with risk factors was assessed by fitting a generalized model, using the General Linear Model function in the R-software; correlations were calculated between all data. RESULTS: HPV was detected in 84% of Cervical Samples, in 24.3% of oral samples and in one urine sample. Only 27% of the HPV-positive results were identical with both PCR DNA assays. 8 male had oral HPV-positive samples different from women cervical samples. In one couple the urine-male sample had the same HPV present in the female-cervical sample. A significant association resulted between women/oral sex practices and men/n. of partners. CONCLUSIONS: This study reports that women (20.4%) with a diagnosis of cervical-HPV and their male partners (30,7%) are at high risk for subclinical oral HPV infection.

Fumonisin-Exposure Impairs Age-Related Ecological Succession of Bacterial Species in Weaned Pig Gut Microbiota.

Pigs are highly affected by dietary mycotoxin contamination and particularly by fumonisin. The effects of fumonisin on pig intestinal health are well documented, but little is known regarding its impact on gut microbiota. We investigate the effects of the fumonisin (FB1, 12 mg/kg feed) on the fecal microbiota of piglets (n = 6) after 0, 8, 15, 22, and 29 days of exposure. A control group of six piglets received a diet free of FB1. Bacterial community diversity, structure and taxonomic composition were carried out by V3⁻V4 16S rRNA gene sequencing. Exposure to FB1 decreases the diversity index, and shifts and constrains the structure and the composition of the bacterial community. This takes place as early as after 15 days of exposure and is at a maximum after 22 days of exposure. Compared to control, FB1 alters the ecological succession of fecal microbiota species toward higher levels of Lactobacillus and lower levels of the Lachnospiraceae and Veillonellaceae families, and particularly OTUs (Operational Taxonomic Units) of the genera Mitsuokella, Faecalibacterium and Roseburia. In conclusion, FB1 shifts and constrains age-related evolution of microbiota. The direct or indirect contribution of FB1 microbiota alteration in the global host response to FB1 toxicity remains to be investigated.

Pathogenicity of three genetically diverse strains of PRRSV Type 1 in specific pathogen free pigs.

Studies from Eastern European countries proved that porcine reproductive and respiratory syndrome virus Type 1 (PRRSV-1) harbours high genetic diversity and that genetically divergent subtypes 2-4 circulate in this area. In the present study, we compared the pathogenicity of two different PRRSV-1 subtype 2 strains and a strain representing PRRSV-1 subtype 1. Four groups of 8-week-old specific pathogen free pigs were either infected with subtype 2 strain ILI6, subtype 2 strain or BOR59, subtype 1 strain 18794, or mock inoculated. The most pronounced clinical signs were observed in pigs infected with BOR59. Pigs from both subtype 2 strain infected groups exhibited significantly elevated mean body temperatures on DPI 2 compared to the other two groups, the difference remaining significant up to DPI 13 for the BOR59 group, only. The pigs in the latter group also displayed significantly highest levels of early viremia together with the most rapid APP response. Overall, the results indicated that BOR59 strain can be considered a highly pathogenic strain, similarly to subtype 3 strains Lena and SU1-bel, while the virulence of the other subtype 2 strain ILI6 was intermediate between BOR59 and subtype 1 strain.

RNA-sequence analysis of gene expression from honeybees (Apis mellifera) infected with Nosema ceranae.

Honeybees (Apis mellifera) are constantly subjected to many biotic stressors including parasites. This study examined honeybees infected with Nosema ceranae (N. ceranae). N. ceranae infection increases the bees energy requirements and may contribute to their decreased survival. RNA-seq was used to investigate gene expression at days 5, 10 and 15 Post Infection (P.I) with N. ceranae. The expression levels of genes, isoforms, alternative transcription start sites (TSS) and differential promoter usage revealed a complex pattern of transcriptional and post-transcriptional gene regulation suggesting that bees use a range of tactics to cope with the stress of N. ceranae infection. N. ceranae infection may cause reduced immune function in the bees by: (i)disturbing the host amino acids metabolism (ii) down-regulating expression of antimicrobial peptides (iii) down-regulation of cuticle coatings and (iv) down-regulation of odorant binding proteins.

RNA-sequence analysis of primary alveolar macrophages after in vitro infection with porcine reproductive and respiratory syndrome virus strains of differing virulence.

Porcine reproductive and respiratory syndrome virus (PRRSV) mainly infects porcine alveolar macrophages (PAMs), resulting in porcine reproductive and respiratory syndrome (PRRS) in pigs. Most of the transcriptomic studies on PAMs infected with PRRSV conducted thus far have made use of microarray technology. Here, we investigated the transcriptome of PAMs in vitro at 12 h post-infection with two European PRRSV strains characterized by low (Lelystad, LV) and high (Lena) virulence through RNA-Seq. The expression levels of genes, isoforms, alternative transcription start sites (TSS) and differential promoter usage revealed a complex pattern of transcriptional and post-transcriptional gene regulation upon infection with the two strains. Gene ontology analysis confirmed that infection of PAMs with both the Lena and LV strains affected signaling pathways directly linked to the innate immune response, including interferon regulatory factors (IRF), RIG1-like receptors, TLRs and PKR pathways. The results confirmed that interferon signaling is crucial for transcriptional regulation during PAM infection. IFN-ß1 and IFN-αω, but not IFN-α, were up-regulated following infection with either the LV or Lena strain. The down-regulation of canonical pathways, such as the interplay between the innate and adaptive immune responses, cell death and TLR3/TLR7 signaling, was observed for both strains, but Lena triggered a stronger down-regulation than LV. This analysis contributes to a better understanding of the interactions between PRRSV and PAMs and outlines the differences in the responses of PAMs to strains with different levels of virulence, which may lead to the development of new PRRSV control strategies.

Evaluation of viral peptide targeting to porcine sialoadhesin using a porcine reproductive and respiratory syndrome virus vaccination-challenge model.

Targeting antigens to professional antigen presenting cells resident at the sites where effective immune responses are generated is a promising vaccination strategy. As such, targeting sialoadhesin (Sn)-expressing macrophages, abundantly present in spleen and lymph nodes where they appear to be strategically placed for antigen capture and processing, is recently gaining increased attention. Previously, we have shown that humoral immune responses to the model antigen human serum albumin can be enhanced by using a porcine Sn-specific monoclonal antibody to target the model antigen to Sn-expressing macrophages. To date however, no studies have been performed to evaluate whether targeted delivery of a pathogen-derived antigen can enhance the pathogen-specific immune response. Therefore, we selected a linear epitope on glycoprotein 4 of porcine reproductive and respiratory syndrome virus (PRRSV), which is known to be a target of virus-neutralizing antibodies. This paper reports on the targeted delivery of this viral peptide to porcine Sn-expressing macrophages and the evaluation of the subsequent immune response in a vaccination-challenge set-up. Four copies of the selected PRRSV epitope were genetically fused to a previously developed porcine Sn-targeting recombinant antibody or an irrelevant isotype control. Fusion proteins were shown to be efficiently purified from HEK293T cell supernatants and subsequently, only Sn-specific fusion proteins were shown to bind to and to be internalized into Sn-expressing cells. Subsequent immunizations with a single dose of the fusion proteins showed that peptide-specific immune responses and neutralizing antibody responses after PRRSV challenge were enhanced in animals receiving a single 500 µg intramuscular dose of the Sn-targeting fusion protein, although correlations between the two read-outs were hard to effectuate. Furthermore, a minor beneficial effect on viral clearance was observed. Together, these data show that viral peptide targeting to porcine Sn-expressing macrophages can improve the anti-viral immune response, although more research will be needed to further explore vaccination potential.

Structural and functional annotation of the porcine immunome.

BACKGROUND: The domestic pig is known as an excellent model for human immunology and the two species share many pathogens. Susceptibility to infectious disease is one of the major constraints on swine performance, yet the structure and function of genes comprising the pig immunome are not well-characterized. The completion of the pig genome provides the opportunity to annotate the pig immunome, and compare and contrast pig and human immune systems. RESULTS: The Immune Response Annotation Group (IRAG) used computational curation and manual annotation of the swine genome assembly 10.2 (Sscrofa10.2) to refine the currently available automated annotation of 1,369 immunity-related genes through sequence-based comparison to genes in other species. Within these genes, we annotated 3,472 transcripts. Annotation provided evidence for gene expansions in several immune response families, and identified artiodactyl-specific expansions in the cathelicidin and type 1 Interferon families. We found gene duplications for 18 genes, including 13 immune response genes and five non-immune response genes discovered in the annotation process. Manual annotation provided evidence for many new alternative splice variants and 8 gene duplications. Over 1,100 transcripts without porcine sequence evidence were detected using cross-species annotation. We used a functional approach to discover and accurately annotate porcine immune response genes. A co-expression clustering analysis of transcriptomic data from selected experimental infections or immune stimulations of blood, macrophages or lymph nodes identified a large cluster of genes that exhibited a correlated positive response upon infection across multiple pathogens or immune stimuli. Interestingly, this gene cluster (cluster 4) is enriched for known general human immune response genes, yet contains many un-annotated porcine genes. A phylogenetic analysis of the encoded proteins of cluster 4 genes showed that 15% exhibited an accelerated evolution as compared to 4.1% across the entire genome. CONCLUSIONS: This extensive annotation dramatically extends the genome-based knowledge of the molecular genetics and structure of a major portion of the porcine immunome. Our complementary functional approach using co-expression during immune response has provided new putative immune response annotation for over 500 porcine genes. Our phylogenetic analysis of this core immunome cluster confirms rapid evolutionary change in this set of genes, and that, as in other species, such genes are important components of the pig's adaptation to pathogen challenge over evolutionary time. These comprehensive and integrated analyses increase the value of the porcine genome sequence and provide important tools for global analyses and data-mining of the porcine immune response.

Pig immune response to general stimulus and to porcine reproductive and respiratory syndrome virus infection: a meta-analysis approach.

BACKGROUND: The availability of gene expression data that corresponds to pig immune response challenges provides compelling material for the understanding of the host immune system. Meta-analysis offers the opportunity to confirm and expand our knowledge by combining and studying at one time a vast set of independent studies creating large datasets with increased statistical power. In this study, we performed two meta-analyses of porcine transcriptomic data: i) scrutinized the global immune response to different challenges, and ii) determined the specific response to Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) infection. To gain an in-depth knowledge of the pig response to PRRSV infection, we used an original approach comparing and eliminating the common genes from both meta-analyses in order to identify genes and pathways specifically involved in the PRRSV immune response. The software Pointillist was used to cope with the highly disparate data, circumventing the biases generated by the specific responses linked to single studies. Next, we used the Ingenuity Pathways Analysis (IPA) software to survey the canonical pathways, biological functions and transcription factors found to be significantly involved in the pig immune response. We used 779 chips corresponding to 29 datasets for the pig global immune response and 279 chips obtained from 6 datasets for the pig response to PRRSV infection, respectively. RESULTS: The pig global immune response analysis showed interconnected canonical pathways involved in the regulation of translation and mitochondrial energy metabolism. Biological functions revealed in this meta-analysis were centred around translation regulation, which included protein synthesis, RNA-post transcriptional gene expression and cellular growth and proliferation. Furthermore, the oxidative phosphorylation and mitochondria dysfunctions, associated with stress signalling, were highly regulated. Transcription factors such as MYCN, MYC and NFE2L2 were found in this analysis to be potentially involved in the regulation of the immune response. The host specific response to PRRSV infection engendered the activation of well-defined canonical pathways in response to pathogen challenge such as TREM1, toll-like receptor and hyper-cytokinemia/ hyper-chemokinemia signalling. Furthermore, this analysis brought forth the central role of the crosstalk between innate and adaptive immune response and the regulation of anti-inflammatory response. The most significant transcription factor potentially involved in this analysis was HMGB1, which is required for the innate recognition of viral nucleic acids. Other transcription factors like interferon regulatory factors IRF1, IRF3, IRF5 and IRF8 were also involved in the pig specific response to PRRSV infection. CONCLUSIONS: This work reveals key genes, canonical pathways and biological functions involved in the pig global immune response to diverse challenges, including PRRSV infection. The powerful statistical approach led us to consolidate previous findings as well as to gain new insights into the pig immune response either to common stimuli or specifically to PRRSV infection.

Impact of genetic variation and geographic distribution of porcine reproductive and respiratory syndrome virus on infectivity and pig growth.

BACKGROUND: The porcine reproductive and respiratory syndrome (PRRS) is a devastating disease for the pig industry. In this study, we analysed the genetic variability of PRRS virus (PRRSV) as well as the relationship between the genetic variability, the geographical and temporal distribution of the PRRSV strains. Moreover, we investigated the association between the glycosylation patterns in PRRSV sequences and pigs growth. RESULTS: The data highlight that PRRSV strains evolve rapidly on individual farms, and temporal evolution of PRRSV is an important factor of genetic variability. Analysis of glycosylation sites in the glycoprotein 5 (GP5) ectodomain revealed that PRRSV isolates had seven combinations of putative N-linked glycosylation sites of which the N37/46/53 sites was found in 79% of the sequences. No significant relationship was found between the genetic variation of the PRRSV strains and the geographic distance. A significant relationship was found between the genetic variation and time of sampling when farm was considered as a factor in the analysis. Furthermore, the commercial semen from artificial insemination centres was not a source of PRRS transmission.The PRRSV having the glycosylation site at position N46 (N46+) were observed to have higher burden on pigs and accordingly the corresponding infected pigs had lower average daily gain (ADG) compared with those infected with PRRSV lacking the glycosylation at N46 (N46-) position site. This study showed that the number of piglets by litter infected by PRRSV was lower for the Landrace breed than for the other studied breeds (Large White, Duroc and Pietrain). CONCLUSIONS: The PRRSV genetic variability which is determined by a local and temporal evolution at the farm level could be considered in a perspective of prevention. Moreover, the association between the PRRSV glycosylation patterns and its virulence could be of interest for vaccine development. The differences of resistance to PRRSV infections among pig breeds might open new horizons for the genetic selection of robustness against PRRSV infection.

Analyses of pig genomes provide insight into porcine demography and evolution.

For 10,000 years pigs and humans have shared a close and complex relationship. From domestication to modern breeding practices, humans have shaped the genomes of domestic pigs. Here we present the assembly and analysis of the genome sequence of a female domestic Duroc pig (Sus scrofa) and a comparison with the genomes of wild and domestic pigs from Europe and Asia. Wild pigs emerged in South East Asia and subsequently spread across Eurasia. Our results reveal a deep phylogenetic split between European and Asian wild boars ∼1 million years ago, and a selective sweep analysis indicates selection on genes involved in RNA processing and regulation. Genes associated with immune response and olfaction exhibit fast evolution. Pigs have the largest repertoire of functional olfactory receptor genes, reflecting the importance of smell in this scavenging animal. The pig genome sequence provides an important resource for further improvements of this important livestock species, and our identification of many putative disease-causing variants extends the potential of the pig as a biomedical model.

Identification of serum proteomic biomarkers for early porcine reproductive and respiratory syndrome (PRRS) infection.

BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) is one of the most significant swine diseases worldwide. Despite its relevance, serum biomarkers associated with early-onset viral infection, when clinical signs are not detectable and the disease is characterized by a weak anti-viral response and persistent infection, have not yet been identified. Surface-enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF MS) is a reproducible, accurate, and simple method for the identification of biomarker proteins related to disease in serum. This work describes the SELDI-TOF MS analyses of sera of 60 PRRSV-positive and 60 PRRSV-negative, as measured by PCR, asymptomatic Large White piglets at weaning. Sera with comparable and low content of hemoglobin (< 4.52 µg/mL) were fractionated in 6 different fractions by anion-exchange chromatography and protein profiles in the mass range 1-200 kDa were obtained with the CM10, IMAC30, and H50 surfaces. RESULTS: A total of 200 significant peaks (p < 0.05) were identified in the initial discovery phase of the study and 47 of them were confirmed in the validation phase. The majority of peaks (42) were up-regulated in PRRSV-positive piglets, while 5 were down-regulated. A panel of 14 discriminatory peaks identified in fraction 1 (pH = 9), on the surface CM10, and acquired at low focus mass provided a serum protein profile diagnostic pattern that enabled to discriminate between PRRSV-positive and -negative piglets with a sensitivity and specificity of 77% and 73%, respectively. CONCLUSIONS: SELDI-TOF MS profiling of sera from PRRSV-positive and PRRSV-negative asymptomatic piglets provided a proteomic signature with large scale diagnostic potential for early identification of PRRSV infection in weaning piglets. Furthermore, SELDI-TOF protein markers represent a refined phenotype of PRRSV infection that might be useful for whole genome association studies.

Using SNP array data to test for host genetic and breed effects on Porcine Reproductive and Respiratory Syndrome Viremia.

BACKGROUND: The effect of breed on Porcine Reproductive and Respiratory Syndrome Viremia (PRRSV) was tested using data collected in 17 Italian commercial pig farms and 1096 genotypes obtained by the PorcineSNP60 BeadChip. A binomial logistic model was used to investigate the relationship between breed-clusters and PRRSV susceptibility. Breed-clusters were defined using the matrix of genomic kinship between all pairs of piglets. RESULTS: Only the contemporary group effect, defined as all piglets reared in the same herd, in the same year and whose samples were collected in the same season, was significant. Sex, age and breed-cluster showed no statistically significant effect on PRRS viremia, although the Landrace and Cross breed-clusters showed the lowest Odds-Ratio CONCLUSIONS: The model failed to detect a significant breed-cluster effect, highlighting the impact of environment and management on PRRS viremia incidence. Incomplete exposure over the observed period may have masked possible breed differences.

Oligonucleotide indexing of DNA barcodes: identification of tuna and other scombrid species in food products.

BACKGROUND: DNA barcodes are a global standard for species identification and have countless applications in the medical, forensic and alimentary fields, but few barcoding methods work efficiently in samples in which DNA is degraded, e.g. foods and archival specimens. This limits the choice of target regions harbouring a sufficient number of diagnostic polymorphisms. The method described here uses existing PCR and sequencing methodologies to detect mitochondrial DNA polymorphisms in complex matrices such as foods. The reported application allowed the discrimination among 17 fish species of the Scombridae family with high commercial interest such as mackerels, bonitos and tunas which are often present in processed seafood. The approach can be easily upgraded with the release of new genetic diversity information to increase the range of detected species. RESULTS: Cocktail of primers are designed for PCR using publicly available sequences of the target sequence. They are composed of a fixed 5' region and of variable 3' cocktail portions that allow amplification of any member of a group of species of interest. The population of short amplicons is directly sequenced and indexed using primers containing a longer 5' region and the non polymorphic portion of the cocktail portion. A 226 bp region of CytB was selected as target after collection and screening of 148 online sequences; 85 SNPs were found, of which 75 were present in at least two sequences. Primers were also designed for two shorter sub-fragments that could be amplified from highly degraded samples. The test was used on 103 samples of seafood (canned tuna and scomber, tuna salad, tuna sauce) and could successfully detect the presence of different or additional species that were not identified on the labelling of canned tuna, tuna salad and sauce samples. CONCLUSIONS: The described method is largely independent of the degree of degradation of DNA source and can thus be applied to processed seafood. Moreover, the method is highly flexible: publicly available sequence information on mitochondrial genomes are rapidly increasing for most species, facilitating the choice of target sequences and the improvement of resolution of the test. This is particularly important for discrimination of marine and aquaculture species for which genome information is still limited.

Sensitive detection and quantification of anisakid parasite residues in food products.

Anisakids are nematodes whose larval stages are often present in fish, molluscs, and crustaceans. Members of the family Anisakidae belonging to the genera Anisakis and Pseudoterranova are implicated in human infections caused by the consumption of raw or undercooked fish. Adequate cooking will kill anisakid larvae, however, killed or inactivated larvae can still cause sensitization and immunoglobulin E-dependent hypersensitivity in human. This work describes the development of DNA-based tests to detect and quantify the presence of Anisakis spp. and Pseudoterranova spp. larvae in fish and fish-derived products, including fish fillets, surimi, fish sticks, canned fish, and baby food. Primers and TaqMan MGB probes recognizing only Anisakis spp. and Pseudoterranova spp. were designed on the first internal transcribed spacer 1 regions of rDNA for a real-time polymerase chain reaction assay. A commercial probe for 18S rDNA was used to detect and quantify the total eukaryotic DNA of the samples. The specificity and sensitivity of the assays were tested using reference samples prepared from mixtures made of Anisakis larvae in different quantity of codfish, and subsequent dilutions. Studies were performed to assess the ability of the test to detect and quantify anisakids in various products. Results showed that this test is able to detect anisakid DNA contained in a proportion of 1:10(5) in 1 ng of total DNA. The high prevalence of anisakids reported in main fishery species was confirmed by frequently detecting anisakids DNA in fish muscle and fish-derived products. A partial correlation was found between the number of larvae present in the viscera and the level of contamination of fish fillets. In conclusion, this molecular test is useful to detect the presence of Anisakis spp. and Pseudoterranova spp. in fish and fish-derived products and to quantify the level of contamination along the food chain, with potential applications for fish farms, fish markets, and food producers.

Gene expression profiling of porcine alveolar macrophages after antibody-mediated cross-linking of Sialoadhesin (Sn, Siglec-1).

Sialoadhesin (Sn) is the prototypic member of the Siglecs, a family of receptors mainly involved in cell-cell interactions. For several Siglecs, but not for Sn, intracellular signaling functions have been described. Because antibody-mediated cross-linking of surface transmembrane proteins is a powerful technique to investigate cell-molecular events, Sn expressed on porcine alveolar macrophages (PAM) was cross-linked with the antibody 41D3, and the expression profiles were compared with mock-treated macrophages by microarray analysis. Gene ontology analysis of 479 differentially expressed transcripts identified gene categories related to membrane localization, signal transduction, receptor and communication activities. Analyses of the human KEGG pathway database identified MAP kinase signaling, regulation of actin cytoskeleton, adipocytokine signaling, and wnt signaling as significantly altered pathways, supporting a role for Sn as intracellular signaling molecule. Real-time PCR of a subset of modulated genes confirmed these results and highlighted the reliability of a short-term cross-linking treatment for transcriptomic analysis of receptor functions.

Genetic analysis of anal atresia in pigs: evidence for segregation at two main loci.

Anal atresia is a relatively common congenital malformation that occurs in about 1 out of 5000 infants, caused by abnormal hindgut development of the embryo, often associated with other developmental anomalies (e.g., Currarino, Townes-Brock, Pallister-Hall syndromes, and VATER association). Genetic analysis in human families is exceedingly difficult due to the multifactorial nature of the trait. In pigs, anal atresia occurs at a higher incidence (0.18%) than in humans. A complete genome scan (165 microsatellite markers) was performed using a backcross pedigree previously obtained by crossing affected animals from a partially inbred line, selected for a high incidence of anal atresia, with an unaffected male of a different breed (Meishan). The data set was analyzed with classical linkage (TWOPOINT) and nonparametric genetic methods (NPL, Non-Parametric Linkage, and TDT, Transmission Disequilibrium Test). Both methods support association of the trait with two loci on Chromosomes 9 and 15. GLI2 (GLI-Kruppel family member GLI2) was identified as a positional candidate gene based on comparative mapping; radiation hybrid mapping confirmed that this locus is located within the QTL region.