A high-density genome-wide approach reveals novel genetic markers linked to small ruminant lentivirus susceptibility in sheep.

Visna/Maedi virus (VMV) is lentiviral disease of sheep responsible for severe production losses. Multiple genomic regions associated with infection were reported indicating genetic complexity. In this study, a combined genome-wide approach using a high-density SNP array has been performed, comparing VMV-infected (n = 78) and non-infected (n = 66) individuals of the Valle del Belice breed. The serological tests showed a seroprevalence of 26%. The comparison among results from different approaches (GWAS, Fisher's exact test and the FST analysis) revealed two association signals: on OAR03 close to the GRIN2B gene and on OAR05 close to the TMEM232 gene. To the best of our knowledge, there has been no previous association between these genes and lentiviral infection in any species. The GRIN2B gene plays a role in pain response, synaptic transmission, and receptor clustering, while TMEM232 is involved in the development of immune-related disorders. The results highlighted new aspects of the genetic complexity related to the resistance/susceptibility to VMV in sheep, confirming that studies on different breeds can lead to different results. The ideal approach for validation of the markers identified in our study is to use samples from a population independent from the discovery population with the same phenotype used in the discovery stage.

Analysis of ddRAD-seq data provides new insights into the genomic structure and patterns of diversity in Italian donkey populations.

With more than 150 recognized breeds, donkeys assume relevant economic importance, especially in developing countries. Even if the estimated number of heads worldwide is 53M, this species received less attention than other livestock species. Italy has traditionally been considered one of the cradles of European donkey breeding, and despite a considerable loss of biodiversity, today still counts nine autochthonous populations. A total of 220 animals belonging to nine different populations were genotyped using the double-digest restriction site associated DNA (ddRAD) sequencing to investigate the pattern of diversity using a multi-technique approach. A total of 418,602,730 reads were generated and successfully demultiplexed to obtain a medium-density SNP genotypes panel with about 27K markers. The diversity indices showed moderate levels of variability. The genetic distances and relationships, largely agree with the breeding history of the donkey populations under investigation. The results highlighted the separation of populations based on their genetic origin or geographical proximity between breeding areas, showed low to moderate levels of admixture, and indicated a clear genetic difference in some cases. For some breeds, the results also validate the success of proper management conservation plans. Identified runs of homozygosity islands, mapped within genomic regions related to immune response and local adaptation, are consistent with the characteristics of the species known for its rusticity and adaptability. This study is the first exhaustive genome-wide analysis of the diversity of Italian donkey populations. The results emphasized the high informativeness of genome-wide markers retrieved through the ddRAD approach. The findings take on great significance in designing and implementing conservation strategies. Standardized genotype arrays for donkey species would make it possible to combine worldwide datasets to provide further insights into the evolution of the genomic structure and origin of this important genetic resource.

Donkeys assume relevant economic importance in several countries worldwide. However, the genetic structure of these populations is less investigated compared to other species. The aim of this study was to investigate the genetic background of nine different Italian donkey populations. A total of 220 animals were genotyped with about 27K markers extracted by the double-digest restriction site associated DNA sequencing. The consistency of the results across different approaches agreed with the demographic history, the origin, and previous results on the nine donkey populations, suggesting that our conclusions are robust. Moreover, the results of the present study highlighted low to moderate levels of admixture and, for some breeds, confirmed the success of proper management conservation plans.

Polymorphisms at Candidate Genes for Fat Content and Fatty Acids Composition: Effects on Sheep Milk Production and Fatty Acid Profile Using Two Dietary Supplementations.

The nutritional value of sheep's milk and its derivatives is influenced by the lipid fraction, which is affected by diet and genetics. This study aimed to explore the genetic variations in the DGAT1 and SCD genes and assessed the impact of the DGAT1 genotype on milk quality in Valle del Belìce sheep, considering diet supplementation with carob pulp and barley grain. Among the potentially polymorphic sites, only DGAT1 g.127 C > A and SCD g.87 C > A showed variability. The DGAT1 genotype did not significantly impact milk yield and composition, except for higher urea content in the CA genotypes than in the CC ones. Carob pulp increased the milk fat content compared to barley grain. Genetic variation in DGAT1 was associated with changes in the milk fatty acid profile; specifically, the CA genotype exhibited higher levels of short-chain fatty acids and lower levels of polyunsaturated fatty acids compared to the CC genotype. Carob pulp supplementation increased saturated fatty acids and reduced unsaturated fractions, leading to milk with higher atherogenic and thrombogenic indices. No significant interaction was found between genotype and diet. This study provides insights into the genetic and dietary factors influencing sheep's milk composition. Further research is needed to understand the impact of these genetic variations on milk production and composition, as well as to determine optimal levels of carob pulp for improving fat percentage and promoting sustainable sheep breeding practices.

Polymorphism at the CSN1S1 Locus and Energy Intake Level Affect Milk Traits and Casein Profiles in Rossa Mediterranea Goats.

A total of twenty-seven Rossa Mediterranea lactating goats, consisting of nine homozygous for strong alleles (AA), twelve heterozygous (AF) and six homozygous for weak alleles (FF) at the CSN1S1 locus, were used to evaluate the effect of genotype, diet and genotype × diet interaction on goat milk traits and casein profile. The goats were used in a 3 × 3 factorial arrangement of treatments, with three genotypes (AA, AF and FF) and three different energy intake levels: high (H), medium (M) and low (L). The diets supplied a complete pelleted feed containing 65% of alfalfa hay, respectively, at 150%, 100% and 70% of the total energy requirements. Milk yield was significantly affected by the genotype and diet: Lower levels were found in FF goats than in AA and AF genotypes (673.7 vs. 934.5 and 879.8 d/g, respectively; p = 0.002) as well as in goats fed with the L diet (651.5 vs. 1041 and 852.9 g/d for H and M diet, respectively, p < 0.001). The genotype influenced the casein profile. Specifically, AA goat milk exhibited higher concentrations of total casein and αs1-casein compared to AF and FF genotypes (for total casein and αs1-casein, respectively: 24.9 vs. 20.4 and 19.8 g/kg, p = 0.001; 7.2 vs. 3.7 and 0.7 g/kg, p < 0.001), while the FF genotype showed higher values for αs2-casein concentrations compared to homozygous AA and heterozygous AF goats (3.1 vs. 2.4 and 2.5 g/kg, respectively, p < 0.001). A significant genotype x diet interaction occurred for αs2-casein levels (g/kg) (p = 0.034) and αs1-casein yields (p = 0.027): The αs2-casein level was not affected by the diet in AA goats, whereas it increased with energy intake in AF and FF genotypes. Conversely, the αs1-casein yield gradually increased with energy intake in AA and AF groups, whereas the diet in FF goats did not modify it. The results demonstrated that high energy input, as well as the strong allele at the CSN1S1 locus, enhanced milk production and casein concentrations. Furthermore, they confirmed the existence of an interaction between αs1-casein polymorphism and diets, influencing the milk casein composition and yield.

Genome-wide population structure, homozygosity, and heterozygosity patterns of Nero Siciliano pig in the framework of Italian and cosmopolitan breeds.

Analysis of genomic data is becoming more and more common for the effective management of livestock breeding programmes, even in the case of local populations. In this work, the genome-wide data of Nero Siciliano pig breed were compared to that of wild boar, Italian local and cosmopolitan breeds to investigate its genetic structure, and runs of homozygosity (ROH) and heterozygosity patterns. The Nero Siciliano has been reported to have the highest rate of genetic diversity among the Italian breeds, and a genetic variability comparable to that of the cosmopolitan breeds. Analyses of genomic structure and relationships underlined its proximity to wild boar, and an internal substructure probably linked to different family lines. The breed showed a low value of inbreeding estimated from ROH, and the highest diversity index among the Italian breeds, even if lower than that of the cosmopolitans. Four ROH islands in three chromosomes (SSC8, SSC11, and SSC14) and one heterozygosity-rich region (SSC1) were identified in Nero Siciliano, highlighting genomic regions related to productive QTL. Across breeds, SSC8 and SSC14 were the chromosomes with most ROH islands, with Mora Romagnola and wild boar showing the highest level of autozygosity. Chromosomes SSC2, SSC6, SSC8 and SSC13 showed the majority of runs of heterozygosity regions, mainly found in the cosmopolitan pig breeds, which reported several genes associated with health-related QTL. The outlined results can help to better identify the genomic profile of this local breed in order to plan matings, maintain adequate internal diversity and exploit the production system.

Alternative Molecular Tools for the Fight against Infectious Diseases of Small Ruminants: Native Sicilian Sheep Breeds and Maedi-Visna Genetic Susceptibility.

Maedi-visna (MV) is a disease caused by small ruminant lentiviruses. It is included in the list of notifiable terrestrial animal diseases due to economic losses and animal welfare harm in the sheep sector. To date, control programs remain the onliest approach to avoiding infection. The allelic variant p.Glu35Lys (E35K) of the TMEM154 gene has been strongly associated with host vulnerability to MV illness. The present study aimed to investigate the association of TMEM154 E35K allele frequencies with MV susceptibility in native Sicilian sheep breeds. More than 400 animals from 14 local sheep were serologically tested and genotyped for the TMEM154 E35K polymorphism. The local breeds displayed different values of MV seroprevalence, with the lowest antibody prevalence in Barbaresca and Pinzirita breeds. TMEM154 protective allele (K35) was less frequent than the risk allele (E35) in Valle del Belìce breed, whereas the other three breeds showed a more balanced alleles distribution. A positive association between seroprevalence and genotype was found in the entire sample set. The risk of infection resulted in more than 3-fold times as high in sheep with EK and EE genotype compared to the KK genotype. Our data could be helpful in establishing selection breeding programs aimed at reducing MV infection in Sicilian sheep farming and encouraging the breeding of native breeds.

Genome-wide survey on three local horse populations with a focus on runs of homozygosity pattern.

Purosangue Orientale Siciliano, Sanfratellano and Siciliano represent the Sicilian equine genetic resource. This study aimed to investigate the genetic diversity, population structure and the pattern of autozygosity of Sicilian horse populations using genome-wide single-nucleotide polymorphism (SNP) data generated with the Illumina Equine SNP70 array. The genotyping data of 17 European and Middle East populations were also included in the study. The patterns of genetic differentiation, model-based clustering and Neighbour-Net showed the expected positioning of Sicilian populations within the wide analysed framework and the close connections between the Purosangue Orientale Siciliano and the Arab as well as between Sanfratellano, Siciliano and Maremmano. The highest expected heterozygosity (He ) and contemporary effective population size (cNe) were reported in Siciliano (He  = 0.323, cNe = 397), and the lowest were reported in Purosangue Orientale Siciliano (He  = 0.277, cNe = 10). The analysis of the runs of homozygosity and the relative derived inbreeding revealed high internal homogeneity in Purosangue Orientale Siciliano and Arab horses, intermediate values in Maremmano and Sanfratellano and high heterogeneity in the Siciliano population. The genome-wide SNP analysis showed the selective pressure on Purosangue Orientale Siciliano towards traits related to endurance performance. Our results underline the importance of planning adequate conservation and exploitation programmes to reduce the level of inbreeding and, therefore, the loss of genetic diversity.

Detection of Mycoplasma agalactiae in Ticks (Rhipicephalus bursa) Collected by Sheep and Goats in Sicily (South-Italy), Endemic Area for Contagious Agalactia.

The aim of this preliminary study was to investigate the presence of Mycoplasma agalactiae (Ma) or other Contagious Agalactia (CA) causative organisms, in hard ticks infesting milking sheep and goats in endemic areas for CA in Sicily (South-Italy). Although there is accumulating evidence to support the role of ticks in the transmission of blood-borne haemoplasmas, information regarding their role in the transmission of CA, remains scarce. Ticks (n = 152) were collected from 25 lactating sheep and goats from three farms with previous outbreaks of CA. Microbiological and biomolecular, as well as serological analysis were performed on milk, tick, and serum samples, respectively. Rhipicephalus bursa species predominated, comprising 84.8% of the sampled ticks. Mycoplasma-like colonies were isolated from 5/56 (8.9%) tick pools and were identified as Ma by specific PCR and 16S rRNA gene sequencing. Unexpectedly, the organism was isolated from R. bursa ticks recovered only from animals whose milk tested negative for the pathogen. This preliminary demonstration suggests the potential role for ticks to act as a reservoir for the organisms, with potential involvement in the spread and maintenance of CA. Further work is required to determine the location of the organisms within the body of the ticks and to assess transmission potential.

Feeding System Resizes the Effects of DGAT1 Polymorphism on Milk Traits and Fatty Acids Composition in Modicana Cows.

The interaction between genetic polymorphism and feeding system on milk traits and fatty acid composition was investigated in Modicana cows. Two DGAT1 K232A genotypes (AK and AA) and two feeding regimes, extensive system (EX) with 8 h of grazing without concentrate (EX) and semi-intensive systems (SI) with 2 h of grazing with concentrate, were investigated. DGAT1 genotype did not influence milk yield and composition. The feeding system affected milk composition: protein was significantly higher in SI and lactose in the EX system. A significant genotype × feeding system interaction was observed: the protein and casein levels of AK cows were higher in the SI compared to the EX system. Milk fatty acids profile, total saturated to total unsaturated fatty acids, n-6 to n-3 ratios, and atherogenic index were affected by the feeding system, improving the healthy properties of milk from animals reared in the extensive system. DGAT1 genotype influenced the fatty acid composition: milk from AA cows had a more favorable fatty acid composition due to lower total saturated fatty acids, saturated to unsaturated ratio, atherogenic index, and higher levels of oleic acid and total unsaturated fatty acids. Furthermore, an interaction genotype x feeding system was observed: the AK milk was richer in short-chain FAs (C4:0-C8:0) and C10:0 only in the EX but not in the SI system. Our data suggest that a high amount of green forage in the diet of Modicana cows can resize the effect of the DGAT1 genotype on milk traits and fatty acids composition.

Ultrasensitive Electrochemical Impedance Detection of Mycoplasma agalactiae DNA by Low-Cost and Disposable Au-Decorated NiO Nanowall Electrodes.

Nanostructured electrodes detecting bacteria or viruses through DNA hybridization represent a promising method, which may be useful in on-field applications where PCR-based methods are very expensive, time-consuming, and require trained personnel. Indeed, electrochemical sensors combine disposability, fast response, high sensitivity, and portability. Here, a low-cost and high-surface-area electrode, based on Au-decorated NiO nanowalls, demonstrates a highly sensitive PCR-free detection of a real sample of Mycoplasma agalactiae (Ma) DNA. NiO nanowalls, synthesized by aqueous methods, thermal annealing, and Au decoration, by electroless deposition, ensure a high-surface-area platform for successful immobilization of Ma thiolated probe DNA. The morphological, chemical, and electrochemical properties of the electrode were characterized, and a reproducible detection of synthetic Ma DNA was observed and investigated by impedance measurements. Electrochemical impedance spectroscopy (EIS) ascribed the origin of impedance signal to the Ma DNA hybridization with its probe immobilized onto the electrode. The electrode successfully discriminates between DNA extracted from healthy and infected sheep milk, showing the ability to detect Ma DNA in concentrations as low as 53 ± 2 copy number µL-1. The Au-decorated NiO nanowall electrode represents a promising route toward PCR-free, disposable, rapid, and molecular detection.

Effect of GH p.L127V Polymorphism and Feeding Systems on Milk Production Traits and Fatty Acid Composition in Modicana Cows.

Growth hormone participates in the regulation of lactation and lipid metabolism. A trial study was conducted to evaluate the effects of genetic polymorphism at GH p.L127V and its interaction with feeding system (extensive, EX; semi-intensive, SI) on milk traits and fatty acids composition in Modicana cows. In the semi-intensive farm (SI) diet consisted of hay, concentrate and 2 h of grazing. In the extensive farm (EX) feeding consisted in 8 h of grazing and hay. The frequencies of LL, LV and VV genotypes were, respectively: 0.64, 0.34, 0.02. GH polymorphism and its interaction with feeding system did not influence milk yield and composition. Cows carrying LL genotype produced milk with lower 6:0 and 8:0 and higher 16:1 c9 and 18:1 c9, total UFA and total MUFA. Feeding significantly affected fatty acids: in EX cows lower SFA and higher PUFA and UFA were found, compared to SI cows. The lower, more favorable atherogenic index of milk from EX system was coherent with the improved healthy characteristics of milk from animals fed almost exclusively on pasture. A significant interaction genotype x feeding system was evident for 18:1, higher in the LL cows only in the EX system, but not in the SI system.

Bovine Papillomavirus 1 Gets Out of the Flock: Detection in an Ovine Wart in Sicily.

A proliferative cauliflower lesion was excised from the udder of a sheep. Histological investigation confirmed the macroscopic classification of the lesion as a papilloma, without any fibroblastic proliferation. PCR revealed the presence of bovine papillomavirus (BPV), which was further confirmed by the identification of a Deltapapillomavirus 4 by Next Generation Sequencing analysis. This was subsequently classified as bovine papillomavirus type 1. Negative staining electron microscopy (EM) analyses produced negative test results for papillomavirus particles. RNA in situ hybridization (ISH) confirmed the presence of BPV-1. The results further confirm the ability of BPVs belonging to the Deltapapillomavirus genus to infect distantly related species and to cause lesions that are different from sarcoids.

Validation of Loop-Mediated Isothermal Amplification (LAMP) Field Tool for Rapid and Sensitive Diagnosis of Contagious Agalactia in Small Ruminants.

Contagious agalactia (CA), an infectious disease of small ruminants, caused by Mycoplasma agalactiae, is responsible for severe losses to dairy sheep production with substantial socioeconomic impacts on small-scale farmers. The diagnosis of CA is still problematic, time-consuming and requires well-equipped labs for confirmation of outbreaks. Therefore, rapid, accurate and cost-effective diagnostic tests are urgently needed. This work aims to validate a novel Loop-Mediated Isothermal Amplification (LAMP) test, based on the p40 target gene, for the detection of M. agalactiae in dairy sheep in order to confirm its potential practical use as a rapid and cheap field test. The LAMP system proposed in this study consists of a portable device composed of real-time fluorometer with the automatic interpretation of results displayed in a tablet. A total of 110 milk samples (90 positives and 20 negatives) were analysed to optimise the analysis procedure and to investigate the efficacy and robustness of the LAMP method. All samples were analysed using LAMP and conventional real-time PCR to compare the diagnostic sensitivity of the methods. The sensitivity of the LAMP was 10-fold higher than that of real-time PCR, with a detection limit up to 103 CFU/ml. The LAMP assay was able to detect M. agalactiae in 81 of 90 (90%, 95%CI 0.84-0.96) positive milk samples compared to 69 (77%, 95%CI 0.59-0.95) positive samples detected by real-time PCR; no positive signal occurred for any of the negative milk samples in either test. Therefore, the LAMP assay was found to be more sensitive than real-time PCR, low-cost, easy to perform, fast and not affected by contamination, indicating its potential as an effective diagnostic tool in the field level for the diagnosis of CA.

Casein haplotype diversity in seven dairy goat breeds.

Selection, drift, gene flow and breeding have extensively shaped the genomic variability of domestic animals. In goat species, several mutations identified within the casein genes have been shown to affect the level of gene expression of milk production traits. The four casein genes - CSN1S1, CSN2, CSN1S2 and CSN3 - are organized in a cluster of 250 kb located in chromosome 6, and due to tight linkage, their genetic variability is well depicted by haplotypes which are transmitted to the progeny. Thirty single nucleotide polymorphisms (SNPs) located within the casein gene cluster were used to characterize the haplotype variability of six southern Italian goat breeds (Girgentana, Maltese, Rossa Mediterranea, Argentata dell'Etna, Messinese, Capra dell'Aspromonte). A representative sample of the Norwegian dairy goat breed (Norsk melkegeit) has been used as an out-group to obtain a weighted measure of genetic diversity in the metapopulation. A total of 54 haplotypes were detected among the seven breeds: 26, 9, 8 and 11 haplotypes were found at CSN1S1, CSN2, CSN1S2 and CSN3 respectively. The number of haplotypes per breed was 14 (Norwegian), 26 (Messinese), 27 (Rossa Mediterranea and Girgentana) and 31 (Maltese, Argentata dell'Etna and Capra dell'Aspromonte). The Maltese breed showed the highest number of private haplotypes, whereas the Norwegian goat recorded the highest number of shared haplotypes. The linkage disequilibrium analysis showed higher levels of association for the SNP pairs within casein loci than SNP pairs between casein loci, likely reflecting low levels of intra-genic recombination. The highest linkage disequilibrium values were found in CSN1S1 and CSN2 genes in all the breeds, except for Argentata dell'Etna and Rossa Mediterranea. The resolution of the haplotype diversity at the casein cluster can be exploited both for selective and conservative plans.

Polymorphism at donkey ß-lactoglobulin II locus: identification and characterization of a new genetic variant with a very low expression.

In the last years, donkey milk had evidenced a renewed interest as a potential functional food and a breast milk substitute. In this light, the study of the protein composition assumes an important role. In particular, ß-lactoglobulin (ß-LG), which is considered as one of the main allergenic milk protein, in donkey species consists of two molecular forms, namely ß-LG I and ß-LG II. In the present research, a genetic analysis coupled with a proteomic approach showed the presence of a new allele, here named F, which is apparently associated with a null or a severely reduced expression of ß-LG II protein. The new ß-LG II F genetic variant shows a theoretical average mass (Mav) of 18,310.64 Da, a value practically corresponding with that of the variant D (∆mass < 0.07 Da), but differs from ß-LG II D for two amino acid substitutions: Thr100 (variant F) → Ala100 (variant D) and Thr118 (variant F) → Met118 (variant D). Proteomic investigation of the whey protein fraction of an individual milk sample, homozygous FF at ß-LG II locus, allowed to identify, as very minor component, the new ß-LG II F genetic variant. By MS/MS analysis of enzymatic digests, the sequence of the ß-LG II F was characterized, and the predicted genomic data confirmed.